PNH Flow

Question 1

What is the triad for

PNH ?

Answer 1

• stem cell disorder, acquired
• Triad:
  
  • intravascular hemolysis
  • peripheral blood cytopenias due to bone marrow failure
  • predisposition to venous thrombosis often at unusual sites
• acquired defect is the loss of GPI anchors by hematopoietic cells

Question 2

What are the clinical scenarios of

PNH presentation?

Answer 2

• Classic PNH
  
  • patients have large clones and intravascular hemolysis
• PNH in the setting of another bone marrow disorder
  
  • AA or MDS
• Subclinical PNH
  
  • small clones are detected in the absence of hemolysis

Question 3

What chromosome is the PIG-A

gene located on ?

Answer 3

• X chromosome
• PIG-A controls the production of normal GPI anchored proteins
• type of mutation in the gene influences if it is a partial or complete loss of the antigen

Question 4

Why are CD55 and CD59 important

in the development of PNH ?

Answer 4

• CD55 - decay accelerating factor (DAF)
• CD59- membrane inhibitor of reactive cell lysis (MIRL)
• these are both complement regulatory proteins and their loss from red cells allows uncontrolled complement activation and intravascular hemolysis
• these are expressed on red cells, leukocytes, and platelets
• although the mechanism is not clear somehow the loss of GPI anchors also leads to thromboses of unusual sites

Question 5

What is the current gold standard for

looking at loss of GPI anchors ?

Answer 5

• flow cytometry
• previous method:
  
  • Ham’s test
  • showed enhanced lysis of PNH red cells in the presence of fresh complement and acidified serum
  • could not detect small clones and was affected by red cell transfusions

Question 6

What is the problem with trying to analyze

loss of GPI anchors in bone marrow specimens ?

Answer 6

• maturation dependent expression of antigens is problematic in the analysis
• CD15, CD16 and CD55
  
  • all of which increase sequentially in myeloid precursors
    
    • particularly neutrophils
  • these markers should be bright on neutrophils and monocytes in the peripheral blood except in MDS which can show dim expression (REMEMBER)

Question 7

Why is FLAER preferred for the

analysis of PNH clones ?

Answer 7

• Aerolysin from bacteria binds the GPI anchor itself and not just the antigen
• so activity is less dependent on maturation stages of the myeloid cells since it doesn’t target the antigen
• specimens must be processed within 48 hours
• PB must be collected in EDTA
• 5000 events must be analyzed to produce the appropriate sensitivity

Question 8

Read p. 204 onward

Question 9

Which antigens are suitable for

analysis of PNH clones on granulocytes?

Answer 9

• CD16, CD24, CD55, CD59 and CD66
• should demonstrate loss of more than one of these markers, so better to evaluate 2 or more

Question 10

Which marker cannot be used to define

the monocytic lineage since it may be lost

in PNH clones ?

Answer 10

• CD14

Question 11

In what circumstances are peripheral blood

monocytes preferred for the screening of PNH clones?

Answer 11

• patients with severe neutropenia
• patients with relative excess of eosinophils to neutrophils (CD13+, CD16-)
• patients with significant left shifted myeloid maturation
  
  • CD13+, CD16dim