Intra- and interlaboratory variability of paroxysmal nocturnal hemoglobinuria testing by flow cytometry following the 2012 Practical Guidelines for high-sensitivity paroxysmal nocturnal hemoglobinuria testing

2013 ◽  
Vol 84B (4) ◽  
pp. 229-236 ◽  
Author(s):  
Iuri Marinov ◽  
Martina Kohoutová ◽  
Vlasta Tkáčová ◽  
Daniel Lysák ◽  
Monika Holubová ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
High Sensitivity ◽  
Interlaboratory Variability ◽  
Practical Guidelines

scholarly journals Technical advances in flow cytometry-based diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria

2016 ◽  
Vol 14 (3) ◽  
pp. 366-373 ◽  
Author(s):  
Rodolfo Patussi Correia ◽  
Laiz Cameirão Bento ◽  
Ana Carolina Apelle Bortolucci ◽  
Anderson Marega Alexandre ◽  
Andressa da Costa Vaz ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Retrospective Study ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Laboratory Data ◽  
High Sensitivity ◽  
Laboratory Diagnosis ◽  
Clone Detection ◽  
Technical Advances ◽  
Sensitive Determination

ABSTRACT Objective: To discuss the implementation of technical advances in laboratory diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria for validation of high-sensitivity flow cytometry protocols. Methods: A retrospective study based on analysis of laboratory data from 745 patient samples submitted to flow cytometry for diagnosis and/or monitoring of paroxysmal nocturnal hemoglobinuria. Results: Implementation of technical advances reduced test costs and improved flow cytometry resolution for paroxysmal nocturnal hemoglobinuria clone detection. Conclusion: High-sensitivity flow cytometry allowed more sensitive determination of paroxysmal nocturnal hemoglobinuria clone type and size, particularly in samples with small clones.

High-Sensitivity Flow Cytometry Testing for Paroxysmal Nocturnal Hemoglobinuria in Children with Cytopenia: A Single Center Study

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2398-2398
Author(s):  
Choladda V. Curry ◽  
M. Tarek Elghetany ◽  
Andrea M. Sheehan ◽  
Alison A. Bertuch ◽  
Ghadir S. Sasa
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Aplastic Anemia ◽  
Immunosuppressive Therapy ◽  
Hemolytic Anemia ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
High Sensitivity ◽  
Deficiency Anemia ◽  
Unknown Etiology ◽  
Flow Cytometry Assay

Abstract Abstract 2398 Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired hematopoietic stem cell disorder characterized by expansion of cells with complete or partial loss of glycosyl phosphatidyl-inositol-anchored proteins. PNH usually presents with one or more of three clinical manifestations: intravascular hemolysis, thrombosis, or acquired bone marrow failure [aplastic anemia (AA) or myelodysplastic syndrome (MDS)]. Flow cytometry has become the gold standard for the diagnosis of PNH, particularly with the recent publication of guidelines for the diagnosis and monitoring of PNH and related disorders in 2010. PNH occurs rarely in children, and, consequently, the published literature regarding PNH in this pediatric population consists only of small case series, making it difficult to extrapolate the frequency of which PNH clones are identified. Moreover, no studies are available on the incidence of PNH clones in children with MDS and acquired aplastic anemia (AAA). We, therefore, sought to determine how frequently a high sensitivity FLAER-based assay, with a sensitivity of 0.01%, would detect PNH clones in children with cytopenias. Method and Results: The study period was from December 2010 to July 2011. PNH testing was performed using a high sensitivity FLAER based assay according to published guidelines using the combination of FLAER/CD64/CD15/CD33/CD24/CD14/CD45 for WBC testing and CD235a/CD59 for RBC testing. There were 31 peripheral blood samples from 29 patients (17 males/12 females) ranging in age from 4 months to 17 years (median, 10 years). All patients were tested for PNH because of cytopenia [pancytopenia (n = 14) and uni- or bicytopenia (n = 15)]. Patients had a mean Hgb of 10.7 gm/dL, mean ANC of 2.66 X103/uL and mean platelet of 115 X103/uL. Review of medical charts revealed the following clinical diagnoses: classic PNH - episodic hemolytic anemia with persistent thrombocytopenia (1), severe AA (SAA, 8), SAA with myelofibrosis (1), MDS (1), Fanconi anemia (1), chronic thrombocytopenia (2), refractory iron deficiency anemia (1), bone marrow suppression likely due to virus/medication (1), parvovirus infection (1), Copper deficiency (1), systemic lupus erythematosus (SLE, 1), and cytopenia of unknown etiology (10). Of note, all patients with AAA had SAA. PNH clones were identified in 6 out of 29 patients (20%): minor clones (<1% PNH population) in 3 patients: average clone sizes 0.12% [range 0.02–0.25] granulocytes (G), 0.51% [0.20–0.99] monocytes (M), and 0.08% [0.04–0.14] red blood cells (RBCs), and major clones (>1% PNH population) in 3 patients: average clone sizes 31.11% [3.98–67.58] G, 31.98% [6.15–71.1] M, and 14.76% [1.19–38.03] RBC, respectively, with ages ranging from 4 to 17 years. Patients who were identified to have minor PNH clones all presented with pancytopenia. Two were diagnosed with SAA; the cause of pancytopenia in the third patient is currently under investigation. None of patients with minor PNH clones had evidence of hemolysis or thrombosis. The three patients with major PNH clones had the following: Classic PNH with hemolytic anemia (1), SAA with PNH clones detected at the time of SAA diagnosis (1), and SAA with PNH clones detected 20 months after immunosuppressive therapy (1). The latter two patients did not have evidence of hemolysis or thrombosis. Of the 10 patients with a diagnosis of SAA or MDS, PNH clones were identified in 4 (40%) patients (2 with minor clones, 2 with major clones). Conclusions: This is the first study to describe the utility of using a standardized high-sensitivity FLAER-based flow cytometry assay to identify PNH clones in children. This is also the first study describing the prevalence of PNH clones in children with MDS and AAA. The identification of a PNH population in 40% of the MDS and AAA cases emphasizes the need for PNH testing in all children with these disorders using a high-sensitivity FLAER based flow cytometry assay. A low sensitivity assay would have missed 2 patients with minor PNH clones. This finding may be of significance considering SAA or MDS patients with PNH clones are more likely to respond to immunosuppressive therapy. Further studies are needed to investigate the prevalence of PNH clones in this setting and its impact on disease manifestations, course, and outcomes in children. Disclosures: No relevant conflicts of interest to declare.

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