Paroxysmal Nocturnal Hemoglobinuria

1. Paroxysmal Nocturnal Hemoglobinuria

2. Paroxysmal Nocturnal Hemoglobinuria Rare acquired hematopoetic stem cell disorder 1-10 per million Most frequent in 3rd decade Asian ancestry RBCs susceptible to complement-mediated lysis Related to lack of cell surface proteins that prevent complement attack PNH is a uncommon acquired hematopoetic stem cell disease with an incidence of approx 1-10 per million. Although it can affect either sex, any age, and any ethnic background, it seems to have a higher prevalence in Asians presenting more frequently in the 3rd decade. The characteristic hemolysis is related to lysis by complement attack which is attributed to a lack of cell surface proteins that normally prevent this from happening.PNH is a uncommon acquired hematopoetic stem cell disease with an incidence of approx 1-10 per million. Although it can affect either sex, any age, and any ethnic background, it seems to have a higher prevalence in Asians presenting more frequently in the 3rd decade. The characteristic hemolysis is related to lysis by complement attack which is attributed to a lack of cell surface proteins that normally prevent this from happening.

3. 1866�first case report by Gull describing nocturnal hematuria 1882�Strubing recognized PNH as a definite syndrome 1925�Enneking coined the name �Paroxysmal Nocturnal Hemoglobinuria� 1930s�Ham identified the role of complement and developed the serum test which is still used for diagnosis 1980s�PNH blood cells found to lack cell surface proteins 1990s�Somatic mutation identified as PIG-A gene History of PNH First described in 1866 by an English MD named Gull who identified the nocturnal and paroxysmal nature of �intermittent hematuria� in a young leather worker, by the way Gull is probably most famous for being a suspect for �Jack the Ripper�. Better known for first describing PNH is Strubing, a nose and throat internist who was later known for his work on laryngospasm, in 1882 �Although multiple case reports had been described in the literature up to this point, it was actually named PNH in 1925 by Enneking �.Further understanding of the mechanism of hemolysis was discovered by Ham who developed the acid serum test used for diagnosis today. He also demonstrated that only a portion of PNH RBCs are abnormally sensitive to complement. First described in 1866 by an English MD named Gull who identified the nocturnal and paroxysmal nature of �intermittent hematuria� in a young leather worker, by the way Gull is probably most famous for being a suspect for �Jack the Ripper�. Better known for first describing PNH is Strubing, a nose and throat internist who was later known for his work on laryngospasm, in 1882 �Although multiple case reports had been described in the literature up to this point, it was actually named PNH in 1925 by Enneking �.Further understanding of the mechanism of hemolysis was discovered by Ham who developed the acid serum test used for diagnosis today. He also demonstrated that only a portion of PNH RBCs are abnormally sensitive to complement.

5. Pathophysiology of PNH Attachment of cell surface proteins transmembrane hydrophobic sequence anchor embedded within the membrane that the protein attaches to Common variable in all missing cell surface proteins is Glycophosphatidylinositol (GPI) anchor

6. GPI anchor Unclear purpose Defective biosynthesis at early step Coded by the PIG-A gene Approx 30 GPI-anchored proteins; 20 shown to be deficient in PNH All vary greatly in function (enzymes, receptors, and complement mediators) The purpose of anchoring cell membrane proteins is not clear. Thought that perhaps the anchoring allows extra mobility of the protein along the cell membrane, or perhaps more importantly that the protein may be easily released from the membrane for transfer between cells. The underlying defect in GPI biosynthesis occurs at an early stage: specifically the addition of N-acetylglucosamine to the Phospoinositol moiety. Although the enzyme mediating this step has not been identified, the gene responsible has been found and is called PIG-A. This defect has been confirmed in over 100 pts with PNH, although the type of mutation differs among pts. Approx 30 GPI anchored proteins are known to exist in humans, and to date 20 have been shown to be lacking in PNH pts. These proteins all vary greatly in function and manifestations of PNH largely lie in results of dysfunction of complement mediation.The purpose of anchoring cell membrane proteins is not clear. Thought that perhaps the anchoring allows extra mobility of the protein along the cell membrane, or perhaps more importantly that the protein may be easily released from the membrane for transfer between cells. The underlying defect in GPI biosynthesis occurs at an early stage: specifically the addition of N-acetylglucosamine to the Phospoinositol moiety. Although the enzyme mediating this step has not been identified, the gene responsible has been found and is called PIG-A. This defect has been confirmed in over 100 pts with PNH, although the type of mutation differs among pts. Approx 30 GPI anchored proteins are known to exist in humans, and to date 20 have been shown to be lacking in PNH pts. These proteins all vary greatly in function and manifestations of PNH largely lie in results of dysfunction of complement mediation.

8. Missing proteins of importance Complement regulating proteins: CD59 (aka MAC inhibitor/protectin) Homologous restriction factor (HRF) CD55 (aka decay accelerating factor) Thrombosis regulating proteins: CD87 (aka urokinase-type plasminogen activator receptor) There are 3 main missing complement regulating proteins that contribute to cell lysis: CD59, also known as membrane attack inhibitor or protectin, which is the dominant protein in preventing lysis. Homologous restriction factor and CD55. These are found on all blood cell types and accounts for the anemia/neutropenia/thrombocytopenia. Thrombosis may also be related to the lack of CD59 on platelets. C9 complexes on the platelet surface are associated with the generation of thrombin, leading to thrombosis. The lack of CD59 allows more C9 complexes to form and more thrombin to be generated. Another factor in increased tendency for thrombosis is the absence of CD87, or urokinase-type plasminogen activator receptor on leukocyte surfaces. This receptor normally binds urokinase, which activates plasminogen to plasmin and mediates thrombolysis. Without this protein, there is less breakdown of clots There are 3 main missing complement regulating proteins that contribute to cell lysis: CD59, also known as membrane attack inhibitor or protectin, which is the dominant protein in preventing lysis. Homologous restriction factor and CD55. These are found on all blood cell types and accounts for the anemia/neutropenia/thrombocytopenia. Thrombosis may also be related to the lack of CD59 on platelets. C9 complexes on the platelet surface are associated with the generation of thrombin, leading to thrombosis. The lack of CD59 allows more C9 complexes to form and more thrombin to be generated. Another factor in increased tendency for thrombosis is the absence of CD87, or urokinase-type plasminogen activator receptor on leukocyte surfaces. This receptor normally binds urokinase, which activates plasminogen to plasmin and mediates thrombolysis. Without this protein, there is less breakdown of clots

9. PNH Cell Types Type I: almost normal cells Type II: intermediate Type III: very sensitive Type II/III cells bind increased C3�excessive number of MAC are formed Can exist in any combination in pts with PNH There are generally 3 hematopoetic cell types found in pts with PNH: type 1 represents nml or near nml cells, type 2 have an intermediate sensitivity to lysis requiring 1/10-1/15th amount complement, and type 3 are very sensitive to lysis requiring only 1/25th the amount of complement needed to lyse. Both type 2 and 3 cells show an increased affinity for C3 in the complement cascade leading to an excess number of MAC. In addition, type 3 cells have also been shown to have an increased hemolytic sensitivity to MAC (ie greater lysis of cells is observed for a given conc of MAC). These 3 cell types can exist in any proportion in a pt. Most of the pathology of PNH can be accounted for by a normal amount of complement activation on or near cells that are very sensitive to its effects.There are generally 3 hematopoetic cell types found in pts with PNH: type 1 represents nml or near nml cells, type 2 have an intermediate sensitivity to lysis requiring 1/10-1/15th amount complement, and type 3 are very sensitive to lysis requiring only 1/25th the amount of complement needed to lyse. Both type 2 and 3 cells show an increased affinity for C3 in the complement cascade leading to an excess number of MAC. In addition, type 3 cells have also been shown to have an increased hemolytic sensitivity to MAC (ie greater lysis of cells is observed for a given conc of MAC). These 3 cell types can exist in any proportion in a pt. Most of the pathology of PNH can be accounted for by a normal amount of complement activation on or near cells that are very sensitive to its effects.

10. Clinical features Highly variable Classic Triad Hemolytic Anemia Bone Marrow failure (thrombocytopenia/neutropenia) Venous Thrombosis Chronic course with acute exacerbations Exacerbations often associated with infection Because of the variability in cell types and expression of abnormal clone, the clinical manefestations are highly variable. PNH is named for its most dramatic manifestation�intermmitent nocturnal hemolysis and morning hemoglobinuria, although this is a bit of a misnomer since it is not always paroxysmal (intermittent) and hemoglobinuria may occur at any time throughout the day. In fact, nocturnal hematuria only occurs in about 25-50% of pts. This is the first of the classic triad of symptoms in PNH. It also typically involves all blood cell lines since it is derived from a hematopoetic stem cell, which leads to neutropenia and thrombocytopenia as well. These processes then lead to PNHs most morbid manifestation: central venous thrombosis (mainly liver/renal/cerebral).Because of the variability in cell types and expression of abnormal clone, the clinical manefestations are highly variable. PNH is named for its most dramatic manifestation�intermmitent nocturnal hemolysis and morning hemoglobinuria, although this is a bit of a misnomer since it is not always paroxysmal (intermittent) and hemoglobinuria may occur at any time throughout the day. In fact, nocturnal hematuria only occurs in about 25-50% of pts. This is the first of the classic triad of symptoms in PNH. It also typically involves all blood cell lines since it is derived from a hematopoetic stem cell, which leads to neutropenia and thrombocytopenia as well. These processes then lead to PNHs most morbid manifestation: central venous thrombosis (mainly liver/renal/cerebral).

11. Hemolytic Anemia Present to some degree in all cases Degree of hemolysis depends on: Proportion of sensitive RBCs Cell type (PNH I-III) Complement activation (ie infection, allergies, transfusion reaction, etc) Other effects of hemolysis: Iron deficiency ATN/ARF during episodes of massive hemolysis CRF Esophageal spasm (achalasia-like sx) Impotence Hemolytic anemia is present to some degree in all cases of PNH and depends upon 3 factors: the proportion of sensitive RBCs (which can range anywhere from 1 to >90%), the ratio of PNH cell types, and the degree of complement activation (which is eleveted in times of stress or immune attack). Other than anemia, these pts can also have varying degrees of iron deficiency, ATN/ARF during acute massive episodes of hemolysis, CRF over time. They can also exhibit dysphagia and impotence thought secondary to the adsorption of NO by free hemoglobin which leads to smooth muscle contraction.Hemolytic anemia is present to some degree in all cases of PNH and depends upon 3 factors: the proportion of sensitive RBCs (which can range anywhere from 1 to >90%), the ratio of PNH cell types, and the degree of complement activation (which is eleveted in times of stress or immune attack). Other than anemia, these pts can also have varying degrees of iron deficiency, ATN/ARF during acute massive episodes of hemolysis, CRF over time. They can also exhibit dysphagia and impotence thought secondary to the adsorption of NO by free hemoglobin which leads to smooth muscle contraction.

12. Bone Marrow Failure Most severe: aplastic anemia More commonly: active BM producing defective cells 2/3 � thrombocytopenia/granulocytopenia

13. Venous Thromboses�the sinister sign 20% incidence in Europe and US (lower in Asians) Mainly central thromboses: Liver (Budd-Chiari) hepatic veins can thrombose in acute crisis or insidiously Tends to persist with periodic exacerbations/remissions Usually ultimately fatal Cerebral Veins/Sinuses Less common Also tends to persist�Poor prognosis Abdominal Veins Renal/Spleen/Stomach/Intestinal LE DVT more common than in general population, but death by PE rare Arterial thrombosis also rare

14. Diagnosis Ham test (acidified-serum lysis test) Gold standard from 1939 until advent of flow cytometry Activation of complement by low pH; PNH cells lyse High specificity Cannot detect varying degrees of RBC sensitivity Flow Cytometry Increased level of sensitivity: allows detection of 0.1% GPI-deficient clones Uses monoclonal Ab to missing proteins (CD55/CD59) and fluorescence of labeled cells to detect certain cell populations May screen RBCs, Platelets, and Lymphocyte components

15. Course and Prognosis Life span estimates 10-15yrs Approx 25% will survive > 25 yrs Spontaneous recovery in ~15% w/o long-term sequelae

16. Course and Prognosis Most common causes of death: consequences of thrombosis (~33%) effects of BM failure (~10%) May be preceded by or lead to the development of aplastic anemia (AA) Incidence from various studies of 25-58% Much less risk of thrombosis�less PNH cells overall Possible �natural gene therapy� producing cells which escape destruction in the setting of AA 3-5% progress to acute leukemia Likely more related to predisposition in pts with AA, not PNH itself

17. Treatment Focus on 3 aspects: Treat anemia Treat and prevent thromboses Modification of hematopoiesis Mainly focused on control of complications rather than interrupting disease process

18. Treating Cytopenias PRBC/Platelet Transfusions Replaces destroyed cells Also suppresses erythropoiesis when done on chronic basis Special transfusion considerations only if necessary Epogen/FeSO4/Folate Expensive, but shown to decrease need for high dose steroids and less transfusions Glucocorticoids Unknown MOA Useful in 50% pts Thought to be related to direct prevention of hemolysis 0.3-1 mg/kg/day Cell Transfusions are indicated for severe or acute exacerbations of anemia. While they are effective by immediately improving oxygen-carrying capacity, they also suppress erythropoiesis over time and decrease formation of complement-sensitive cells in acute situations. An actual increase in PNH-cell hemolysis may occur during or after a transfusion secondary to complement activation from foreign leukocytes, simulating an acute hemolytic reaction. This prompted the customary use of saline-washed PRBCs to prevent this problem, however the practice has been deemed no longer necessary by a group who reviewed 38 yrs of PNH transfusion data given the rarity of such reactions and the waste of red cells, time, and money washing involves. Pts with a hx of febrile transfusion reactions, demonstrable leukocyte Ab, or a hx of hemolysis with unwashed cells should receive leukocyte-depleted PRBCs. Otherwise, it is best to use group-specific, fresh red cells.Cell Transfusions are indicated for severe or acute exacerbations of anemia. While they are effective by immediately improving oxygen-carrying capacity, they also suppress erythropoiesis over time and decrease formation of complement-sensitive cells in acute situations. An actual increase in PNH-cell hemolysis may occur during or after a transfusion secondary to complement activation from foreign leukocytes, simulating an acute hemolytic reaction. This prompted the customary use of saline-washed PRBCs to prevent this problem, however the practice has been deemed no longer necessary by a group who reviewed 38 yrs of PNH transfusion data given the rarity of such reactions and the waste of red cells, time, and money washing involves. Pts with a hx of febrile transfusion reactions, demonstrable leukocyte Ab, or a hx of hemolysis with unwashed cells should receive leukocyte-depleted PRBCs. Otherwise, it is best to use group-specific, fresh red cells.

19. Treatment and Prevention of Clots Prevention Prophylactic anticoagulation for pts w/o contraindications Treatment IV/Oral anticoagulants Thrombolytics: TPA/Streptokinase/Urokinase

20. Modifying Hematopoiesis Immunosuppresants Better response in pts with hypoplastic marrow than hemolysis Mixed results: antithymocyte globulin response rates 0-63%; cyclosporin not effective Bone Marrow Transplant Currently most curative and optimal Tx High risk of morbidity/mortality (10-20%) Risk:benefit considering pts with lesser sx No controlled studies for ethical reasons Gene therapy