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Case study (39) – Paroxysmal Nocturnal Hemoglobinuria (PNH)

Case study (39) – Paroxysmal Nocturnal Hemoglobinuria (PNH)

Case study (39) – Paroxysmal Nocturnal Hemoglobinuria (PNH) 

A 56-year-old woman presenting with increasing tiredness and malaise. 

An in-depth investigation of her iron deficiency was conducted a year ago, and no cause was found. 

She has subsequently noticed episodes of pain associated with the passage of dark urine. 


Hemoglobin (Hb) 9.5 g/dL 

Mean corpuscular volume (MCV) 72 fL 

White blood cells (WBC) 4.1 X 109/L 

Platelets 113 X 109/L 


Q1. Comment on the blood film and blood count. 

Q2. A specialized test is performed on her serum and cells. There are nine tubes, as follows: 

1. Patient cells with patient serum (unacidified). 

2. Patient cells with patient serum (acidified). 

3. Patient cells and patient serum (acidified and heated). 

4. Patient cells with donor serum (unacidified). 

5. Patient cells with donor serum (acidified). 

6. Patient cells and donor serum (acidified and heated). 

7. Donor cells with donor serum (unacidified). 

8. Donor cells with donor serum (acidified). 

9. Donor cells and donor serum (acidified and heated). 

Q3. What is the diagnosis? How is the diagnosis confirmed? 

Q4. What are the pathogenesis and natural history of this disease? 


A1. The blood film shows hypochromic microcytic red cells which are consistent with iron deficiency. 

There is a disproportionate degree of anisocytosis and polychromasia. 

A2. This is an illustration of Ham’s test and demonstrates that her red cells have increased sensitivity to lysis by complement present in her serum. 

Heat inactivates complement, whereas low pH activates complement. 

Therefore, lysis of her cells occurs to a small extent in the presence of her serum, while the donor serum is exaggerated due to the presence of acidified serum, and It does not happen when the complement is disabled. 

Donor cells are not lysed, even in the presence of patient serum. 

The intravascular hemolysis leads to the presence of hemosiderin, which can be stained by Perl’s reaction on the urine deposit. See the image below. 

The only other disorder that may give a positive Ham’s test is a rare congenital dyserythropoietic anemia (CDA type II, hereditary erythrocyte multinuclearity with positive acidified serum [HEMPAS] test, Image below). 

A3. Paroxysmal nocturnal hemoglobinuria (PNH). 

Ham’s test is no longer used for diagnosis but is included here because it illustrates hemolysis. 

The diagnosis was confirmed by flow cytometry analysis of blood cells, which proved that the expression of CD55 and CD59 was reduced (see below). 

A4. This is an acquired clonal disorder that arises due to acquired mutations of the PIG-A gene in hemopoietic stem cells. 

Affected cells are lack glucosyl-phosphatidylinositol-anchored proteins (GPI-APS), which can cause cell membrane defects, thereby impairing the inactivation of complement and increasing complement-mediated lysis. 

Many patients have defective platelet function and a thrombotic tendency, and presentation with hepatic vein thrombosis (Budd–Chiari syndrome) is well recognized. Fluorescence-labeled aerolysin (FLAER) analysis revealed GPI-AP-deficient hematopoietic cells, which can be used to assess the size of abnormal clones. 

The standard diagnostic test for PNH uses flow cytometry to prove that the expression of CD 55 and CD 59 on red blood cells is reduced. 

A diagram of the ham test is given to illustrate the pathological abnormality, namely the ham test obsolete. 

Patients with PNH may develop pancytopenia with aplastic anemia, and a proportion goes on to develop acute myeloid leukemia. 

Patients with significant hemolysis benefit from treatment with eculizumab, a monoclonal antibody that inhibits the activation of terminal complement components by binding to C5. 

Supportive therapy with blood transfusion may also be required. 

The transfused cells should be filtered to remove contaminating leukocytes, as the transfusion of such leukocytes may lead to sensitization to human leukocyte antigen (HLA) antigens, which can lead to complement 
activation and further hemolysis. 

Severely affected patients should consider allotransplantation.