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Paroxysmal Nocturnal Hemoglobinuria (PNH) - High Sensitivity by FLOW

Test Code

FLOW PNH

Test Synonyms

PNH Screen, high sensitivity analysis for PNH, CD59, FLAER, GPI deficiency

Associations

Coombs negative hemolytic anemia, myelodyspastic syndromes with unilineage dysplasia and unexplained hemoglobinuria, aplastic anemia, cytopenia or thrombosis;

Soliris® (Eculizumab)

Methodology

Flow cytometry, multiparameter analysis
Screen WBC, if any positive finding auto-reflex for RBC component

Turnaround Time

1 day

Specimen Requirements

2.0 mL (min. 0.5 mL) heparin whole blood, EDTA accepted
2.0 mL (min. 0.5 mL) heparin bone marrow aspirate*, EDTA accepted

*Although not a preferred sample type, bone marrow samples are acceptable for detecting most cases of clinically symptomatic disease but are not ideal for detecting very small PNH-like clones, <1-2%

Specimen Stability
Whole blood stable for 48 hours at room temperature
Storage & Handling

Ship at ambient temperature; Ship in a Styrofoam container with a cool/refrigerated pack (Do not allow cool pack to directly contact sample)

Causes for Rejection

Specimen >48 hours old prior to testing; Specimens stored at incorrect temperature; Non-viable specimens; Specimens in inappropriate anticoagulant; Too few cells; Excessive hemolysis; Specimen clotted

Reference Range

Normal = no evidence of PNH population

Positive = > 0.01% PNH population

Description

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematopoietic stem cell disorder characterized by a somatic mutation of the PIG-A gene leading to a deficiency of the cell membrane anchor glycosylphosphatidylinositol (GPI) and a deficiency of GPI linked proteins. PNH has three distinct clinical features; complement-mediated and primarily intravascular hemolysis, thrombotic tendency and underlying bone marrow failure.  The International PNH Interest Group lists three main categories of presentation: classic PNH with hemolytic and thrombotic episodes, PNH in the context of other primary disorders and subclinical PNH.  Deficiency in the GPI-anchor membrane protein CD59 (membrane inhibitor of reactive lysis) is used to identify PNH red blood cells (RBC) with identification of types I, II and III (III = totally lacking CD59, II = partially deficient, I = normal, CD59+). 
A novel reagent, fluorescent aerolysin (FLAER) binds specifically to GPI-anchors and is used to evaluate WBC PNH clones.  PNH populations will have no reactivity with the FLAER reagent. Lineage specific (CD15, CD33) monoclonal antibodies will be incorporated into gating algorithm, in addition to GPI-anchor linked proteins CD24 and CD14 for the WBC evaluation to increase specificity and sensitivity.

References
  1. Southerland et al. (2007). Diagnosing PNH with FLAER and multiparameter flow cytometry. Cytometry. 72B:167–177.
  2. Borowitz MJ, et al. Guidelines for the Diagnosis and Monitoring of Paroxysmal Nocturnal Hemoglobinuria and Related Disorders by Flow Cytometry. Cytometry. 78B: 211-230 (2010)
  3. Parker C. et al. Diagnosis and management of paroxysmal nocturnal hemoglobinuria. Blood. 106: 3699-3709 2005
  4. Richards SJ et al. Recent advances in the diagnosis monitoring and management of patients with paroxysmal nocturnal hemoglobinuria. Cytometry. 72B: 291-298 2007
Trademarks

Soliris is a a registered trademark of Alexion