Quantitative analysis of the expression of glycosylphosphatidylinositol‐anchored proteins during the maturation of different hematopoietic cell compartments of normal bone marrow

Hernández-Campo, Pilar; Almeida, Júlia; Matarraz, Sergio; Santiago, M. de Jesús Gallegos; Sánchez, María Luz; Órfão, Alberto

doi:10.1002/cyto.b.20143

Cited by 60 publications

(52 citation statements)

References 19 publications

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“…The presence of cells with CD59 loss/low expression was not related to immature myeloid cells in the peripheral blood, a finding in keeping with the observation by Hernandez-Campo et al that there is no significant change in CD59 expression throughout myeloid maturation. 31 We back-traced the cells with CD59 loss/low expression and found, at least in some cases, that these cells clustered in the junction between monocytes and granulocytes ( Figure 2A3). It is noteworthy that monocytes often exhibit significantly dimmer expression of CD59 than granulocytes, 17 , were more prominent in CMML or RAEB in their series 19 and concluded that the detection of large numbers of PNH + cells was associated with a poor prognosis.…”

Section: Cd66bmentioning

confidence: 97%

“…It has been shown that the expression of GPI-anchored proteins varies throughout neutrophil and monocyte maturation. 17,31,32 The level of CD55 expression is low in early myeloid precursors until the intermediate stages of maturation, and increases in bands and granulocytes. The expression of CD16 is detectable on metamyelocytes, reaching highest levels on bands/mature neutrophils.…”

Section: Cd66bmentioning

confidence: 99%

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Detection of paroxysmal nocturnal hemoglobinuria clones in patients with myelodysplastic syndromes and related bone marrow diseases, with emphasis on diagnostic pitfalls and caveats

Wang¹,

Pozdnyakova

Jorgensen³

et al. 2009

Haematologica

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BackgroundThe presence of paroxysmal nocturnal hemoglobinuria clones in the setting of aplastic anemia or myelodysplastic syndrome has been shown to have prognostic and therapeutic implications. However, the status of paroxysmal nocturnal hemoglobinuria clones in various categories of myelodysplastic syndrome and in other bone marrow disorders is not well-studied. Design and MethodsBy using multiparameter flow cytometry immunophenotypic analysis with antibodies specific for four glycosylphosphatidylinositol-anchored proteins (CD55, CD59, CD16, CD66b) and performing an aerolysin lysis confirmatory test in representative cases, we assessed the paroxysmal nocturnal hemoglobinuria-phenotype granulocytes in 110 patients with myelodysplastic syndrome, 15 with myelodysplastic/myeloproliferative disease, 5 with idiopathic myelofibrosis and 6 with acute myeloid leukemia. ResultsParoxysmal nocturnal hemoglobinuria-phenotype granulocytes were detected in nine patients with low grade myelodysplastic syndrome who showed clinicopathological features of bone marrow failure, similar to aplastic anemia. All paroxysmal nocturnal hemoglobinuria-positive cases demonstrated loss of the four glycosylphosphatidylinositol-anchored proteins, with CD16 - CD66b-clones being larger than those of CD55 -CD59 -(p<0.05). Altered glycosylphosphatidylinositol-anchored protein expression secondary to granulocytic hypogranulation, immaturity, and/or immunophenotypic abnormalities was present in a substantial number of cases and diagnostically challenging. ConclusionsThese results show that routine screening for paroxysmal nocturnal hemoglobinuria clones in patients with an intrinsic bone marrow disease who show no clinical evidence of hemolysis has an appreciable yield in patients with low grade myelodysplastic syndromes. The recognition of diagnostic caveats and pitfalls associated with the underlying intrinsic bone marrow disease is essential in interpreting paroxysmal nocturnal hemoglobinuria testing correctly. In our experience, the CD16/CD66b antibody combination is superior to CD55/CD59 in screening for subclinical paroxysmal nocturnal hemoglobinuria because it detects a large clone size and is less subject to analytical interference.

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Section: Cd66bmentioning

confidence: 97%

Section: Cd66bmentioning

confidence: 99%

Detection of paroxysmal nocturnal hemoglobinuria clones in patients with myelodysplastic syndromes and related bone marrow diseases, with emphasis on diagnostic pitfalls and caveats

Wang¹,

Pozdnyakova

Jorgensen³

et al. 2009

Haematologica

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“…This is a fluorochrome-conjugated inactive variant of the bacterially derived channel-forming protein aerolysin, which binds specifically to GPI-anchors. FLAER may offer significant advantages as a reagent for PNH testing; in contrast with antibodies against many of the GPI-linked antigens normally studied (25,27,28), its binding is less sensitive to the maturational stage of the cells. FLAER can also be used in multicolor combinations with monoclonal antibodies to GPI-linked and non-GPI antigens for the detection of PNH clones (29).…”

Section: Diagnosis Of Pnhmentioning

confidence: 99%

“…Similarly, granulocytes from aged samples (>48-h old) may show altered FSC/SSC scatter characteristics due to poor viability and can also show increased nonspecific binding of antibodies. Another potential pitfall with granulocyte analysis is the presence of immature forms that have significantly weaker expression of some GPI-linked antigens when compared with more mature forms (25,28). In patients with granulocytopenia and relatively high proportions of eosinophils, it may be difficult to construct a pure granulocyte gate unless appropriate markers are used in multicolor analysis.…”

Section: Additional Factors Affecting Interpretationmentioning

confidence: 99%

Guidelines for the diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria and related disorders by flow cytometry

Borowitz

Craig

DiGiuseppe

et al. 2010

Cytometry Part B Clinical

273

429

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Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematopoietic stem cell disorder characterized by a somatic mutation in the PIGA gene, leading to a deficiency of proteins linked to the cell membrane via glycophosphatidylinositol (GPI) anchors. While flow cytometry is the method of choice for identifying cells deficient in GPI-linked proteins and is, therefore, necessary for the diagnosis of PNH, to date there has not been an attempt to standardize the methodology used to identify these cells.Methods: In this document, we present a consensus effort that describes flow cytometric procedures for detecting PNH cells.Results: We discuss clinical indications and offer recommendations on data interpretation and reporting but mostly focus on analytical procedures important for analysis. We distinguish between routine analysis (defined as identifying an abnormal population of 1% or more) and high-sensitivity analysis (in which as few as 0.01% PNH cells are detected). Antibody panels and gating strategies necessary for both procedures are presented in detail. We discuss methods for assessing PNH populations in both white blood cells and red blood cells and the relative advantages of measuring each. We present steps needed to validate the more elaborate high-sensitivity techniques, including the need for careful titration of reagents and determination of background rates in normal populations, and discuss technical pitfalls that might affect interpretation.Conclusions: This document should both enable laboratories interested in beginning PNH testing to establish a valid procedure and allow experienced laboratories to improve their techniques. V C 2010 Clinical Cytometry Society

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“…Pig-a mutation can be measured rapidly using small samples of peripheral blood and flow cytometry, which accounts for the popularity of this assay (Dobrovolsky et al, 2010). Pig-a encodes a protein involved in the synthesis of glycosylphosphatidylinositol (GPI) anchors that link a specific subset of proteins to the cell surface, including the antigens CD59, CD55, CD24, and CD48 (Kawagoe et al, 1994;Hernandez-Campo et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

Persistence of red blood cells with <i>Pig-a</i> mutation in <i>p53</i> knockout mice exposed to X-irradiation

et al. 2014

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-The Pig-a mutation assay is becoming one of the major experimental procedures used to assess in vivo genotoxicity. The assay allows simple in vivo analysis and enables gene mutations in the hematopoietic system to be measured using high throughput flow cytometry. Previously, we demonstrated that X-irradiation increased the Pig-a mutant frequencies in red blood cells (RBCs) of mice in a radiation dose-dependent manner. In this study, to understand how RBCs with Pig-a mutation induced by X-irradiation persist, we compared Pig-a mutant frequencies between irradiated C57BL/6J (p53 +/+ ) mice and irradiated p53 homozygous knockout (p53 -/-) mice by using the RBC Pig-a assay. After the peak in radiationinduced Pig-a mutant frequencies, a gradual decrease in mutant frequencies in irradiated p53 -/-mice was observed, while irradiated p53 +/+ mice had a rapid decrease, which suggests that RBCs with Pig-a mutation are eliminated normally in irradiated p53 +/+ mice but not in irradiated p53 -/-mice due to lack of p53 function. In addition, we also found that the p53 function affected the regulation of Pig-a mutagenesis in aging mice. Our results suggest that p53 function, distinct types of mutation, and the life span of RBCs play key roles in the persistence of Pig-a mutation in the hematopoietic system of RBCs after irradiation.

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Quantitative analysis of the expression of glycosylphosphatidylinositol‐anchored proteins during the maturation of different hematopoietic cell compartments of normal bone marrow

Abstract: Conclusion: Overall, these results provide a detailed map GPI-AP expression during normal hematopoietic differentiation, which could serve as a basis for the identification and characterization of changes occurring in PNH. q

Cited by 60 publications

References 19 publications

Detection of paroxysmal nocturnal hemoglobinuria clones in patients with myelodysplastic syndromes and related bone marrow diseases, with emphasis on diagnostic pitfalls and caveats

Detection of paroxysmal nocturnal hemoglobinuria clones in patients with myelodysplastic syndromes and related bone marrow diseases, with emphasis on diagnostic pitfalls and caveats

Guidelines for the diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria and related disorders by flow cytometry

Persistence of red blood cells with <i>Pig-a</i> mutation in <i>p53</i> knockout mice exposed to X-irradiation

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