1. Paroxysmal Nocturnal Hemoglobinuria (PNH)
Section 5 HA
Paroxysmal Nocturnal Hemoglobinuria (PNH)
Definition
Etiology and pathogenesis
Characteristics of PNH （clinical and lab）
Diagnostic process

2. Definition
PNH is a clonal disorder of hematopoietic stem cells caused by a somatic mutation of the pig-A gene on the short arm of the X chromosome . And the patients with it may present either with a HA( with intravascular hemolysis and episodes of nocturnal hemoglobinuria) or pancytopenia with a dysplastic or aplastic marrow morphology.
GPI - glycosylphosphatidylinositol glycan

3. Etiology and pathogenesis of PNH

4. mutations of pig-A gene in HSC
Marrow damage
complete or partial failure in the production of GPI anchor protein
CD59 : membrane inhibitor of reactive lysis,
CD55（ decay accelerating factor
CD58
CD16
CD14
Loss of several GPI-anchored membrane proteins
anemia
increased sensitivity to C-mediated lysis
Intravascular hemolysis
hemoglobinuria
Neutrophils
decrease
infection
thrombosis
Platelet falls, bleeding
complement-driven activation of platelets, impaired fibrinolysis
Hypercoagulabe state
pancytopenia

5. Clinical features of PNH
Severity of anemia is variable .
Severe anemia：fatigue, weakness, pallor, dyspnea
2. Dark urine : paroxysmal or on awakening or no.
(hemolysis episode after infection, transfusion, acid food or drugs, stress, fatigues or after aplastic or hypoplastic anemia.)
PNH I : GPI-negative cells(normal)
PNH II : Partial deficiency
PNH III:Complete deficiency
Complement activation

6. Clinical features of PNH
3. Splenomegaly in some patients
4. Venous thrombosis (hepatic, portal, splenic mesenteric veins) or DIC
5. Major bleeding or infection.
Some die of thrombotic diseases, some die of leukemia, some develop(or revert to AA.

7. usually hypochromic and normochromic anemia
Lab findings of PNH
Blood:
Ret serum bilirubin
RBC, Hb
usually hypochromic and normochromic anemia
dimorphic
WBC : neutropenia, low LAP score or absence.
BPC: fall, abnormal platelet function

8. Hypochromic anemia of PNH

9. hyperplasia or hypoplasia (aspiration site)
Lab findings of PNH
2. Bone marrow
hyperplasia or hypoplasia (aspiration site)
hypochromic and normochromic erythrone
Iron stain is often absent.(iron deficiency)

10. PNH: hypercellularity

11. Bone marrow of PNH: erythroid hyperplasia

12. bone marrow of PNH

13. Hypochromic, polychromic, stippling

14. Why the patien with hypochromic anemia?
A. intermittent hemoglobinuria
hemosiderinuria
B. sideropenia (secondary to iron deficiency)
C. marrow insufficiency

15. 3. Urine: sometimes hemoglobinuria，URO +
Lab findings of PNH
3. Urine: sometimes hemoglobinuria，URO +
constant hemosiderinuria (Rous test +)
4. Specific tests
--- Sugar water test: screening test for PNH
--- Ham test: definitive test for PNH
5. Immunophenotype：decreased CD55, CD59

16. Urine of PNH patient in the morning

17. 3. Urine: URO +

19. 結果

20. 【Method】 SUGAR WATER TEST Red cells suspension(patient)
control serum ( or same blood type)
room temperature 1h
sucrose
no hemolysis
exclude PNH
PNH(lysis>5%)
hemolysis
MA, IHA or some leukemia

21. HAM’S TEST
【Principle】 The complement present in serum is responsible for lysis of PNH cells with sensitivity to acidifiction.
patient’s red cells suspension
acidified pH6.5 , 37℃1h
mixed with fresh complement
( same type control serum or patient’s own serum)
lysis: no lysis:
PNH normal or lack of PNH cells

22. Control tubes for excluding false negative
Red cells suspension +
inactivated serum (56℃, 30’)
pH6.5 , 37℃1h
no lysis:

23. 流式细胞仪检测PNH细胞群

25. Progressive Studies
1. kinetic studies with 51Cr labeled ,the red cells show a double population:
A. with a short half-life
B. with a subnormal survival
2. A cytometric assay of CD16 and CD66b on granulocytes in patients with equivocal red cell
3. GPI anchor protein and PIG-A gene.

26. Diagnosis for PNH： Clinical featrues Plus two of the following or
Sugar water test: (+)
Ham test: (+)
Rous test +
one of the following
> twice +,
Hemoglobinuria +
evidence of intravascular hemolysis
Exclude HS,IHA,G-6PD deficiency and PCH
Plus
exclude AA.(exp. hypoplasia)

27. Case assay:
A 30-year-old male came to see the physician because of increasing fatigue over the previous few months.
PE : a pale but otherwise normal-appearing adult male, the liver and spleen slightly enlarged.
The patient reported noticing that his first urine of the morning was occasionally brown.

28. Case assay:

29. CBC: Hb:85 g/L Hematocrit 0.25 l/L RBC 2.6×1012/L WBC 4.4×109/L
Case assay:
CBC:
Hb:85 g/L
Hematocrit 0.25 l/L
RBC 2.6×1012/L
WBC 4.4×109/L
Blood smear
Ret 13%

30. Bone marrow figure

31. Case assay:
Rous test

32. Case assay:
Questions:
1. What is the most probably diagnosis of this patient? List your evidence of the diagnosis.
2. If you want to have an exact diagnosis of this patient, which tests are supposed to be done and what are the expected results?
3. Can you explain the following results with the patient?
Serum iron: 8.1umol/L, TIBC: 66umol/L

33. Questions :
What is the etioloy of PNH?
How to diagnose PNH?
3.How to differentiate PNH and AA?
4. How to differentiate PNH and IDA?

34. AA and PNH: AA-PNH syndrome AA crisis Typical PNH AA atypical
Clinical features of AA
Ham test positive or Hburia,Rous test positive