Flow cytometric instruments generally rely on optical signals for the analysis of particles which pass through a focused flow cell. Usually, calibration and/or analysis of a reference control product to confirm instrument operating conditions are required prior to performing a particle analysis, in order to ensure accurate and reliable assay results.
Quality control has long been a necessary and routine procedure in clinical hematology. Accuracy in the counting of various types of blood cells is dependent, in part, upon the use of adequate control products and methods of using the control products. With the numerous types of equipment for particle counting now available, quality control by the use of control products is necessary, since the possibility of instrument malfunctioning is ever present. The traditional method of maintaining a quality control program for automatic particle counting equipment has consisted of providing fresh human blood as a whole blood standard. However, this fresh blood is usable for only one day, therefore, various manufactured control products which have longer product lifetime have been developed.
Commonly used particles in a control product simulate or approximate the types of particles or cells that are intended to undergo analysis. Consequently, these particles have been frequently referred to as analog particles. The analog particles should be selected or made so that they have certain characteristics that are similar to those of the particles or cells to be analyzed in the instruments. Exemplary characteristics and parameters include similarities in size, volume, surface characteristics, granularity properties, light scattering properties and fluorescence properties.
Various commercial reference control products are now available, which use various processed or fixed human or animal blood cells as analogs of human blood cells. U.S. Pat. No. 5,512,485 (to Young, et al) teaches a hematology control comprising several white blood cell analogs made of processed and fixed animal red blood cells. U.S. Pat. Nos. 6,187,590 and 5,858,790 (to Kim, et al) teach a hematology control comprising a nucleated red blood cell (NRBC) analog made of lysed and fixed avian or fish red blood cells. U.S. Pat. Nos. 6,406,915, 6,403,377, 6,399,388, 6,221,668, and 6,200,500 (to Ryan, et al) teach a hematology control comprising a NRBC analog derived from avian blood cells. U.S. Pat. No. 6,448,085 (to Wang, et al) teaches a hematology control comprising a nucleated red blood cell (NRBC) analog derived from chicken blood and fixed human blood with nucleated red blood cells. This prior art teaches measurement of cell size and fluorescent intensity of the control material to facilitate a determination of nucleated red blood cell staging. U.S. Pat. Nos. 6,653,137 and 6,723,563 (to Ryan) teach methods of making and using a hematology reference control which contains a nucleated red blood cell component made by lysing and removing cytoplasm from reptile or fish blood cells.
In addition to the use of blood cell analogs for control purposes, synthetic microspheres have also been used in flow cytometry control products. More specifically, U.S. Pat. No. 6,074,879 (to Zelmanovic, et al) teaches a method using synthetic spherical particles for calibrating and standardizing a flow cytometric instrument for particle analysis. The prior art specifically teaches using particles having average mean particle diameters and refractive index essentially reproducible of the particles or cells to be assayed. U.S. Pat. No. 6,521,729 (to Zelmanovic, et al) further teaches a hematology calibration and reference control which comprises fluorine-containing spherical particles having an average mean particle diameter of about 5 to 5.5 μm and a refractive index of about 1.4.
On the other hand, several detection methods for measuring nucleated red blood cells in a blood sample on a flow cytometric instruments have been reported. U.S. Pat. No. 5,879,900 (to Kim, et al) teaches a method of differentiating nucleated red blood cells by measuring two low angles of light scatter and a fluorescence signal of a blood sample under lysing condition. U.S. Pat. Nos. 5,874,310 and 5,917,584 (to Li, et al) teach a method of differentiating nucleated red blood cells by measuring two angles of light scatter signals of a blood sample under lysing condition.
Currently, reference controls for nucleated red blood cells taught by prior art are limited to the use of stabilized, processed or fixed blood cells as the nucleated red blood cell analog particles. It is known that processes for manufacturing these blood cells for a control product are complex and costly, and the analog particles have limited stability and are prone to lot-to-lot variation.
Therefore, it is desirable to use synthetic particles as nucleated red blood cell analog in a reference control composition, wherein the synthetic particles simulate the properties of nucleated red blood cell population under specific reaction and detection conditions.