Exam 1 Hematology

Hematologic System

• The hematologic system includes the formation of the Erythrocyte-Megakaryocyte lineage (EMk) which differentiates into erythrocyte progenitors and Megakaryocyte progenitors through stimulation by thrombopoietin (TPO) and Stem Cell Factor (CSF).
• Thrombopoietin stimulates the controlled fragmentation of megakaryocytes, resulting in the release of >1000 platelets per cell (avg 5-10k).

Platelets

• Each platelet is ~3 micrometers in size.
• Platelets stick to damaged blood vessels using a glycoprotein called Von Willebrand Factor, which connects to a protein complex on the platelet surface called GP Ib-IX.
• Platelets secrete ADP, thromboxane A2, and serotonin, which activate other platelets to become sticky and join in.
• Platelet IIb/IIIa protein increases the binding of platelets to fibrinogen and thus platelets sticking together.

Coagulation Cascade

• The coagulation cascade involves multiple factors, including:
  • Fitzgerald Factor (High molecular weight kininogen)
  • Fletcher Factor (Prekallikrein)
  • Thromboplastin
  • Factor I (Fibrinogen)
  • Factor II (Prothrombin)
  • Factor III (Tissue Thromboplastin)
  • Factor IV (Calcium)
  • Factor V (Proaccelerin)
  • Factor VII (Proconvertin)
  • Factor VIII (Antihemophilic factor A)
  • Factor IX (Antihemophilic factor B)
  • Factor X (Stuart factor)
  • Factor XI (Antihemophilic factor C)
  • Factor XII (Hageman factor)

Coagulation Pathways

• There are two main coagulation pathways:
  • Tissue Factor Pathway: Factors I, II, V, VII, X
  • Contact Activation Pathway: Factors I, II, VIII, IX, X, XI, XII
  • Common Pathway: I, II, V, X

Inhibitors of Coagulation

• Thrombin binds to thrombomodulin on the surface of endothelial cells and converts Protein C to its active form.
• Protein C, along with Protein S, its cofactor, serve many functions of anticoagulation, including:
  • Breakdown of Factor Va
  • Breakdown of Factor VIIIa
  • Blocking the inhibition of TPA
• Antithrombin III, sometimes simply called antithrombin, mainly targets proteases of the Contact Activation pathway: Factor IX, Factor X, Factor XI, and Factor II.

Thrombocytopenia and Thrombocytosis

• Thrombocytopenia: a platelet count of less than 150,000, symptoms include petechial hemorrhages, excessive bleeding, easy bruising, and sometimes fatigue.
• Thrombocytosis or thrombocythemia: a platelet count of above 450,000, symptoms include chest pain, claudication, syncope, thrombosis, and may also cause petechial hemorrhages or purpura.

Case Study

• A 1-year-old baby presents with a lifetime history of nosebleeds and bloody stool, and a PT is normal, PTT is prolonged, and Factor VIII levels return at 18% of normal.
• The diagnosis is likely Hemophilia A, and Factor VIII levels should be raised to 50-100% of normal before procedures.

Hematopoiesis

• Hematopoietic stem cells become multi-potent progenitors, which lack self-renewal capacity
• Interleukin 7 stimulates progression to lymphocytic lineage
• Stem Cell Factor primarily stimulates myeloid lineage
• Thrombopoietin stimulates platelet development

Lymphoid Lineage

• Common lymphoid progenitor differentiates into B-cell progenitor and T/NK cell progenitors
• Interleukin 4 stimulates B-cell growth
• Interleukin 2 stimulates T-cell growth
• Interleukin 15 stimulates Natural Killer cell growth
• Lymphocytic dendritic cells require multiple factors, including interleukins, Tumor Necrosis Factor-alpha, and Flt-3 ligand

Myeloid Lineage

• Common myeloid progenitor differentiates into granulocyte-macrophage and Erythrocyte-Megakaryocyte lineages
• Granulocyte-macrophage lineage is stimulated by Stem Cell Factor and granulocyte-macrophage colony-stimulating factor
• Erythrocyte-Megakaryocyte lineage is stimulated by interleukin 3, Stem Cell Factor, and thrombopoietin

Formed Elements

• Hematopoietic stem cells reside in bone marrow niches: osteoblastic and vascular
• There are three formed elements in the blood: erythrocytes, leukocytes, and thrombocytes

Leukocytes

• Neutrophils: 40-60% of circulating leukocytes, first responders, engage in phagocytosis, contain digestive enzymes and cytotoxic proteins
• Eosinophils: 2-4% of circulating leukocytes, named due to eosin staining, release granular contents, activated in response to parasites, allergens, and cancer cells

Erythrocytes

• Red blood cell size is measured by mean corpuscular volume (MCV)
• MCV is calculated as Hematocrit divided by RBC count
• Macrocytosis: high MCV, microcytosis: low MCV
• Mean corpuscular hemoglobin (MCH) measures hemoglobin amount
• MCH is calculated by Hemoglobin divided by RBCs
• Mean corpuscular hemoglobin concentration (MCHC) measures hemoglobin concentration within the cell
• Hypochromia: low MCHC, hyperchromia: high MCHC

Iron

• Heme groups contain iron atoms, essential for oxygen binding
• Ferritin stores excess iron inside cells, produced in response to iron presence
• Transferrin mediates iron transport in the plasma
• Iron deficiency affects ferritin and transferrin levels
• Total Iron Binding Capacity (TIBC) measures iron needed to bind all transferrin

Natural Immunity

• Physical barriers: acidity in the stomach, skin barriers, and mucosal linings
• Certain white blood cells: neutrophils, mast cells, and macrophages that nonspecifically target microorganisms
• Cytokines:
  • Interleukins: stimulate lymphocyte proliferation and maturation in response to pathogens
  • Interferons: kill viruses and activate macrophages to kill phagocytosed organisms
  • Colony-stimulating factors: stimulate granulocyte, monocyte, and macrophage proliferation and maturation
  • Tumor necrosis factors: stimulate dendritic cell proliferation and acute inflammation
  • TGF-beta: promotes wound healing
• Lysozymes: enzymes that cleave bacterial cell walls
• Complement system:
  • Small liver-produced proteins that circulate as inactive precursors (zymogens)
  • Activated complement proteins trigger pathways leading to bacterial cell wall disruption, phagocytosis, and inflammation
• Mannose-binding lectin (MBL) pathway:
  • MBL binds to mannose and glucose residues on bacterial cell walls
  • Activates Mannose Binding Lectin Associated Serine Protease (MASP)
  • MBL and MASP join to activate C4, C2, and C3 convertase

Complement System

• Classical pathway:
  • Triggered by antigen-antibody complex
  • C1 activates C4, which is then cleaved into C4a and C4b
  • C14b and C2a combine to form C3 convertase
  • C3 convertase breaks C3 into C3a and C3b
  • C5 convertase forms and breaks C5 into C5a and C5b
  • MAC (C5b6789) forms and ruptures bacterial cell walls
• Alternative pathway:
  • Triggered by bacterial endotoxins, yeast cell wall antigens, and cobra venom
  • Factor C3b combines with Factor B, activating an open site on Factor B
  • Factor C3bBb forms and acts as C3 convertase
• Lectin Mannose pathway:
  • Relies on MBL synthesized in the liver
  • Activates C4, C2, and C3 convertase
  • Forms MAC (C5b6789)

Functions of Complement System

• Cell lysis: MAC ruptures bacterial cell walls
• Opsonization: C3b activates neutrophils and macrophages to phagocytose pathogens
• Chemotaxis: C5a attracts neutrophils and macrophages to areas of concern
• Mast cell activation: C3a, C4a, and C5a activate mast cells to release histamine and serotonin

Macrophages

• Phagocytose many bacteria and damaged cells
• Secrete toxic enzymes that kill bacteria
• Can form granulomas in chronic inflammation
• Microglia are brain phagocytic cells that regenerate from the yolk sac

Dendritic Cells

• Reside in mucosa, skin, spleen, and lymph nodes
• Present antigens to T-cells to activate them
• Capable of phagocytosis

Eosinophils

• Release substances toxic to parasites

Basophils

• Release heparin, histamine, and serotonin to increase vascular permeability

Mast Cells

• Release similar substances to basophils
• Can phagocytose and present antigens

Natural Killer Cells

• Lymphocyte-like cells that kill virus-containing cells
• Express activation receptors that recognize virus-infected cells and tumor cells without requiring prior immune recognition

Anemia Learning Objectives

• Compare and contrast various anemias according to pathophysiology
• Differentiate the etiology, clinical presentation, diagnostic evaluation, and management of anemias
• Test the intrinsic and extrinsic causes of hemolysis
• Debate the etiology and management of the causes of normocytic anemia
• Use a pragmatic approach to patients with anemia
• Relate the history, physical examination, and testing of anemia in patients
• Illustrate general principles regarding treatment of anemias
• Differentiate the anemias relating to blood loss, including both acute and chronic
• Differentiate the autoimmune hemolytic anemias

Marrow Hypoplasia

• Primary anemias tend to be normocytic, but patients have a low reticulocyte count and pancytopenia
• Bone marrow biopsy is the gold standard of diagnosis
• Patients who have purely red cell aplasia/hypoplasia generally have a T-cell autoimmunity, or some other direct toxicity to erythrocyte precursors

Aplastic Anemia

• Inherited forms usually present in the first decade of life, occurring due to abnormal ribosomal biogenesis, DNA repair, or telomere length
• Acquired forms are extremely rare but usually due to autoimmune cytotoxic T-cells targeting the hematopoietic stem cells
• Drugs associated with acquired AA are beta-lactam antibiotics, sulfonamides, NSAIDs, anticonvulsants, gold salts, and antithyroid medications
• Diagnosis is confirmed by bone marrow aspirate, biopsy, iron stain, and flow cytometry
• It is important to differentiate this from other conditions that may cause pancytopenia, such as Myelodysplastic Syndromes (MDS), Large granular lymphocytic leukemia, and Paroxysmal Nocturnal Hemoglobinuria
• Treatment for those who are younger and who have a suitable donor is allogeneic hematopoietic stem cell transplant (HSCT)
• For those who are over the age of 50, or in whom no suitable donor is found, treatment is immunosuppression with antithymosite globulin, cyclophosphamide, and prednisone
• EPO stimulates erythrocyte formation
• Eltrombopag is a bone marrow stimulant that may be used in patients ineligible for HSCT who have chronically low platelets

Fanconi Anemia

• The most frequently reported syndrome of bone marrow failure
• Often presents with congenital defects such as a short stature, developmental delay, and underlying endocrinopathies
• Primarily autosomal recessive, Fanconi typically presents as a pancytopenia, often around the age of 3-5
• Diagnosis is made by chromosome breakage testing
• The only permanent cure is HSCT, though some patients may respond to hematopoietic growth factors
• Most patients eventually develop AML or other cancers

Myelodysplastic Syndromes

• Primarily occur in patients over the age of 60, with the average age of diagnosis being 71
• Characterized by ineffective hematopoiesis and peripheral cytopenia
• Most cases are idiopathic, with the remainder due to exposure to an alkylating agent or radiation
• Patients with MDS often die due to complications of bone marrow failure or progression to acute myelogenous leukemia (AML)
• CBC and peripheral smear will show pancytopenia and dysplastic findings, with macrocytosis or nucleated RBCs as common findings
• It is important to rule out drug-induced cytopenia and B12/folate deficiency
• There are six current classifications: MDS with multilineage dysplasia (MDS-MLD), etc.

Anemia and EPO

• In some cases of anemia, increased EPO stimulates TPO receptors, which may lead to increased thrombopoiesis.
• This is not uncommon in iron deficiency anemia.

Smoking and Thrombogenesis

• Smoking causes increased inflammation, leading to increased thrombogenesis.
• Smokers have increased levels of von Willebrand factor, resulting in significant hypercoagulability.
• No treatment is needed unless platelets exceed 1,000,000/μL.

Case Study: 1-year-old Baby with Nosebleeds and Bloody Stool

• The patient presents with a lifetime history of nosebleeds and bloody stool, which is concerning for a bleeding disorder.
• Initial workup includes PT/PTT, CBC, and platelet count, which returns normal, except for a prolonged PTT.
• Differential diagnosis includes a coagulation factor deficiency, such as hemophilia A or B.
• Factor VIII levels return at 18% of normal, which is consistent with hemophilia A.
• Factor VIII levels should be raised to 80-100% of normal before procedures.

Platelets and Thrombopoietin

• Thrombopoietin stimulates the formation of the Erythrocyte-Megakaryocyte lineage (EMk).
• EMk cells differentiate into Megakaryocyte progenitors, which undergo controlled fragmentation, releasing 1,000-5,000 platelets per cell.
• Thrombopoietin is released by the kidney and liver in response to low platelets.

Platelet Function

• Platelets stick to damaged blood vessels using von Willebrand Factor, which connects to a protein complex on the platelet surface called GP Ib-IX.
• Platelets secrete ADP, thromboxane A2, and serotonin, which activate other platelets to become sticky and join in.
• Platelet IIb/IIIa protein increases the binding of platelets to fibrinogen, leading to platelet aggregation.

The Coagulation Cascade

• The coagulation cascade involves the following factors:
• Factor I (Fibrinogen)
• Factor II (Prothrombin)
• Factor III (Tissue Thromboplastin)
• Factor IV (Calcium)
• Factor V (Proaccelerin)
• Factor VII (Proconvertin)
• Factor VIII (Antihemophilic factor A)
• Factor IX (Antihemophilic factor B)
• Factor X (Stuart factor)
• Factor XI (Antihemophilic factor C)
• Factor XII (Hageman factor)
• There is no Factor VI.

Coagulation Pathways

• The coagulation cascade involves two pathways: the Tissue Factor pathway and the Contact Activation pathway.
• The Tissue Factor pathway involves Factors III, VII, IX, and X.
• The Contact Activation pathway involves Factors I, II, VIII, IX, X, XI, and XII.

Steps to Hemostasis

• The steps to hemostasis include:
  1. Vessel constriction and release of Tissue Factor and Factor III.
  2. Platelet adhesion and activation, releasing thromboxane A2 and ADP.
  3. Coagulation cascade activation.
  4. Fibrinolysis, mainly caused by plasmin, which begins to cleave the fibrin mesh.

Inhibitors of Coagulation

• Thrombin binds to thrombomodulin on the surface of endothelial cells, converting Protein C to its active form.
• Protein C, along with Protein S, serves as an anticoagulant, breaking down Factors Va and VIIIa, and blocking the inhibition of TPA.
• Antithrombin III targets proteases of the Contact Activation pathway, including Factors IX, X, and XI.
• Heparin enhances the activity of Antithrombin III.

PT/PTT

• PT evaluates the Tissue Factor pathway.
• PTT evaluates the Contact Activation pathway.
• INR (International Normalized Ratio) is a measure of the PT, used to standardize results.

Vitamin K

• Vitamin K is essential for the synthesis of coagulation factors, including Factors II, VII, IX, and X.
• A deficiency in Vitamin K may cause a reduction in the production of these factors.

Thrombocytopenia and Thrombocytosis

• Thrombocytopenia is defined as a platelet count <150,000, characterized by petechial hemorrhages, excessive bleeding, easy bruising, and fatigue.
• Causes include bone marrow suppression, immune disorders, and infections.
• Thrombocytosis is defined as a platelet count >450,000, characterized by chest pain, claudication, syncope, thrombosis, and petechial hemorrhages.
• Causes include anemia, infections, cancers, and genetic mutations.

Platelet Function Disorders

• Various platelet function disorders include:
• Bernard-Soulier Disease
• Immune Thrombocytopenic Purpura
• Gray Platelet Syndrome
• Chédiak-Higashi Syndrome
• Thrombocytopenia with Absent Radius syndrome
• Heřmanský–Pudlák Syndrome
• Wiskott-Aldrich Syndrome
• Glanzmann’s Thromboasthenia