When pigs fly: Immunomagnetic separation facilitates rapid determination of Pig-a mutant frequency by flow cytometric analysis

Abstract

In vivo mutation assays based on the Pig-a null phenotype, that is, the absence of cell surface glycosylphosphatidylinositol (GPI) anchored proteins such as CD59, have been described. This work has been accomplished with hematopoietic cells, most often rat peripheral blood erythrocytes (RBCs) and reticulocytes (RETs). The current report describes new sample processing procedures that dramatically increase the rate at which cells can be evaluated for GPI anchor deficiency. This new method was applied to blood specimens from vehicle, 1,3-propane sultone, melphalan, and N-ethyl-N-nitrosourea treated Sprague Dawley rats. Leukocyte- and platelet- depleted blood samples were incubated with anti-CD59-phycoerythrin (PE) and anti-CD61-PE, and then mixed with anti-PE paramagnetic particles and Counting Beads (i.e., fluorescent microspheres). An aliquot of each specimen was stained with SYTO 13 and flow cytometric analysis was performed to determine RET percentage, RET:Counting Bead ratio, and RBC:Counting Bead ratio. The major portion of these specimens were passed through ferromagnetic columns that were suspended in a magnetic field, thereby depleting each specimen of wild-type RBCs (and platelets) based on their association with anti-PE paramagnetic particles. The eluates were concentrated via centrifugation and the resulting suspensions were stained with SYTO 13 and analyzed on the flow cytometer to determine mutant phenotype RET:Counting Bead and mutant phenotype RBC:Counting Bead ratios. The ratios obtained from pre- and post-column analyses were used to derive mutant phenotype RET and mutant phenotype RBC frequencies. Results from vehicle control and genotoxicant-treated rats are presented that indicate the scoring system is capable of returning reliable mutant phenotype cell frequencies. Using this wild-type cell depletion strategy, it was possible to interrogate ≥ 3 million RETs and ≥ 100 million RBCs per rat in approximately 7 minutes. Beyond considerably enhancing the throughput capacity of the analytical platform, these blood-processing procedures were also shown to enhance the precision of the measurements.

Effective use of the <i>Pig-a</i> gene mutation assay for mutagenicity screening: measuring CD59-deficient red blood cells in rats treated with genotoxic chemicals

“…Immunomagentic methods for depletion of wild-type RBCs (Dertinger et al, 2011b) or enrichment of RETs (Kimoto et al, 2011a) show promise in this regard. Dertinger et al developed a system for depletion of CD59-positive RBCs Vol.…”

Le gènePIG-A, nouveau marqueur de mutagenèse

“…Son intérêt réside, en partie, dans les faibles volumes de sang nécessaires pour l'examen permettant ainsi des analyses itératives, par exemple sur le même animal dans les études de toxicologie à administrations réitérées. Les protocoles appliqués actuellement chez le rongeur permettent d'analyser en quelques minutes plus d'un million d'érythrocytes ou de réticulocytes à partir d'un prélèvement sanguin de 100 µl [27]. Si le principe du test reste inchangé, le protocole a progressivement été amélioré afin d'augmenter ses performances tout en limitant le nombre d'événements totaux analysés.…”

“…Un champ magnétique permettra de retenir les cellules marquées fixées sur les billes. L'analyse sera réalisée soit sur les cellules retenues [4,30], soit sur les cellules non marquées, et donc libres, qui seront récupérées dans l'éluat [27].…”

The <i>in vivo</i> Pig-a gene mutation assay is applied to study the genotoxicity of procarbazine hydrochloride in Sprague-Dawley rats

“…Both were incubated for 20 min. Then, the samples were washed and mixed together; thus, the mixture was able to provide sufficient numbers of RBCs, and a full range of FITC fluorescence intensities were useful for optimizing PMT voltages and fluorescence compensation settings (Dertinger et al, 2011). Moreover, the accuracy of flow cytometry detection was assessed by evaluating the linear correlation between the expected CD59-negative value and the theoretical value; the measurements were set to 1.…”

“…The two phenotypes can be distin-guished and detected by indirect immunofluorescence staining (Kimoto et al, 2012). Presently, the Pig-a gene has been utilized successfully to investigate the MF in many studies (Bhalli et al, 2011;Dobrovolsky et al, 2012;Dertinger et al, 2011). The literature data indicated that the absence of GPI is the leading cause of paroxysmal nocturnal haemoglobinuria (PNH) (Phillips et al, 2001).…”