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

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

Etiology and pathogenesis of PNH

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

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

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.

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

Hypochromic anemia of PNH

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)

PNH: hypercellularity

Bone marrow of PNH: erythroid hyperplasia

bone marrow of PNH

Hypochromic, polychromic, stippling

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

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

Urine of PNH patient in the morning

3. Urine: URO +

結果

【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

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

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

流式细胞仪检测PNH细胞群

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.

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)

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:

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%

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

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