PNH Flow Flashcards

1
Q

What is the triad for

PNH ?

A
  • 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
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2
Q

What are the clinical scenarios of

PNH presentation?

A
  • 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
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3
Q

What chromosome is the PIG-A

gene located on ?

A
  • 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
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4
Q

Why are CD55 and CD59 important

in the development of PNH ?

A
  • 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
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5
Q

What is the current gold standard for

looking at loss of GPI anchors ?

A
  • 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
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6
Q

What is the problem with trying to analyze

loss of GPI anchors in bone marrow specimens ?

A
  • 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)
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7
Q

Why is FLAER preferred for the

analysis of PNH clones ?

A
  • 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
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8
Q

Read p. 204 onward

A
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9
Q

Which antigens are suitable for

analysis of PNH clones on granulocytes?

A
  • CD16, CD24, CD55, CD59 and CD66
  • should demonstrate loss of more than one of these markers, so better to evaluate 2 or more
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10
Q

Which marker cannot be used to define

the monocytic lineage since it may be lost

in PNH clones ?

A
  • CD14
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11
Q

In what circumstances are peripheral blood

monocytes preferred for the screening of PNH clones?

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