Cell Population Data As a Predictor of Stem Cell Aphaeresis Efficiency

Autologous Hematopoietic Stem Cell Transplantation (AHSCT) is a necessary component of treatment for many oncohaemathological diseases. For success of AHSCT a sufficient quantity of Hematopoietic Stem Cells (HSC) are needed. The estimation of the quantity of HSC post aphaeresis is vital. The procedure of aphaeresis is time consuming and expensive but the CD34+ count (equivalent to HSC) measured by traditional flow cytometry, in peripheral blood can predict the quality of the aphaeresis product but is much more expensive than a complete blood count. It has been proposed that morphological changes of myeloid cells detected by some hematological analyzers could reflect the processes of bone marrow stimulation and may provide useful information for the prediction of the efficiency of stimulation and expected outcome of CD34+ stem cells.

Materials and methods: Nine patients, 7 with multiple myeloma and 2 with Hodgkins disease were studied after informed consent: 8 female, 1 male, age range 21 to 59. Patients received the standard dose G-CSF, protocol-driven chemotherapy +G-CSF (dose=5 mcg/kg/day). Initial mobilization typically processed 10–12 liters of blood. The Beckman Coulter Cellular Analysis System DxH800 performs Flow Cytometric Digital Morphology analysis of leukocytes with measurement of cell volume (impedance), internal complexity and nucleo/cytoplasm ratio (cell conductivity in the radio-frequency current) and granularity (measurement of 5 angles of light scatter). All these measurements (Mean and Standard Deviations (SD)) are reported as numerical values, called Cell Population Data (CPD). Analysis of CD34+ stem cells was performed on FC500 Flow Cytometer with Stem Kit (Beckman Coulter) using the single-platform ISHAGE protocol. All patients were followed-up daily, starting from the day before G-CSF administration.

For patients, responding to therapy (8 from 9 included in the study) an increase in Ne CPD - Neutrophil Mean Volume (NeMV), and Neutrophil SD Volume (NeSDV) was seen from 2 to 4 days before the increase in CD34+ count in peripheral blood. The calculated parameter, Ne Immaturity Index (ImmNeIndex), using the formula (NeMV × NeSDV)/100 was introduced for the analysis. Patients that responded to the stimulation had an increase in NeMV greater than 15%, increase in NeSDV more than 60% and increase in ImmNeIndex more that 85% compared to the values pre- treatment. There was significant correlation between CD34+ and NeMV (correlation coefficient = 0.491, significance level P=0.0011, n=41), CD34+ and NeSDV (correlation coefficient = 0.418, significance level P=0.0065, n=41) and CD34+ and Imm Ne Index (correlation coefficient = 0.489, significance level P=0.0012, n=41). Patients were classified into 3 groups according to %CD34+ cells in peripheral blood at the end of one cycle of stimulation with NeMV, NeSDV and ImmNeIndex (Table 1), and this estimation can give information for the best time for harvest of CD34+ cells after aphaeresis.

ROC curve analysis with MedCalc Software (Mariakerke, Belgium) showed that patients with % CD34+ cells higher than 0.2% in peripheral blood can be detected with NeMV (AUC 0. 903, cut-off 162, sensitivity 93%, specificity 89%) and Imm Ne Index (AUC 0.847 cut-off >41.43, sensitivity 93%, specificity 70%).

Cell Population Data available from Beckman Coulter Cellular Analysis System DxH800 are able to detect morphological changes in the cell size of neutrophils and anisocytosis of neutrophil population. These morphological features follow the stimulation of the bone marrow activity and the production of immature cells, including CD34+ cells. Changes in CPD were detectable 2–4 days before the observed increase in CD34+ count in the peripheral blood and can improve the management of patients. These parameters available on the routine haematological analyser without any additional cost or time can be used as additional criteria in the laboratory to make the decision about timing of aphaeresis for patients after the G-CSF stimulation.

No relevant conflicts of interest to declare.