4/17/2018 Paroxysmal Nocturnal Paroxysmal Nocturnal Hemoglobinuria Hemoglobinuria: • PNH was first reported in the medical literature in the latter half of the 19th century. Understanding the Diagnosis, Complications • It was so named because of the mistaken belief that hemolysis (red and Treatment Options blood cell break down) and subsequent hemoglobinuria (free hemoglobin in urine) occurred: • Intermittent episodes (paroxysmal) Iberia Romina Sosa, MD, PhD • Greater frequency during the night (nocturnal). Assistant Professor of Medicine Baylor College of Medicine April 21, 2018 Epidemiology PNH Stem Cell • • PNH originates from a defect in a The prevalence is estimated to be between 0.5-1.5 per million multipotent hematopoietic stem cell. people in the general population. • PNH is believed to affect males and females in equal numbers, • It can arise de novo or in the setting of an although some studies show a slight female preponderance. underlying bone marrow failure disorder • The disorder has been described in many ethnic groups and such as has been identified in all areas of the world. • Aplastic Anemia • It may occur with greater frequency in individuals of Southeast Asia or • Myelodysplasia the Far East who experience greater rates of aplastic anemia. • Primary Myelofibrosis • It can affect any age group. • The median age at diagnosis is during the 30s. Disease Mechanism • PIG-A gene is found in the X chromosome • A single hit is enough to generate the PNH phenotype. • The disease begins with the GPI anchored protein • Males only have one X chromosome expansion of the hematopoietic EIN Ethanolamine phosphate • Females undergo lyonization (chromosome inactivation). stem cell that has severe deficiency P or absence of GPI—a glycolipid • The mechanism for the mutations moiety that anchors >150 different Glycan Core is unknown. proteins. Glucosamine • Typical mutagenic events such as PI Phosphotidylinositol radiation, chemotherapy, etc. rarely exist. • GPI deficiency is the result of a somatic mutation in PIG-A gene Cell membrane • To cause PNH, the stem cell needs • Phosphotidylinositol glycan anchor to undergo clonal selection and Red blood cell biosynthesis, class A expansion. 1 4/17/2018 PIG-A PIG-C • PIG-A gene is responsible for the first step PIG-H PIG-P RBC in the synthesis of GPI anchor that PIG-Q PIG-Y ER DPM2 Hematopoietic attaches a subset of proteins to the cell stem cell Hemolysis surface. Monocytes • Leads to a deficiency in complement inhibitory proteins: CD55 and CD59 which leads to chronic complement mediated PMNs hemolysis of the GPI deficient red blood Expansion of mutant clone cells. Lymphocytes Immune attack ? Platelets Alternative Pathway Classification of PNH C3 C5 Classical + C3b Classification Example Clinical Findings Lab Findings Bone marrow C6 C7 C8 C9 Pathway C3b findings C3 convertase C5b C5 convertase Classic PNH Dark/red urine Elevated LDH, Cellular marrow with Lectin Thrombotic reticulocyte count and erythroid hyperplasia complications of indirect bilirubin No karyotypic Pathway Amplification CD55 unusual sites Low haptoglobin abnormalities PNH in the setting of PNH/Aplastic Anemia Variable Consistent with Abnormal bone CD59 another bone marrow PNH/refractory Hemolysis hemolysis marrow morphology disorder anemia- MDS Nonrandom karyotypic abnormalities PNH-subclinical in the PNH-sc/Aplastic No hemolysis Normal hemolysis labs Abnormal bone setting of another Anemia Small populations of marrow findings bone marrow disorder GPI-associated protein deficient cells Classic PNH Clinical Manifestations: Signs and Symptoms • Characterized by signs and symptoms of hemolysis or red blood cell breakdown. • Fatigue -80% • Other complications: • Symptoms directly attributed to anemia or the destruction of red blood cells: • Iron deficiency anemia • Shortness of breath • Shortness of breath -64% • Headaches • Fatigue • Hemoglobinuria (Free hemoglobin • Confusion • Symptoms indirectly associated to the release of free hemoglobin: in urine) -62% • Dysphagia • Abdominal pain -44% • Abdominal pain • Bone marrow suppression – 44% • Erectile dysfunction • Pulmonary hypertension • Erectile dysfunction -38% • Renal dysfunction • Chest pain -33 % • Hypercoagulability or tendency to clot • Thrombosis – 16 % • Renal Failure -14 % 2 4/17/2018 Laboratory Abnormalities Additional Laboratory Abnormalities • Anemia (low red blood cell counts) • Hypocellular, normocellular or hypercellular bone marrow often with erythroid • Note: possible to have hemolysis without anemia if the bone marrow is able to hyperplasia compensate. • may see erythroid dysplasia • Increased reticulocyte count • Iron deficiency: • Increased lactate dehydrogenase (LDH) and indirect bilirubin • Low serum iron, low ferritin, incrased transferrin, absent bone marrow iron • Decreased haptoglobin • Creatinine and blood urea nitrogen may be increased (BUN) from acute and/or • Free serum hemoglobin with pink/red serum chronic renal damage • Hemoglobinuria with pink/red urine, positive dipstick for heme and negative • Liver function tests may be abnormal due to hepatic or portal vein thrombosis sediment for red blood cells • Mild reduction in blood counts may occur due to splenomegaly caused by • Negative direct antiglobulin (Coombs or DAT) portal vein thrombosis or splenic vein thrombosis Dysphagia Smooth Muscle Dystonia: Mechanism Pulmonary Hypertension Shortness of breath Abdominal Pain Nitric Oxide Chronic Kidney Disease Erectile dysfunction Nitric Oxide Scavenging: Hemoglobinuria NO depletion impairs regulation of Releases free hemoglobin Oxyhemoglobin reacts with NO to smooth muscle tone: Thrombosis form methemoglobin and nitrate. Vascular constriction Pulmonary Hypertension Anemia Shortness of Breath Fatigue Thrombosis Thrombosis in PNH: Mechanism • It leads to severe morbidity and • It is poorly understood. it is the most common cause of • Platelet activation secondary to: mortality in PNH. • The absence of complement regulatory proteins in platelets leads to prothrombotic • Venous thrombosis is more microparticles. common than arterial • Free hemoglobin leads to scavenging of NO. thrombosis. • Complement activation leads to increased inflammatory proteins: IL6, IL8, • It may occur in any PNH patient tumor necrosis factor a but those with large percentage • Defective fibrinolysis resulting from deficiency or absence of GPI linked of PNH cells (>50% WBC) are at proteins such as: greatest risk. • Urokinase type plasminogen activator • 10 year risk of thrombosis for • Heparan sulfate those with >50% clone: 44% • Tissue factor pathway inhibitor compared to 5.8% on those patients with <50%. 3 4/17/2018 Thrombosis in PNH: Evaluation Who should get screened for PNH? • Patients with hemoglobinuria. • No evidence to support routine imaging or laboratory testing. • Patients with Coombs negative intravascular hemolysis (especially with • Clinical suspicion! concurrent iron deficiency) • Rely on history and physical examination • Patients with venous thrombosis at unusual sites: • • Budd-Chiari (hepatic vein) Baseline d-dimer may be useful to direct history taking to elicit • Mesenteric or portal veins symptoms of thrombosis • Cerebral veins • Neurologic symptoms, abdominal pain, leg swelling • Dermal veins • Increased abdominal girth, large spleen and liver, neurologic deficits on exam • Patients with aplastic anemia diagnosis • Patients with refractory anemia MDS • Patients with episodic dysphagia (difficulty swallowing) or abdominal pain with evidence of intravascular hemolysis How do you diagnose PNH? Types of PNH cells • Red blood cells are defined by the • Flow cytometry analysis using antibodies abundance of GPI anchored proteins on directed against GPI-associated proteins: the surface: • CD55, CD59 • PNH Type I cells: normal levels • Quantification of at least two GPI-AP • PNH Type II cells: partial absence • PNH Type III cells: complete absence • FLAER (fluorescent labeled aerolysin): • Knowing the percentage and type of • Takes advantage of binding of a bacterial protein: aerolysin to GPI anchor. deficient RBC can be helpful in • Useful for the analysis of white blood cells but not red blood cells management of anemia. • Other information to get from flow: • Discrete populations with different degrees of deficiency • A word about transfusion: It is unlikely to obscure the diagnosis BUT it can affect the • Percentage of cells that are abnormal calculation of proportion of cells with normal expression of GPI-AP. • Ham and sucrose tests • Best to perform flow before transfusion or during a (minimum 30 days) period of transfusion abstinence. • Largely abandoned as diagnostic assays Charles Parker et al. Blood 2005;106:3699-3709 • It is recommended that at least two independent flow cytometry Testing of RBC to assess severity reagents be used (FLAER and antibodies against monoclonal proteins) of disease in at least two cell lineages (i.e. RBC and WBC). • Assessment of the RBC population can be • WBC (monocytes, granulocytes) are the optimal cell type for assessing useful to determine severity of disease. the PNH clone. • In general: • The life span of WBC is normal • Patients with high Type III RBC have clinically • Unaffected by RBC transfusion. apparent hemolysis. PNH types Clone Size in granulocytes • A patient may have diagnosis of PNH but if Classic PNH 40-99% the percentage of Type III RBC is low, Acquired Aplastic Anemia 0.1-10% hemolysis may not be clinically significant. MDS <1% • If RBC have partial deficiency (type II), Healthy Individuals