diagnosis of paroxysmal nocturnal hemoglobinuria

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diagnosis of paroxysmal nocturnal hemoglobinuria
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outline

• introduction
• paroxysmal nocturnal hemoglobinuria (PNH)
• clinical indications for testing
• guidelines for diagnosis of PNH
• standard sensitivity testing
• leukocyte assays
• red cell assays
• high sensitivity testing
• examples

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PNH

• 1.3 new cases per million persons per year
• stem cell disorder (affects all blood cell
  lineages)
• acquired mutation of X-linked PIGA gene
• deficiency of proteins normally linked to the
  cell membrane by GPI anchor
• partial deficiency type II cells
• complete deficiency type III cells
• deficiency of CD55 and CD59 leads to
  complement-mediated hemolysis

Borowitz MJ, et al. Cytometry B 201078B211-230.
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PNHclinical features

• hemolysis
• thrombosis
• bone marrow failure

Borowitz MJ, et al. Cytometry B 201078B211-230.
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indications for PNH testinghemolysis

• intravascular (hemoglobinuria or elevated plasma
  hemoglobin)
• accompanied by
• iron deficiency or
• abdominal pain or esophageal spasm or
• thrombosis or
• granulocytopenia and or thrombocytopenia
• other acquired Coombs-negative non-schistocytic,
  non-infectious hemolytic anemia

Borowitz MJ, et al. Cytometry B 201078B211-230.
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indications for PNH testingthrombosis

• unusual sites
• hepatic veins (Budd-Chiari syndrome)
• other intraabdominal veins (portal, splenic,
  splanchnic)
• cerebral sinuses
• dermal veins
• with signs of accompanying hemolytic anemia
• with unexplained cytopenia

Borowitz MJ, et al. Cytometry B 201078B211-230.
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indications for PNH testingbone marrow failure

• suspected or proven aplastic or hypoplastic
  anemia
• refractory cytopenia with unilineage dysplasia
• other cytopenias of unknown etiology after
  adequate work up

Borowitz MJ, et al. Cytometry B 201078B211-230.
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diagnosis

• standard sensitivity assays
• 5000-10,000 cells to achieve 1 sensitivity
• classic PNH
• high sensitivity assays
• 250,000 cells to achieve 0.01 sensitivity
• small clones in patients with bone marrow failure
  disorders
• aplastic anemia
• refractory cytopenia with unilineage dysplasia

Borowitz MJ, et al. Cytometry B 201078B211-230.
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diagnosis

• white blood cell assays
• best estimate of clone size
• granulocyte assays performed in all cases
• monocyte assays provide confirmation
• lymphocytes not suitable
• red blood cell assays
• better than granulocyte assays for detecting
  partial deficiency (type II cells)
• performed in cases where granulocyte clone
  detected or in all cases

Borowitz MJ, et al. Cytometry B 201078B211-230.
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diagnosisleukocyte assays antibodies

• CD16
• absent from eosinophils, lost in MDS, polymorphic
  variants not recognized by some antibodies
• best combined with another antigen
• CD14
• expressed on monocytes
• commonly used to detect monocyte clones
• absent from immature monocytes and dendritic
  cells so small negative populations cannot be
  interpreted as clones
• CD66b
• FLAER
• binds to GPI anchor
• may be most useful reagent for detecting monocyte
  and granulocyte clones

Borowitz MJ, et al. Cytometry B 201078B211-230.
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diagnosisleukocyte assays

• gating
• FSC/SSC or 45/SSC can be used
• lineage markers help identify pure populations
• analysis
• helpful to have positive and negative cells in
  gate
• desirable to use 2 markers and set quadrants
• type II populations identified occasionally
  clinical significance unknown

Borowitz MJ, et al. Cytometry B 201078B211-230.
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Borowitz MJ, et al. Cytometry B 201078B211-230.
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PNH granulocyte population 23.5
PNH granulocyte population 97
Borowitz MJ, et al. Cytometry B 201078B211-230.
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PNH granulocyte population 60.9 54.8 type III
cells 6.1 type II cells
Borowitz MJ, et al. Cytometry B 201078B211-230.
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diagnosisRBC assays antibodies

• CD59
• high level of expression
• CD55
• less abundant
• not suitable for use as single marker
• may not add to analysis of CD59

Borowitz MJ, et al. Cytometry B 201078B211-230.
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diagnosisRBC assays

• gating
• log/log displays of forward and side scatter to
  identify RBCs
• gating with glycophorin A may improve isolation
  of RBCs
• aggregation a problem
• analysis
• single color histograms for populations large
  enough to form separate peak
• dot plots for small populations

Borowitz MJ, et al. Cytometry B 201078B211-230.
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diagnosisRBC assays identification of type II
cells

• compare to type I cells in specimen
• wash well
• transfusion may yield 2 type I populations
• positive and negative (unstained cells) control
  can be used to identify expected positions of
  type I and III cells

Borowitz MJ, et al. Cytometry B 201078B211-230.
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Borowitz MJ, et al. Cytometry B 201078B211-230.
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Borowitz MJ, et al. Cytometry B 201078B211-230.
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Borowitz MJ, et al. Cytometry B 201078B211-230.
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Borowitz MJ, et al. Cytometry B 201078B211-230.
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diagnosishigh sensitivity assays

• RBCs
• glycophorin A to purify red cells
• CD59 advantage of adding CD55 not studied
• causes of false positives fragmented RBCs
  (glycophorin A), carryover of unstained control
  (run first)
• leukocytes
• lineage markers for gating
• more than one GPI-linked antigen
• FLAER helpful
• CD14 not acceptable as single marker because
  monocyte gate will contain dendritic cells
• commonly used combinations CD24 and FLAER for
  granulocytes, CD and 14 FLAER for monocytes

Borowitz MJ, et al. Cytometry B 201078B211-230.
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Borowitz MJ, et al. Cytometry B 201078B211-230.
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Background rate in normal person (2/315,617).
Small PNH clone (0.013).
Borowitz MJ, et al. Cytometry B 201078B211-230.
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• Specimen Peripheral blood
• 
  
  
  
• Test Immunophenotypic analysis was performed
  using gating antibodies CD45, CD33, GPI-linked
  antibodies CD14, CD24, as well as fluorescent
  Aerolysin (FLAER)
• 
  
• CELL TYPE POPULATION
  DEFICIENT
• 
  
• Monocytes FLAER/CD14 Deficient
  
• 
  
• Granulocytes FLAER/CD24 Deficient
  
• 
  
• no evidence of deficiency
• INTERPRETATION
• Monocytes No evidence of decreased or absent
  expression of FLAER or CD14
• Granulocytes No evidence of decreased or absent
  expression of FLAER or CD24
• Red Blood Cells Test not performed when WBC
  testing is negative
• Comment Flow cytometric analysis does not show
  any evidence of a PNH clone. These findings do
  not support diagnosis of PNH. Clinical
  correlation is recommended.

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• Specimen Peripheral blood
• 
  
  
  
• Test Immunophenotypic analysis was performed
  using gating antibodies CD45, CD33, GPI-linked
  antibodies CD14, CD24, as well as fluorescent
  Aerolysin (FLAER)
• 
  
• CELL TYPE POPULATION
  
  DEFICIENT
• 
  
• Monocytes FLAER/CD14 Deficient
  
  60
• 
  
• Granulocytes FLAER/CD24 Deficient
  
  59.8
• 
  
• RBC Type II (partial
  CD59 deficient)
  21.6
• RBC Type III (complete
  CD59 deficient)
  1.1
• RBC PNH Clone size
  (Type II and Type III combined)
  22.7
• 
  
• 77.2 CD59 positive RBC detected (normal-Type I)
• INTERPRETATION