Evaluation of hematopoietic stem cells in peripheral blood and apheresis product on the hematological analyzer Sysmex XN (XN-HPC): a clinical case

Summary

Background

The routine method for evaluation of hematopoietic stem cells (HSC) is flow cytometry using labeled antibodies and vital dye. Serious disadvantages of this method include the difficulty of standardization, the lack of automation, the high labor costs of qualified personnel, and the relative high cost of consumables. Frequent evaluation of HSC in peripheral blood (PB) and apheresis products are a high clinical need test. The development of standardized, automated methods of HSC evaluation is an actual challenge. Sysmex-XN is a clinical hematological analyzer which has already had all the necessary facilities for standardizing the evaluations conducted on it. Currently, the ability to evaluate the number of HSCs on Sysmex-XN in a WPC channel in the XNHPC mode has been developed. The evaluation of HSC by
this method is at research stage, but in the future it can be introduced into clinical practice in addition to the routine method in the case of obtaining comparable data. Aim of this study was to compare the data of the quantitative evaluation of HSC in peripheral blood and apheresis product after mobilization with G-CSF by two methods, as well as labor and financial costs.

Materials and methods

As a standard, the HSC counting method was used: counting of leukocytes in the Goryaev’ chamber and flow cytometry with markers CD45/CD34/7AAD (BD, San Jose, USA) using BD FacsCalibur and the ISHAGE protocol. A new method is an automated method for calculating HSC, available on Sysmex XN analyzers (XN-HPC) in the WPC channel, based on measuring the size, structure, and fluorescence intensity of cells. Samples of PB and apheresis product were taken for research from same specimens. The HSCs were collected on a COBE Spectra separator.

Results

We report a clinical case of the patient G., 2 years 4 months, height 85.5 cm, weight 15 kg, with the diagnosis of medulloblastoma. The treatment of relapse has to include highdose chemotherapy with autologous hematopoietic stem cell transplantation. Harvesting HSCs of PB was performed after stimulation of G-CSF in a dose of 10 μg/kg once for 5 days. Apheresis was performed on the fifth day of mobilization. The volume of perfusion was 2900.0 ml, 230.0 ml of concentrate was obtained. The comparative results of the studies are presented in the tables: (1) the quantitative evaluation of HSC in the PB, (2) the estimation of the amount of HSC in the apheresis concentrate, (3) the financial and labor costs evaluation (without depreciation of equipment and utility costs).

Conclusions

The presented clinical case demonstrates that the results of quantitative evaluation of HSC on the Sysmex-XN analyzer are comparable with the results of the routine method, while labor and financial costs are at least 3 times lower. The speed of obtaining the result and the relatively low cost make it possible to propose the use of quantitative evaluation of HSC on the Sysmex-XN analyzer as an additional screening method, the results of which will subsequently be validated by the standard method of investigation. The introduction of a new screening method for evaluation of HSCs will lead to optimization of the peripheral blood HSC harvesting protocols and, as a consequence, to obtaining HSC transplants of better quality. Nevertheless, to use the new method in clinical practice, it is necessary to conduct comparative studies on the evaluation of HSC in the PB and apheresis products from a broad cohort of donors/patients.

Keywords

Hematopoietic stem cells, peripheral blood, HSC harvesting, apheresis concentrate, flow cytometry, Sysmex-XN, quantitative HSC screening.

Table 1. Evaluation of HSCs in peripheral blood

Table 2. The evaluation of HSCs in apheresis concentrate

Table 3. Evaluation of time expenses and financial costs