Introduction

Assays for cell counting using flow cytometry and calibrated fluorescent particles are fast and accurate. The single platform method that enumerates T cells by counting the identifier cells in a precise known cell volume or an internal “peak” of a known number of fluorescent particles calibrated by flow cytometry is simple and efficient. These assays allow T-cell counting during anti-T-cell globulin treatment of heart, lung, and kidney transplant patients.

Additionally, labs can determine absolute CD4 and CD8 T cell counts to estimate HIV disease progression with the single platform method. Calibrated fluorescent particles and flow cytometry are also used to count platelets in a wide range of murine models of platelet disorders. It is also various other cell types can be counted with flow cytometry and calibrated fluorescent particles. Accurate quantification of blood cells is crucial for the accurate determination of treatment procedures during the clinic watch out. Flow cytometry and calibrated fluorescent particles have improved cell counting methods on predicates methods. In the past, multiplatform methods have been used to quantify T cells.

The method uses percentages of cell markers from the flow cytometer and white blood cell count, percentage of lymphocytes, and absolute lymphocyte count from a haematology analyzer to enumerate T cells in the blood. Substantial variations of the data have been obtained between different laboratories using multiplatform methods. These variations are due to an error created in each independent measurement that is multiplied in consecutive steps during calculations.

The single platform CD4 and CD8 T-cell determination techniques are claimed to be an acceptable alternative to the multiplatform method. Single platform tests using a known number of reference particles resulted in significant and substantial improvements in precision from laboratory to laboratory and within a laboratory over cross-platform methods for T-cell counting.

The method for absolute cell counting employing flow cytometry and calibrated fluorescent particles is known as the metric method of absolute count ratio. This method uses only light scattering parameters for the cell gate on the flow cytometer. The forward angle light scattering measures the size of the cells. Side scatter, right-angle light dispersion, measures the granularity of the cells. The cells viewed under these parameters simultaneously produce a distinct group of cells in the dot plot. During T-cell counting, a gate is placed around the lymphocytes as they are sampled is displayed with the forward angle light scattering parameters versus the right angle light scattering parameters with the flux cytometer.

The number of lymphocytes is then measured as events are monitored on a dot plot of CD3-FITC versus CD4-RPE. The total number of calibration fluorescence particles is then counted when the RPE-Cy5 vs angle light scattering parameters are closed. The absolute CD4 + T cell count equals the ratio of CD4 + cells counted with the number of counted calibrated particles, multiplied by the concentration of the calibration particles considering that the concentration of the calibration particles is known. Other types of cells can be counted using similar parameters of the flow cytometer.

SPHEROTM AccuCount particles are designed to be used as reference particles during cell enumeration. The absolute number of cells can be determined using the SPHEROTM AccuCount particles since the number of particles per mL is known. SPHEROTM AccuCount particles are very easy to use and cost-effective.

Process

To obtain accurate absolute cell counts, SPHEROTM AccuCount particles are used in conjunction with cytometry. SPHEROTM AccuCount particles have a concentration of approximately 1×106 particles / mL. The actual concentration is indicated in the technical data sheet of the product. The first step during sample preparation consists of adding the monoclonal antibody to 100 µL of the test sample. The sample is then incubated, lysed, washed, and resuspended in 1 to 2 ml phosphate-buffered saline, 0.1 M, pH 7.4. If staining and lysis are not necessary, add a known volume of the test sample in 1 to 2 ml of phosphate-buffered saline.

AccuCount Particle Wash with the sample before analysis is discouraged because a reduction in the number of reference particles will happen. Then add exactly 50 µL of the AccuCount particle to the suspension. Accuracy during pipetting of AccuCount particles is absolutely critical. The sample is then analyzed by flow cytometry. Bill and the cell population is selected on the fluorescence and/or side scatter channel. Record the number of events for AccuCount particles and test samples. The absolute cell count is determined by the following equation.

CALCULATION:

(A / B) x (C / D) = Number of cells per µL

Where:

A = number of events for the test sample
B = number of events for AccuCount particles
C = number of AccuCount particles per 50 µL
D = volume of the test sample used initially in µL

Conclusion

SPHEROTM AccuCount Particles are used to obtain absolute cell counts accurately and quickly. SPHEROTM AccuCount particles are used as reference particles during cell enumeration. T cell counts using flow cytometry and calibration fluorescence particles provide better reproducibility and agreement for replicate samples compared to cross-platform methods. The precision of absolute T-cell counts from a single platform over cross-platform methods increases with careful and accurate pipette measurements. The use of fluorospheres for routine clinical treatments. Evaluation of absolute CD4 and CD8 cell counts is recommended.