Flow Cytometric Analysis of Erythropoietic Abnormality: Changes in the Cell Maturity Index of Reticulocytes and Retic Distribution Index Are Useful as Indicators of Erythropoietic Toxicity in Non-Clinical Studies

Abstract

-We performed a flow cytometric (FCM) analysis of the maturity of reticulocytes using peripheral blood obtained from rats administered 5-fluorouracil (5-FU) at 10, 50 and 100 mg/kg or acethylphenyl hydrazine (APHZ) at 1 and 3 mg/kg to clarify whether the FCM method is useful for assessing toxicity. In the 5-FU-administered rats, a decrease and recovery of the immature reticulocyte fraction (Cell Maturity Index, CMI; Retic Distribution Index, RDI) was observed more rapidly (several days prior to changes in the reticulocyte ratio), and sensitively regarding dose-dependency (clear changes were observed at 10 mg/kg, whereas the reticulocyte ratio was only slightly affected). In addition, there was good agreement between the microscopic results obtained by counting Heilmyer's reticulocyte maturation groups, especially for type I and II, and CMI/RDI assessed by the FCM method after the administration of 50 and 100 mg/kg of 5-FU, the dose at which clear changes were obtained with the microscopic method. In the APHZ-administered rats, a dose-dependent increase in CMI/RDI coinciding with the enhancement of reticulocyte production was observed. The results suggested that the automated FCM method could be a useful and valuable tool to assess and predict impairments of erythropoiesis, especially for CMI and RDI, and could help in the diagnosis of hematological disorders in experimental animals.

Development of an in vivo gene mutation assay using the endogenous Pig‐A gene: I. Flow cytometric detection of CD59‐negative peripheral red blood cells and CD48‐negative spleen T‐cells from the rat

“…In our FCM assay, we initially gated a single cell population by light scatter to eliminate platelets from subsequent consideration. Our cytogram settings and gating strategy for RBCs are the same as previously reported by Kakiuchi et al [2006]. As shown in Figure 1A (left), the relatively low forward and side scatter of platelets clearly differentiated them from RBCs and WBCs.…”

The Liquid Culture Filtrates of Entomogenous fungus Paecilomyces tenuipes and Its Glycoprotein Constituent Protects against Anemia in Mice Treated with 5-Fluorouracil

“…The column was subsequently eluted with H 2 O (500 ml) to afford 100 fractions of 5 ml and the absorbance was monitored at 220 nm. After lyophilization, some of the eluted fractions (fractions [20][21][22][23][24][25] were combined and lyophilized to give Fraction II (PGF: 31.9 mg). This method effectively extracted PGF, which represented the active fraction isolated from PTCF.…”

“…In the present experiment, oral injection of culture medium alone could not protect against 5-FU-induced hematotoxicity, even with the same PTCF regimen and a higher dose (Ͻ100 mg/kg/d; data not shown). Since EPO is known to act as a stimulatory signal for the proliferation and differentiation of hematopoietic progenitors, especially in erythroid lineages, [4][5][6] this cytokine was used as a positive control 20) and also in the present experiment. Subcutaneous injection of mrEPO (5 U/mouse) produced a significant improvement of erythrocytopenia and slightly improved the leukocytopenia induced by 5-FU (Fig.…”

“…3C). Since the peripheral reticulocyte count reflects erythropoiesis, 18,20) we also enumerated peripheral reticulocytes in 5-FU-treated mice. The number of reticulocytes in peripheral blood was dramatically reduced on day 5, and then quickly increased to 142% (relative to the normal level) by 16 d after 5-FU injection (Fig.…”

Laboratorio De Radiopatología De La Autoridad Regulatoria Nuclear

“…El IMR se basa en el análisis por citometría de flujo de poblaciones reticulocitarias de sangre periférica a través de la correlación entre la intensidad de fluorescencia obtenida con un colorante específico (naranja de tiazol) y el contenido de ácido ribonucleico (RNA), definiendo poblaciones de baja, media y alta fluorescencia. La presencia de reticulocitos de alta fluorescencia puede ser considerada como una expresión temprana de actividad eritropoyética, y ésta un indicador de la actividad hematopoyética global [1,2]. En un estudio de 27 pacientes sometidos a ICT como condicionamiento para el trasplante de médula ósea se observó que el corrimiento hacia la región de alta fluorescencia a partir de reticulocitos con alto contenido de RNA y verificado a través del IMR, antecede de modo significativo al método convencional de recuento manual o automatizado de reticulocitos totales, neutrófilos y plaquetas en sangre periférica [3], constituyéndose en un método cuantitativo y de fácil implementación para la detección temprana de reactivación de la médula ósea.…”