A flow cytometric method for determination of the blood neutrophil fraction in rats

Abstract

Determination of the proportion of neutrophils in the peripheral blood is important for diagnostic purposes in medicine and for evaluating new drugs in the pharmaceutical industry. To measure the neutrophil concentration in rat blood, a fast and accurate flow cytometric method was developed. Rat neutrophils were quantified by using primary antibodies that recognize the RP1 antigen and secondary antibodies conjugated with fluorescein isothiocyanate. The flow cytometric method was calibrated by comparing cytometric results with data from a manual differential count. The results obtained by these 2 methods correlated with a Pearson correlation coefficient of 0.91 and were in agreement according to subsequent statistical analysis. To confirm the usefulness of the method in preclinical applications, the production of neutrophils in rats was stimulated by pegfilgrastim. Blood samples were taken at predetermined time points, and the pharmacodynamic profile was determined. These results confirmed that the flow cytometric method for neutrophil quantification is accurate and much faster than the manual microscopic method. Moreover, the flow cytometric method is easy to use, suggesting that it could become the method of choice for preclinical applications.

Figure 1.

Representative flow cytometric analysis of rat peripheral blood leukocytes by forward and side scatter characteristics. Region A is the main leukocyte population.

Figure 2.

Cells were treated with primary antibodies specific for rat neutrophils and FITC-conjugated secondary antibodies. The histogram shows the green fluorescence (530 nm bandpass) of the rat leukocytes (narrow line, 2 peaks). Cells untreated with primary antibodies were used as a negative control (wide line, 1 peak).

Figure 3.

Correlation between flow cytometric and microscopic methods. A line-fitted plot of regression analysis comparing the microscope-determined percentage of neutrophils to flow cytometric-determined percentage of neutrophils, r = 0.91.

Figure 4.

A difference plot in which the difference between the 2 methods was plotted against the mean value of the methods. Dotted lines represent 1.96 SD above and below the mean (95% prediction interval).

Figure 5.

Three-dimensional histogram of the green-fluorescence of rat leukocytes after induction with pegfilgrastim. Samples were obtained from an untreated rat (0 h) and at various times after induction.

Figure 6.

Pegfilgrastim-induced pharmacodynamic profile. Each point on the graph represents the absolute neutrophil count (mean ± 1 SD; n = 8). Solid circles, flow cytometry; open circles, microscopy.