Análise da expressão de moléculas ancoradas ao glicosilfosfatidilinositol (GPI) em diferentes linhagens celulares, em pacientes com anemia aplásica e síndrome mielodisplásica

Abstract

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired hematopoietic stem cell disorder, caused by genetic alterations that compromises the synthesis of glycosylphosphatidylinositol (GPI) anchor and results in deficiency of linked proteins. The laboratory diagnosis is based on the detection of blood cells deficient in GPI-anchored proteins by flow cytometry. Small PNH clones have been detected in patients with aplastic anaemia (AA) and myelodysplastic syndrome (MDS) and their presence has been associated to favorable response to immunosuppressive therapy. The use of sensitive assays for detection of small PNH clones is a current demand. Our goals were to develop a sensitive assay for detection of GPI-anchored proteins deficient cells, by flow cytometry, in the Laboratory Medicine Service of Hospital das Clínicas/UFMG and to investigate the presence of PNH clones in AA and MDS patients. Peripheral blood samples from 20 AA patients and 30 MDS patients were analyzed by flow cytometry using monoclonal antibodies to CD16, CD24, CD55 and CD59 (neutrophils); CD14 and CD55 (monocytes); CD55 and CD59 (erythrocytes). Fluorescent aerolysin (FLAER) was used to analyze neutrophils and monocytes.Lineage markers were used to select the cell populations of interest. The control group was represented by peripheral blood samples of 20 adult volunteers. PNH cells were detected in 5 (25%) AA patients. They were clearly distinct from normal cells in the three analyzed cell populations. The deficient GPI-anchored protein expression was detected by all combinations of reagents used and the proportions of PNH cells varied from 0.14 to 94.84% of the analyzed events. Clinical and laboratory evidence of intravascular hemolysis was present in only one patient, suggesting a case of AA that has evolved to classic PNH. PNH cells were not detected in blood samples of MDS patients. However, hypogranular neutrophils and/or immature monocytes were observed in 53% of the patients resulting in overlap between these two populations on the SSC/CD45 plot. In addition, single decreased expression/loss of CD14, CD16 and/or CD55 was observed on immature and dysplastic cells in 50% of these samples, which however was not interpreted as PNH cells, based on defined clone positivity criteria. Thereby, it was possible to identify the technical challenges and to define the procedures to evaluate samples of MDS patients. Concerning the FLAER performance, neutrophils and monocytes showed bright staining resulting in a clear distinction between normal cells and GPI deficient cells. The results showed total agreement between FLAER staining and GPI-anchored proteins expression. CONCLUSION: Multiparameter flow cytometry analysis offers high sensitivity and accuracy in the detection of subclinical PNH clones in the setting of AA and MDS. FLAER showsexcellent performance in the detection of PNH neutrophils and monocytes and may be used in a routine laboratory for screening of PNH clones.