scholarly journals Standardized high-sensitivity flow cytometry testing for paroxysmal nocturnal hemoglobinuria in children with acquired bone marrow failure disorders: A single center US study

2017 ◽  
Vol 94 (4) ◽  
pp. 699-704 ◽  
Author(s):  
Rachel E. Donohue ◽  
Andrea N. Marcogliese ◽  
Ghadir S. Sasa ◽  
M. Tarek Elghetany ◽  
Alka A. Redkar ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Bone Marrow Failure ◽  
High Sensitivity ◽  
Single Center

Establishment of a flow cytometry assay for detecting paroxysmal nocturnal hemoglobinuria-type cells specific to patients with bone marrow failure

2018 ◽  
Vol 97 (12) ◽  
pp. 2289-2297
Author(s):  
Kohei Hosokawa ◽  
Chiharu Sugimori ◽  
Ken Ishiyama ◽  
Hiroyuki Takamatsu ◽  
Hideyoshi Noji ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Bone Marrow Failure ◽  
Flow Cytometry Assay

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.

Paroxysmal Nocturnal Hemoglobinuria: An Atypical Clinical Case Report and Proof of the Disease in Blood and BONE Marrow with FLOW Cytometry

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5166-5166
Author(s):  
Fabienne Pineau-Vincent ◽  
Pierre Lemaire ◽  
Habib Ghnaya ◽  
Guillaume Direz ◽  
Mohamed Kaabar ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Case Report ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Blood Smear ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Chromatin Condensation ◽  
Clinical Signs ◽  
Pnh Clone

Abstract Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired disease, associated with hemolytic anemia and bone marrow failure. The cellular abnormality is a mutation in the phosphatidylinositol glycan class (PIG A) resulting in a deficiency of glycosylphosphadityl-inositol (GPI)-anchored complement regulatory proteins, including CD 55 and CD59, on the surface of blood cells. Case report We report the case of a French, 81 year-old-man, who was admitted to our institution with an unusual clinical presentation. He had a rheumatologic monitoring in the context of polyarthritis associated with anemia (98g/L). No hemolytic events were noticed and there was no notion of either transfusion. Biological results showed hemolytic regenerative anemia (98g/L) with 136G/L of reticulocytes, neutrophil polynuclears (4.2G/L) without degranulation and nevertheless rare degranulation cells, no blasts, normal level of platelets (258G/l), increase of LDH (Nx3), low haptoglobin (0.07g/L), negative direct Coombs test. The cytology aspect of medullar cells associated dysgranulopoiesis with degranulation of myeloid lineage and abnormal chromatin condensation, dyserythropoiesis, dysmegacaryopoiesis, in favor of a multilineage dysplasia without blasts. The marrow karyotype was normal. Due to the morphological results observed on the blood smear and their dissociation with the medullary cytology, flow cytometry (FC500) for GPI‘s expression study was performed. The used antisera were: CD55, CD59, CD14, CD16, CD24, CD66b, CD157, no FLEAR was tested. Results TableBloodBone marrowMononuclear cells CD14 FL378% intermediar cells70% negative cellsNeutrophil cells CD16 PE56% intermediar cells56% negative cellsNeutrophil cells CD66b FITC57% negative cells70% negative cellsGranular cells CD24 PE49% negative cells62% negative cellsRed cells CD55 FITC10% negative cells11% negative cellsRed cells CD59 FITC12% negative cells12% negative cells Figure 1 Blood Figure 1. Blood Figure 2 Bone Marrow Figure 2. Bone Marrow The confirmation was obtained by using CD157PE antisera on bone marrow with 70% negative mononuclear and granular cells. The results confirmed the PNH clone’s presence in the blood and also in bone marrow, and the results of flow cytometry could explain the cytological aspect of neutrophil polynuclear cells. It is rare to explore the expression of GPI molecules in bone marrow and there is no publication about the PNH clone whose identification required bone marrow cells for the confirmation of abnormalities in blood. Thus, the apoptosis in the bone marrow of the defective myeloid cells would explain the difference of granularity of polynuclear cells between bone marrow and blood smear. Conclusion The significance of this observation is related to the search of a PNH clone when cytological dissociation is observed between the peripheral blood and bone marrow, associated with biological hemolysis arguments (increased LDH and decreased haptoglobin). It is well known that 6 at 8% of myelodysplasia had PNH clone; the originality of this case report is the initial clinical signs and the laboratory proof of PNH in the blood and the bone marrow. This observation was submitted at the national reference center of PNH in France (St Louis Hospital - Hematology Department - Professor SOCIE) and the treatment by eculizumab was introduced. Disclosures No relevant conflicts of interest to declare.

Clinical significance of a minor population of paroxysmal nocturnal hemoglobinuria–type cells in bone marrow failure syndrome

Blood ◽  
2002 ◽  
Vol 100 (12) ◽  
pp. 3897-3902 ◽  
Author(s):  
Hongbo Wang ◽  
Tatsuya Chuhjo ◽  
Shizuka Yasue ◽  
Mitsuhiro Omine ◽  
Shinji Nakao
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Clinical Significance ◽  
Blood Cells ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Bone Marrow Failure ◽  
Refractory Anemia ◽  
Severe Thrombocytopenia ◽  
Minor Population ◽  
A Minor

A minor population of blood cells deficient of glycosylphosphatidylinositol (GPI)–anchored membrane proteins is often detected in patients with aplastic anemia (AA), though the clinical significance of such paroxysmal nocturnal hemoglobinuria (PNH)–type cells remains unclear. To clarify this issue, we studied 164 patients with myelodysplastic syndrome (MDS) for the presence of CD55−CD59− granulocytes and red blood cells using sensitive flow cytometry. Among the different subgroups of MDS, a significant increase (ie, at least 0.003%) of PNH-type cells was detected in 21 of 119 patients with refractory anemia (RA); this frequency (17.6%) of RA patients with increased PNH-type cells (PNH+ patients) was much lower than what we previously reported (52.0%) for AA patients. PNH+ RA patients had distinct clinical features compared with RA patients without increased PNH-type cells (PNH− patients), such as less pronounced morphologic abnormality of blood cells, more severe thrombocytopenia, lower rates of karyotypic abnormality (4.8% vs 32.8%) and of progression to acute leukemia (0% vs 6.2%), higher probability of response to cyclosporine therapy (77.8% vs 0%), and higher incidence of HLA-DR15 (90.5% vs 18.5%). These data indicate that the presence of a minor population of PNH-type cells suggests a benign type of bone marrow failure, probably caused by an immunologic mechanism. To choose an appropriate therapy, peripheral blood should be tested using sensitive flow cytometry for the presence of PNH-type cells in all patients with bone marrow failure before treatment.

High sensitivity flow cytometry to detect small population of PNH clone in bone marrow failure syndrome in Japan

Immunobiology ◽  
2016 ◽  
Vol 221 (10) ◽  
pp. 1186
Author(s):  
Yasutaka Ueda ◽  
Jun-ichi Nishimura ◽  
Chiharu Sugimori ◽  
Kohei Hosokawa ◽  
Yuji Yonemura ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Bone Marrow Failure ◽  
High Sensitivity ◽  
Small Population ◽  
Bone Marrow Failure Syndrome ◽  
Pnh Clone

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.

scholarly journals Management of thrombosis in paroxysmal nocturnal hemoglobinuria: a clinician’s guide

2016 ◽  
Vol 8 (3) ◽  
pp. 119-126 ◽  
Author(s):  
Morag Griffin ◽  
Talha Munir
Keyword(s):  
Bone Marrow ◽  
Median Survival ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Bone Marrow Failure ◽  
Paroxysmal Nocturnal Haemoglobinuria ◽  
Orphan Disease ◽  
Life Threatening

Paroxysmal nocturnal haemoglobinuria (PNH), an ultra-orphan disease with a prevalence of 15.9 per million in Europe, is a life-threatening disorder, characterized by haemolysis, bone marrow failure and thrombosis. Patients with PNH prior to the availability of eculizumab had a median survival of between 10 and 22 years, with thrombosis accounting for 22–67% of deaths. 29–44% of patients had at least one thrombosis. This paper provides a clinician’s guide to the diagnosis, management and complications of PNH, with an emphasis on thrombosis.

Diagnosis of paroxysmal nocturnal hemoglobinuria in peripheral blood and bone marrow with six-color flow cytometry

2013 ◽  
Vol 7 (1) ◽  
pp. 99-111 ◽  
Author(s):  
Hai-Su Yang ◽  
Min Yang ◽  
Xiaoyu Li ◽  
Sorina Tugulea ◽  
Henry Dong
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Peripheral Blood ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Color Flow
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