1. Paroxysmal Nocturnal Hemoglobinuria

2. Relationship between AA and PNH

3. Late clonal hematologic diseases
Important and as yet unexplained complication
in the clinical course of aplastic anemia
often years after successful immunosuppressive therapy
Paroxysmal nocturnal hemoglobinuria
approximately 9% of patients *
Myelodysplasia and Acute myelogenous leukemia
cumulative incidence rate - 16%, 10 yrs after treatment **
* Br J Haematol 1989;73:121-6
** NEJM 1993;329:1152-7

4. PNH
GPI anchor defect

6. PIG-A mutation (X-chromosome)

7. PNH acquired stem cell disorder Sx triad predominant symptom
intravascular hemolysis
thrombosis
deficient hematopoiesis
predominant symptom
AA-PNH
PNH-AA

8. intravascular hemolysis
DAF, decay-accelerating factor MIRL, membrane inhibitor of reactive lysis

9. GPI-anchored protein defect
CD55 (DAF)
inhibit the assembly of C3 and C5 convertase
thereby regulating the complement cascade at the C3 step
CD59 (MIRL)  more important
limit the polymerization of C9 in membrane C5b-9 complex
absent or deficient in the blood cells
increased sensitivity of red cells to complement-mediated lysis
subsequently to intravascular hemolysis and hemoglobinuria

10. thrombosis incidence - Europe, America (40%) > Japan (5%)
m/c – hepatic, portal, mesenteric, cerebral vein
cause – not clear, multifactorial
platelets
lack the complement defense proteins, CD55 and CD59
inhibited to prevent excess complement activation
formation of MAC complexes
CD59 is not present to prevent the conversion of C9
more vesicles are budded from the platelets
resulting in a marked increase in prothrombinase activity

11. localization of the clot to the vessel wall may occur
complement activation and the generation of membrane attack complexes on the defective blood cells
stimulate endothelial cells to express tissue factor
aggregated platelets to adhere
particularly in areas where the flow of blood is sufficiently slow
delayed fibrinolysis
fibrinolysis
promoted by the activation of plasminogen to plasmin
catalyzed by urokinase-like plasminogen activators
This reaction is localized at the cell surface by urokinase plasminogen activator receptor (UPAR) which is present on monocytes but not on platelets
Monocytes are thought to infiltrate thrombi and initiate fibrinolysis
UPAR is GPI-linked and is therefore deficient in PNH monocytes

12. deficient hematopoiesis
still poorly understood
50% of aplastic anemia have GPI(-) hematopoietic cells
particularly during and after recovery in antithymocyte globulin
many pts with PNH develop aplastic anemia as the final stage of the disease
Young-Luzzatto hypothesis
GPI(-) hematopoietic stem cells exist in very small numbers in the bone marrow of many healthy persons
growth disadvantage in the normal marrow environment
when the marrow is affected by aplastic anemia (autoimmune reaction against marrow precursor)
defective precursors are thought to be less suppressed than normal GPI(+) precursor
become the dominant source of hematopoiesis
cause of selection and expansion of the PNH clone is unclear

13. another hypothesis in PNH marrow
decrease in apoptosis of the nuclear precursors
dominance of the GPI(-) clone

14. Diagnosis suspected laboratory tests
unexplained hemolytic anemia or pancytopenia
intravascular hemolysis
recurrent venous thrombosis (hepatic, IVC, portal, cerebral vein)
laboratory tests
to demonstrate intravascular hemolysis
elevated serum LDH
elevated reticulocyte count
low~absent serum haptoglobin
hemoglobinuria
hemosiderinuria

15. Ham test acidified serum lysis test positive result false positive
PNH red cells are incubated
in tubes to fresh acidified serum, unacidified serum, and heated acidified serum
lysis is determined by optical density
positive result
lysis in acidified serum >1%
lesser degrees of hemolysis may occur with unacidified serum
hemolytic properties of the serum are destroyed by heating
false positive
congenital dyserythropoietic anemia type II
HEMPAS (Hereditary Erythroblastic Multinuclearity with a Positive Acidified Serum lysis test)
false negative
operator error
presence of a small population of complement-sensitive cells

16. Sucrose lysis test more sensitive, less specific
reducing ionic strength of sucrose solution
enhancing the binding of complement to the red cells
PNH cells develop membrane defect
sucrose molecules enter the red cells
produce osmotic lysis
lysis detected by optical density
positive result
lysis >5%
false positive
in some other disorders, such as agnogenic myeloid metaplasia
false negative
operator error
complement-sensitive cells is low due to transfusion

17. Flow cytometry using monoclonal antibodies
analyze the expression of the GPI-anchored proteins CD55 and CD59
deficient populations that comprise more than 1% of the red cells can be identified by this technique
type
PNH I - nearly normal expression of GPI-anchored protein
PNH II - intermediate expression of GPI-anchored protein
PNH III - no expression of GPI-anchored protein

19. Subclinical PNH lack of hemolytic feature
affected RBC are undetectable
despite the existence of affected progenitors in the BM and pph blood
eventually progress to apparent PNH

21. Treatment Treatment for anemia Glucocorticoids
usual adult dose of prednisone is mg/d ( mg/kg/d) given daily during hemolysis and changed to alternate days during remission
about 70% of adult patients experience improvement in hemoglobin levels
Replacement of nutritional iron
necessary to prevent development of iron deficiency
because of increased loss of iron from the hemolysis, and the increase in iron urinary excretion
Iron replacement can stimulate reticulocytosis
that can trigger hemolysis by releasing a new cohort of complement-sensitive cells
can be prevented by adding prednisone during replacement therapy
Folic acid at 1 mg/d also is recommended
because of the high cellular turnover rate
Androgenic hormones
stimulation of erythropoiesis has been successful in patients with moderate decrease in red cell production
short(1-2mon) trial  if not effective, drug should not be used
Supportive care for severe anemia
blood transfusion using leuko-depleted packed RBCs to prevent alloimmunization

22. Management of thrombotic complications
using heparin emergently, then maintenance using an oral anticoagulant
Sometimes, heparin can exacerbate the thrombotic problem
possibly by activating complement
prevented using inhibitors of cyclooxygenase system, such as aspirin, ibuprofen, and sulfinpyrazone
Bone marrow hypoplasia
treated most effectively with bone marrow transplantation
if there is no suitable donor available, antithymocyte globulin (ATG) has been used in the treatment of aplastic anemia