Myeloproliferative Disorders (MPDs) Overview

Overview of Myeloproliferative Disorders (MPDs)

• Clonal hematopoietic stem cell disorders caused by genetic mutations, leading to excessive production of erythrocytes, granulocytes, and platelets
• Types include Chronic Myelogenous Leukemia (CML), Polycythemia Vera (PV), Essential Thrombocythemia (ET), and Primary Myelofibrosis (PMF)
• Common features include affecting middle-aged or older individuals, chronic disorders that may progress to acute leukemia, splenomegaly in 60-100% of patients, and similar cytogenetic abnormalities

Chronic Myelogenous Leukemia (CML)

• Genetic abnormality is the Philadelphia chromosome (Ph) due to t(9;22) translocation, forming the BCR/ABL1 fusion gene
• Clinical course involves a chronic phase (3-4 years, <10% blasts), an accelerated phase (6-18 months, 10-19% blasts), and a blast phase (3-4 months, >20% blasts)
• Lab findings include leukocytosis (WBC >300 × 10^9/L), neutrophilia, basophilia, eosinophilia, thrombocytosis and low Leukocyte Alkaline Phosphatase (LAP) score
• Treatment includes Imatinib (Gleevec) (a tyrosine kinase inhibitor targeting BCR/ABL), and bone marrow/stem cell transplantation is curative

Polycythemia Vera (PV)

• Genetic mutation: JAK2 V617F mutation in 90-97% of patients
• Symptoms: Headaches, dizziness, ruddy cyanosis, and splenomegaly
• Lab findings include increased RBC count, HGB, HCT, normal or increased O2 saturation, decreased EPO, and elevated LAP score
• Treatment includes therapeutic phlebotomy to reduce red cell mass, hydroxyurea to control cell proliferation, and Ruxolitinib (Jakafi) as a JAK2 inhibitor

Essential Thrombocythemia (ET)

• Key feature is persistent thrombocytosis (platelets >450 × 10^9/L)
• Symptoms include vascular occlusion, bleeding, headache, and dizziness
• Lab findings include elevated platelets and abnormal platelet morphology, normal red cell mass, and absence of significant fibrosis
• Treatment includes hydroxyurea to reduce platelet count, with good prognosis if thrombocytosis is controlled

Primary Myelofibrosis (PMF)

• Pathophysiology: Abnormal megakaryocytes stimulate fibrosis, leading to bone marrow failure and extramedullary hematopoiesis
• Symptoms: Splenomegaly, hepatomegaly, anemia, and thrombocytopenia
• Lab findings include dry tap on bone marrow aspiration, fibrosis on bone marrow biopsy, and teardrop cells, immature granulocytes, and NRBCs in peripheral blood
• Treatment includes JAK2 inhibitors (e.g., Ruxolitinib), and stem cell transplantation is the only curative option
• Prognosis: Average survival ~5 years

Key Diagnostic Tests

• CML: Requires presence of the Philadelphia chromosome (t(9;22)) and BCR/ABL fusion gene
• PV: Requires JAK2 V617F mutation, increased RBC mass, and decreased EPO
• ET: Persistent thrombocytosis, exclusion of other MPDs
• PMF: Bone marrow biopsy showing fibrosis, extramedullary hematopoiesis

Prognosis

• CML: Median survival is 3-4 years, <30% survive to 5 years
• PV: Risk of transformation to acute leukemia
• ET: Good prognosis if controlled
• PMF: Poor prognosis, average survival ~5 years

Treatment Highlights

• CML treatment includes Imatinib (Gleevec) and bone marrow transplant
• PV treatment includes Phlebotomy, hydroxyurea, and ruxolitinib
• ET treatment includes Hydroxyurea to control platelets
• PMF treatment includes JAK2 inhibitors and stem cell transplant

Key Mutations

• CML: BCR/ABL fusion gene (Philadelphia chromosome)
• PV/ET/PMF: JAK2 V617F mutation

Important Lab Findings

• CML: Low LAP score, high WBC, basophilia
• PV: High RBC mass, low EPO, high LAP score
• ET: High platelets, normal RBC mass
• PMF: Fibrosis on biopsy, teardrop cells in peripheral blood

Hematologic Neoplasms Overview

• Involves malignant diseases with abnormal proliferation of white blood cells in the bone marrow or lymphoid tissue
• Types include:
  • Leukemia: Can be acute (rapid progression) or chronic (slow progression), and myeloid or lymphoid
  • Lymphoma: Malignant neoplasm of lymphoid tissue, originating in the reticuloendothelial and lymphatic systems
• Etiology:
  • Acquired genetic mutations
  • Chromosomal translocations (e.g., t(9;22) in CML, t(8;14) in Burkitt lymphoma)
  • Oncogenes and loss of tumor suppressor genes lead to uncontrolled cell proliferation
  • Multi-hit theory: Multiple mutations are needed for malignancy.

Leukemia Characteristics

• Leukemic cells are functionally and structurally abnormal, with arrested differentiation and unregulated proliferation
• Normal cells mature fully and have regulated proliferation
• Diagnosis based on CBC, symptoms, and bone marrow analysis
• Treatment:
  • Chemotherapy: Targets rapidly dividing cells (both normal and malignant)
  • Radiation therapy: Damages DNA to kill cells
  • Targeted therapy: Monoclonal antibodies, tyrosine kinase inhibitors (e.g., for CML)
  • Stem cell transplantation: Syngeneic, allogeneic, or autologous
  • Supportive care: Blood transfusions, cytokines, antibiotics

Myelodysplastic Syndrome (MDS)

• A group of clonal disorders characterized by ineffective hematopoiesis, leading to peripheral blood cytopenias despite a hypercellular bone marrow
• Pathogenesis includes mutations in myeloid progenitor cells (GEMM cells) affecting erythroid, myeloid, and megakaryocytic cell lines, with dysplastic maturation and abnormal function of blood cells
• Clinical features include pancytopenia, dysplastic changes in blood cells, and risk of transformation to acute leukemia (especially AML)
• Diagnosis:
  • Bone marrow biopsy: Shows dysplasia and <20% blasts
  • Cytogenetic studies: Chromosomal deletions are common
  • Flow cytometry: CD markers help differentiate from leukemia
• Classification:
  • FAB classification: Based on morphology and % blasts in bone marrow
  • WHO classification: Incorporates molecular and cytogenetic changes
• Treatment:
  • Supportive care: Transfusions, erythropoietin (EPO)
  • Chemotherapy: For higher-risk MDS
  • Stem cell transplantation: Potentially curative for some patients

Key Genetic and Molecular Concepts

• Oncogenes are mutated genes that promote cell division
• Tumor suppressor genes normally inhibit malignant transformation, and their loss leads to uncontrolled growth
• Chromosomal Abnormalities:
  • Translocations are common in leukemia
  • Deletions are common in MDS
• Altered gene expression leads to arrested differentiation and uncontrolled proliferation

Normal vs. Leukemic Cells

• Normal cells mature fully, have regulated proliferation, and are functionally normal
• Leukemic cells have arrested maturation, unregulated proliferation, and abnormal function

Remission and Cure

• Remission requires the absence of disease symptoms, normal blood counts, and no evidence of leukemia in the bone marrow
• Cure requires remission for over 4 years with no return of disease and no therapy, achievable in some leukemias

Classifications of Leukocyte Neoplasms

• FAB Classification: Based on morphology and cytochemical stains
• WHO Classification: Emphasizes molecular and cytogenetic changes
• Major Categories:
  • Myelodysplastic: Defective maturation, cytopenias
  • Myeloproliferative: Increased immature myeloid cells
  • Acute vs. Chronic Leukemia: Based on maturity and clinical course

Dysplasia in MDS

• Dyserythropoiesis: Abnormal RBC morphology
• Dysmyelopoiesis: Abnormal neutrophils
• Dysmegakaryocytopoiesis: Abnormal megakaryocytes and platelets

Differential Diagnosis

• MDS must be distinguished from other conditions causing dysplasia

Key Points to Remember

• Leukemia and MDS are both clonal disorders with genetic mutations leading to abnormal cell proliferation and differentiation
• MDS is characterized by dysplasia and cytopenias, with a risk of transforming into acute leukemia
• Treatment focuses on eradicating malignant cells and providing supportive care
• Classifications are crucial for diagnosis, therapy, and prognosis

Normal Hematopoiesis

• Medullary Hematopoiesis: Occurs in the bone marrow (BM)
• Extramedullary Hematopoiesis: Occurs outside the BM
• Fetal Hematopoiesis Stages:
  • Mesoblastic: Yolk sac, produces primitive erythroblasts
  • Hepatic: Fetal liver, produces RBCs, WBCs, and megakaryocytes
  • Myeloid/Medullary: Bone marrow becomes the primary site after birt
• BM Changes with Age:
  • Infants: All red marrow
  • Adults: Long bones contain yellow marrow
  • Yellow marrow can reactivate in response to hemolysis or bleeding

Bone Marrow Structure & Microenvironment

• Components: Involves trabecular bone, vascular sinuses, extravascular cords, and stroma
• Erythroid Islands: Erythroblasts mature near sinuses, supported by macrophages
• Myeloid Cells: Located deeper in the cords

Erythropoiesis

• Stages: Pronormoblast → Basophilic normoblast → Polychromatic normoblast → Orthochromic normoblast → Reticulocyte → Erythrocyte
• Control:
  • Erythropoietin (EPO): Stimulates red cell production in response to hypoxia
  • Hypoxia Triggers: Low RBC count, abnormal hemoglobin, lung function, high altitude
  • Excess Precursors: Removed by apoptosis

Bone Marrow Examination

• Indications: Hematological diseases, Systemic diseases, and Unexplained organomegaly or cytopenias
• Specimen Types: Aspirate and Core Biopsy
• Collection Sites:
  • Adults: Posterior superior iliac crest
  • Children (<2 yrs): Upper tibia
• Needles: Jamshidi needle for biopsy/aspiration

Bone Marrow Sampling Procedure

• Steps: Informed consent, local anesthesia, small incision -Biopsy taken first, then aspirate -Aspirate volume is 1-2 mL collected, slides made at bedside -Biopsy: Core placed in formalin for histology
• Dry Tap: No aspirate due to dense marrow. Use touch preps from biopsy

Bone Marrow Evaluation

• Aspirate Evaluation: -Cellularity: Assess spicules -Differential Count: 300-500 nucleated cells counted
  • M:E Ratio: Normal = 1.5:1 to 3.3:1 -Iron Stores: Uses perls' Prussian Blue stain -Megakaryocytes: Assess number per low power field (LPF)
• Biopsy Evaluation -Cellularity: Normal = 50% hematopoietic cells, 50% fat -Hypercellularity: Increased hematopoietic cells -Hypocellularity: Increased fat cells

Bone Marrow Report

• Components: Patient history, peripheral blood data, Bone marrow differential count, cellularity, M:E ratio, and Iron stores
• Normal Values: HGB, HCT, MCV, MCH, WBC, platelet count

Bone Marrow Failure

• Definition: Reduction or cessation of blood cell production
• Examples: Aplastic Anemia and other Disorders

Key Stains & Techniques

• Wright's Giemsa: For aspirate smears
• Perls' Prussian Blue (PPB): For iron stores
• H&E Stain: For biopsy sections

Common Findings

• Hypercellular Marrow indicates increased hematopoietic cells
• Hypocellular Marrow indicates increased fat cells
• Megaloblastic Anemia: Abnormal RBC precursors
• Plasma Cells: May indicate multiple myeloma

Key Concepts to Remember

• Hematopoiesis Sites: Fetal vs. adult
• BM Microenvironment: Stroma, erythroid islands, myeloid cells
• Erythropoiesis Control: EPO, hypoxia, apoptosis
• BM Sampling: Aspirate vs. biopsy, dry tap, touch preps
• M:E Ratio: Normal range and significance
• Iron Stores: PPB stain, hemosiderin
• BM Failure: Pancytopenia, aplastic anemia

Quantitative Terms

• Monocytosis: Absolute monocyte count > 1.0 × 10^9/L in adults, > 3.5 × 109/L in neonates
• Monocytopenia: Absolute monocyte count < 0.2 × 10^9/L
• Lymphocytosis: Absolute lymphocyte count > 4.5 × 10^9/L in adults, > 10.0 × 10^9/L in children
• Lymphopenia: Absolute lymphocyte count < 1.0 × 10^9/L in adults, < 2.0 × 109/L in children

Monocytes

• Monocyte Morphology: Mature monocytes
• Monocytosis: Often seen after neutropenia or overwhelming infections, indicating recovery
• Monocytopenia: Rare, associated with conditions like aplastic anemia, chemotherapy, Hairy

Qualitative Changes

• Reactive changes during infection or recovery include nuclear contortion, increased cytoplasmic volume, and phagocytic activity

Lymphocytes

• Age-Related Differences: Children have higher lymphocyte counts than adults
• Lymphocytopenia: Age-dependent,
• Lymphocyte Morphology: Small resting lymphocytes can transform into active cells upon antigen interaction
• Reactive Lymphocytes: Seen in infections like Infectious Mononucleosis (IM), characterized by large, pleomorphic cells with abundant blue cytoplasm and irregular nuclei

Infectious Mononucleosis (IM)

• Caused by Epstein-Barr Virus (EBV) infections
• Symptoms: Sore throat, lymphadenopathy, fever, fatigue, splenomegaly, hepatomegaly
• Lab Findings: Elevated WBC, absolute lymphocytosis, reactive lymphocytes
• Heterophile Antibody Test (Monospot): Detects IgM antibodies

Immunodeficiency States

• T-Cell Disorders: DiGeorge's Syndrome, AIDS
• B-Cell Disorders: Bruton's Agammaglobulinemia, IgA Deficiency
• Combined B & T Cell Disorders: Severe Combined Immunodeficiency (SCID)

Non-Malignant Disorders

• Pelger-Huët Anomaly: Hyposegmented neutrophils, are autosomal dominant and asymptomatic
• Alder-Reilly Anomaly: Metachromatic granules in leukocytes resembling toxic granulation
• Chediak-Higashi Syndrome: Giant lysosomes in granular cells, dysfunctional leukocytes
• May-Hegglin Anomaly: Thrombocytopenia, giant platelets, Döhle body-like inclusions in leukocytes

Reactive Changes in Leukocytes

• Granulocytes: Left shift, Döhle bodies, toxic granulation, vacuoles, degranulation
• Monocytes: Immature forms, contorted nuclei
• Lymphocytes: Cell enlargement, increased basophilic cytoplasm, pleomorphism

Key Lab Tests

• Flow Cytometry: For diagnosing immunodeficiency disorders
• Serum Protein Electrophoresis & Immunofixation: For immunoglobulin levels
• CBC & Smear: Often shows lymphopenia in immunodeficiency states

Artifacts

• Smudge Cells: Seen in chronic lymphocytic leukemia (CLL)
• Leukocyte Artifacts: Necrotic cells, denuded nuclei, anti-coagulant changes

Summary of Key Disorders

• Infectious Mononucleosis: EBV, reactive lymphocytes, heterophile antibodies
• Immunodeficiency Disorders: T-cell, B-cell, and combined deficiencies
• Non-Malignant Leukocyte Disorders: Pelger-Huët, Alder-Reilly, Chediak-Higashi, May-Hegglin

Age Related Changes in Hematology

• Pediatric Population: Newborns have higher WBC counts, neutrophil percentages, and absolute counts
• Geriatric Population: Slightly lower WBC, RBC, HGB, and PLT counts; slightly higher MCV

Quantitative Neutrophil Changes

• Benign Causes: Stress, trauma, exercise, epinephrine release
• Pathological Causes: Infections, increased bone marrow production, release from storage pool
• Absolute Neutrophilia: Increased neutrophils, often with a left shift
• Leukemoid Reaction: Reactive leukocytosis with marked left shift
• Leukoerythroblastic Reaction: Immature neutrophils and nucleated RBCs

Neutropenia

• Causes: Increased destruction, decreased production, bone marrow suppression, or depletion of storage pool
• Associated with conditions like aplastic anemia, acute leukemias, and certain infections

Eosinophilia & Basophilia

• Eosinophilia : Allergies, parasitic infections, autoimmune disorders, malignancies
• Basophilia : Hypersensitivity, chronic infections, hypothyroidism, myeloproliferative disorders

Qualitative Granulocyte Disorders

• Acquired Abnormalities: Hypersegmentation or hyposegmentation
• Inherited Abnormalities: Pelger-Huët Anomaly, Alder-Reilly Anomaly, May-Hegglin Anomaly, and Chediak-Higashi Syndrome

Chronic Granulomatous Disease (CGD)

• Rare, X-linked or autosomal recessive
• Neutrophils cannot kill ingested bacteria due to NADPH oxidase deficiency
• Recurrent bacterial and fungal infections, granuloma formation

Reporting WBC Morphology

• Report toxic changes and abnormal granules using quantitative terms

Key Terms to Remember

• Left Shift: Increase in immature neutrophils
• Leukemoid Reaction: Extreme leukocytosis mimicking leukemia
• Leukoerythroblastic Reaction: Immature RBCs and neutrophils, often indicating bone marrow pathology
• Toxic Changes: Granulation, vacuoles, Döhle bodies in response to infection/inflammation

Clinical Correlations

• Neutrophilia: Bacterial infection
• Eosinophilia: Allergies, parasitic infections
• Basophilia: Chronic inflammation, myeloproliferative disorders
• Neutropenia: Bone marrow suppression, severe infections

Leukocyte Categories

• Mature leukocytes are categorized based on their precursor cells, specific function, site of maturation, and morphology
• Cell surface markers are used to identify cell types by species, lineage, maturation stage, and activation state

Leukopoiesis (WBC Production)

• Hematopoietic Stem Cells are pluripotent cells that self-renew, differentiate, or undergo apoptosis
• Progenitor Cells:
  • Common Myeloid Progenitor gives rise to granulocytes, monocytes, erythrocytes, and platelets
  • Common Lymphoid Progenitor gives rise to lymphocytes
• Cytokines regulate differentiation, growth, and production of leukocytes

Neutrophils

• Development: Matures in bone marrow and is stimulated by G-CSF
• Function: Phagocytosis of bacteria, fungi, and foreign material
• Morphology stages include :myeloblast, promyelocyte, myelocyte, metamyelocyte, band neutrophil, and segmented neutrophil
• Pools: Bone marrow, blood, connective tissue

Eosinophils

• Function: Involved in allergic reactions, parasitic infections, and immune regulation
• Granules: Contain Major Basic Protein, Eosinophil Cationic Protein, and Peroxidase
• Degranulation: Can occur via classical exocytosis, piecemeal degranulation, or cytolysis.

Basophils

• Function: Involved in hypersensitivity reactions and allergic inflammation.

Monocytes/Macrophages

• Development: Derived from Granulocyte-Monocyte Progenitor and stimulated by M-CSF
• Function:
  • Innate Immunity: Phagocytosis, cytokine production, and nitric oxide synthesis
  • Adaptive Immunity: Act as antigen-presenting cells to activate T and B cells
  • Housekeeping: Removal of debris, dead cells, and synthesis of proteins

Lymphocytes

• Types:
  • B Cells produce antibodies
  • T Cells: Cell-mediated immunity
  • NK Cells exhibit innate immunity
• Development:
  • B Cells mature in bone marrow and migrate to lymphiod tissue
  • T Cells: Develop in the thymus migrate to secondary lymphoid organs
  • Nk celss develope in bone marrow or thymus
• Function:
  • B cells transform into plasma cells to produce antibodies
  • T cells help in immune responses, CD8+ T cells kill infected cells
  • NK cell kill cells lacking self antigens

T Cell Subsets

• CD4+ T Cells: -TH1: Fight intracellular pathogens
  • TH2: Fight extracellular parasites and is involved in allergies -TH17: Fight extracellular bacteria and fungi
  • Treg maintain self-tolerance
• CD8+ T Cells are cytotoxic T cells, kill infected or abnormal cells

Automated Cell Counting

• Principles: Impedance and flow cytometry
• Scatterplots: Used to differentiate cell populations based on size and granularity
• Cytochemical Staining: Identifies specific cell types

Key Concepts

• Cluster of Differentiation Markers are used classify leukocytes
• Cytokines: Regulate leukocyte production and function
• Phagocytosis: Key function of neutrophils and macrophages
• Immune Regulation: Role of T cells, B cells, and NK cells in adaptive and innate immunity

Morphological Changes

• Neutrophil Maturation: From myeloblast to segmented neutrophil
• Monocyte Development: Monoblast → promonocyte → monocyte

Granules

• Neutrophils: Primary and secondary granules
• Eosinophils: Contain Major Basic Protein, Eosinophil Cationic Protein
• Basophils: Contain histamine and cytokines

Life Span & Circulation

• Neutrophils: Short-lived, circulate between blood and tissues
• Lymphocytes: Recirculate between blood and lymphoid tissues, long-lived