Paroxysmal Nocturnal Hemoglobinuria (PNH)

Section 5 HA

Definition

Etiology and pathogenesis

Characteristics of PNH （ clinical and lab ）

Diagnostic process

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.

Definition

GPI - glycosylphosphatidylinositol glycan

Etiology and pathogenesis of PNH

mutations of pig-A gene in HSC

complete or partial failure in the production of GPI anchor protein

Loss of several GPI-anchored membrane proteins

CD59 : membrane inhibitor of reactive lysis, CD55 （ decay accelerating factor CD58CD16CD14

Neutrophilsdecrease

infection

anemia

increased sensitivity to C-mediated lysis

Intravascular hemolysis

hemoglobinuria

Marrow damage

pancytopenia

thrombosis

Platelet falls, bleeding

complement-driven activation of platelets, impaired fibrinolysis

Hypercoagulabe state

Clinical features of PNH

1. 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.)

Complement activation

PNH I : GPI-negative cells(normal)

PNH II : Partial deficiency

PNH III:Complete deficiency

3. Splenomegaly in some patients

Clinical features of PNH

Some die of thrombotic diseases, some die of leukemia, some develop(or revert to AA.

4. Venous thrombosis (hepatic, portal, splenic mesenteric veins) or DIC

5. Major bleeding or infection.

1. Blood:

• Ret serum bilirubin

• RBC, Hb

usually hypochromic and normochromic anemia

dimorphic

• WBC : neutropenia, low LAP score or absence.• BPC: fall, abnormal platelet function

Lab findings of PNH

Hypochromic anemia of PNH

2. Bone marrow

hyperplasia or hypoplasia (aspiration site) hypochromic and normochromic erythrone

• Iron stain is often absent.(iron deficiency)

Lab findings of PNH

PNH: hypercellularity

Bone marrow of PNH: erythroid hyperplasia

bone marrow of PNH

Hypochromic, polychromic, stippling

A. intermittent hemoglobinuria

hemosiderinuria

B. sideropenia (secondary to iron deficiency)

C. marrow insufficiency

Why the patien with hypochromic anemia?

3. Urine: sometimes hemoglobinuria， URO + constant hemosiderinuria (Rous test +)

Lab findings of PNH

5. Immunophenotype ： decreased CD55, CD59

4. Specific tests --- Sugar water test: screening test for PNH --- Ham test: definitive test for PNH

Urine of PNH patient in the morning

3. Urine: URO +

結果

SUGAR WATER TEST

【 Method 】

control serum ( or same blood type)

Red cells suspension(patient)

sucrose room temperature 1h

no hemolysis

exclude PNHPNH(lysis>5%)

hemolysis

MA, IHA or some leukemia

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

mixed with fresh complement

( same type control serum or patient’s own serum)

acidified pH6.5 , 37℃1h

lysis: no lysis:

PNH normal or lack of PNH cells

Red cells suspension

+

inactivated serum (56℃, 30’)

pH6.5 , 37 1h℃

no lysis:

Control tubes for excluding false negative

流式细胞仪检测 PNH 细胞群

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.

Progressive Studies

Clinical featrues

Plus

Plus exclude AA.(exp. hypoplasia)

Diagnosis for PNH ：

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

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.

Case assay:

Case assay:

CBC:Hb:85 g/LHematocrit 0.25 l/LRBC 2.6×1012/LWBC 4.4×109/L

Blood smear

Ret 13%

Bone marrow figure

Case assay:

Rous test

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

Questions :

1.What is the etioloy of PNH?

2.How to diagnose PNH?

3.How to differentiate PNH and AA?

4. How to differentiate PNH and IDA?

AA and PNH:

AA-PNH syndrome

PNHAA

Typical

atypical

AA crisis

Clinical features of AA

Ham test positive or Hburia,Rous test positive