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
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
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
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
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
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)
- all of which increase sequentially in myeloid precursors
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
8
Q
Read p. 204 onward
A
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
10
Q
Which marker cannot be used to define
the monocytic lineage since it may be lost
in PNH clones ?
A
- CD14
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