Specimen

Peripheral Blood

Collection Medium:

Minimum:

Adult or pediatric: 10ml; yellow top tube (ACD-A)

Delivery Instructions:

Deliver to laboratory immediately after collection.

Testing Schedule:

0800-1630 Monday through Friday.  For additional services, 
contact Clinical Pathology Resident on-call at pager #3404.

Analytic Time:

2 days

Reference Range:

An interpretative report will be provided by the pathologist.

Comments:

Ham's acid hemolysin and sucrose lysis tests have been replaced by flow 
cytometric testing for glycosylphosphatidyl inositol (GPI)-anchored 
proteins CD55 and CD59, and aerolysin binding.  These proteins are not 
expressed on PNH blood cells and their lack of expression is determined 
by flow cytometric assay.

The channel-forming toxin, aerolysin, and its inactive precursors, 
proaerolysin, bind selectively with a high affinity to the GPI anchor 
itself.  The lack of CPI anchor on blood cell surface will decrease the 
ability of fluorescently labeled protein aerolysin (FLAER) to bind to 
nucleated blood cells in patients with PNH.

Paroxysmal nocturnal hemoglobinuria (PNH) is a stem cell disorder in 
which the affected cells are deficient in GPI-anchored proteins. 
GPI-anchored proteins include a number of important molecules on the 
surfaces of blood cells of all lineages.  These include CD55 
(decay-accelerating factor, DAF) and CD59 (membrane inhibitor of 
reactive lysis, MIRL) which protect against accidental activation of 
the complement system and cell lysis.

Determination of CD55 and CD59 must be performed on fresh whole blood.  
Both monocytes and granulocytes are analyzed for CD55/CD59 expression 
and aerolysin bindings. Granulocytes are the most sensitive population 
in which to detect GPI-anchored protein deficiency.  Two additional 
markers are performed for gating purposes, CD45 (leukocyte common 
antigen) and CD33 (myeloid antigen).

Results are issued as a Bone Marrow (H-6) report interpreted by a 
pathologist. The number of GPI-anchored protein deficient cells can 
vary widely from case to case.  Those patients with the highest 
relative numbers of GPI-anchored protein deficient cells are most 
likely to have classical PNH symptoms, while those with small relative 
numbers are more likely to present with aplastic anemia or 
myelodysplastic syndrome.  About 20-25% of patients with aplastic 
anemia and MDS have been found to demonstrate small clones of PNH 
cells, so studies for PNH may also be indicted in patients with these 
diagnoses.

REFS:
1)Richards, S et al. Application of Flow Cytometry to the Diagnosis of 
Paroxysmal Noctural Hemoglobinuria. Cytometry 2000; 42:223-233.
2)Dunn, D, et al. Paroxysmal Nocturnal Hemoglobinuria in Patients with 
Bone Marrow Failure Syndromes. Ann Int Med 1999; 131:401-408.
3) Brodsky RA, et al. Improved detection and characterization of 
paroxysmal nocturnal hemoglobinuria using fluorescent aerolysin.  Am J 
Clin Pathol 2000; 114:459-66.

Methodology:

Flow Cytometry

CPT Code:

Technical:     88184 x1 and 88185 x3
Professional:  88187 - 26

See Additional Information:
Specimens Requiring Immediate Delivery