Hematopoietic progenitor cells may serve as surrogates for CD34(+) cells in PBSCT. However, further investigations are required to verify this.

Background A rapid enumeration of CD34+ cells is expected to facilitate a safe and efficient PBSC collection for a successful transplantation. We previously reported a novel enumeration method of hematopoietic progenitor cells (designated as HPC) using an automated hematology analyzer, XN-series model (Sysmex Corporation, Japan), which does not require monoclonal antibody. This method seemed very promising in a pilot study in a single institute (ASH meeting #1922, 2011). Hence, we conducted a multicenter study to confirm the results in a larger number of subjects in various situations. In this study, we used an up-graded analyzer which had been installed with a revised analytical program. This new program employs an automatically-adjusted gating to fit the individual variety, whereas the former used a fixed gating for detecting HPC. We planned to divide the study into 2 parts; the first is to confirm the outcome of the previous single-institute study and to develop a model to predict CD34+ cell counts from HPC, and the second is to validate the model. Here we show the results of the first part of the study. Patients and Method PB or PBSC samples were taken from G-CSF- and/or chemo-mobilized subjects (25 patients undergoing PBSCT and 25 healthy donors) in 5 institutes. Samples were divided into 2 tubes; one is for in-house assay in which HPC and CD34+ cells were counted in each institute within a few hours without any manipulation, and another was shipped to the central laboratory (SRL, Inc., Japan) where CD34+ cell count was examined. HPC and central labo-assayed CD34+ cell counts were compared with each other and used in a correlation analysis. HPC and in-house CD34+ cells were used in the analysis of kinetics. This study was approved by IRB, and an informed consent was obtained from each participant before apheresis. Results Five PBSC samples (2.3%) were estimated to be invalid for HPC assay because of difficulty in identifying HPC, and were excluded from the analysis. There were good correlations between HPC and CD34+ cell counts in all samples (n=211; R2 0.9576, slope 1.0678, Fig.1), in PB (n=87; R2 0.7631, slope 0.8878) and PBSC (n=124; R2 0.9501, slope 1.0689), in autologous PBSCT patients (n=116; R2 0.9759, slope 1.0596) and donors (n=95; R2 0.7914, slope 1.1381). However, HPC and CD34+ cell counts differ more than 3 times around the critical concentrations for making decision in some samples. Concerning their kinetics in PB during mobilization, which was observed in 2 donors and 9 patients, HPC showed a very similar trend as CD34+ cells, and the timing of appearance and increase in PB was almost concordant in most cases (Fig. 2). Conclusions In this multicenter study, we confirmed not only there was a good correlation between HPC and CD34+ cell counts, but also both HPC and CD34+ cell counts in PB showed a very similar trend during mobilization, as was observed in our previous single-institute study. However, a few samples were estimated as invalid for HPC assay, and there were a few more samples in which HPC and CD34+ cell counts differ significantly. Therefore, it remains to be elucidated how much HPC could be helpful in the clinical setting for determining the optimal timing of collection, for predicting the number of CD34+ cells in the apheresis product, and for determining the optimal blood volume to be apheresed. We are currently underway of the later validation part of this study to evaluate these issues. Disclosures: Tanosaki: Sysmex Corporation: Automated hematology instruments were provided on loan and the cost for CD34+ cell counts were owed to Sysmex Corporation, Kobe, Japan in this study. Other.

Paralysis of the mental nerve is one of the principal complications of surgery of the mandibular canal and mental foramen region. The position of mental foramen can be clearly depicted on CT scans. The mental foramen is bilaterally located at the mandibular premolar region and appears as a dimple on the bone surface. However, several reports have described an accessory mental foramen (AMF). We examined CT pictures taken from patients with implants for missing mandibular teeth to detect variations of the AMF. The results were follows: 1) AMFs were present in 28 patients (24.6 %). 2) Unilateral AMFs were found in 24 patients, and bilateral AMFs in 4 patients. 3) Among patients with unilateral AMFs, 21 had AMFs with one foramen, and 3 had AMFs with two foramens. Among patients with bilateral AMFs, 2 patients had one foramen on each side, and 2 had two foramens on one side. 4) The position of AMF relative to that of the mental foramen was as follows: 18 foramens were superior mesial, 8 were superior distal, 6 were inferior mesial, and 5 were inferior distal. These results suggest that one quarter of patients with missing mandibular teeth may have AMFs around the mental foramen.

1922 The number of infused CD34+ cells is crucial to the success of peripheral blood stem cell transplantation (PBSCT). Although counting CD34+ cells currently depends solely on flow cytometry technology, the complexity of the procedure and the high cost of reagents (including monoclonal antibodies) are the main disadvantages. The SYSMEX SE-9000 (SE) and XE-2100 (XE) automated hematology analyzers quickly estimate the number of immature cells, referred to as hematopoietic progenitor cells (HPCs), at very low cost. The number of peripheral blood SE/XE-determined HPC (SE/XE-HPC) is used to determine the optimal timing of peripheral blood stem cell (PBSC) collection. However, SE/XE-HPCs are limited as a substitute for CD34+ cells because they are likely to be affected by co-existing immature cells (e.g. immature granulocytes), resulting in overestimation of the HPC count. Therefore, we developed a new technology for counting HPCs. The assay's mechanism is based on finely-tuned hemolysis reactions and chemical staining with a specific dye, and does not require monoclonal antibodies. The assay is followed by a flow cytometry-based optical detection technique that differs from the SE or XE former types, which use the electrical radiofrequency/direct currency impedance detection method. This modified program has been installed into an revised model of an automated hematology analyzer, the XN Prototype (SYSMEX corporation, Kobe, Japan), which enables us to cost-effectively obtain the number of new, marked HPCs, designated as 'XN-determined HPC (XN-HPC)', within 4 minutes using small (200 μL) samples. The purpose of this study is to evaluate the XN-HPC in comparison with CD34+ cells, and this is the first report of the results. Between 2008 and 2011, a total of 87 blood or G-CSF-mobilized apheresis samples were taken from healthy donors (n=20) or patients undergoing autologous PBSCT (n=5) at the National Cancer Center Hospital, Japan. Next, CD34+ cells and XN-HPCs were analyzed in the same samples. XN-HPCs were counted using the XN Prototype, and CD34+ cells were quantified using a flow cytometer (FACSCalibur, BD, New Jersey, USA) using the dual platform method according to the International Society of Hematology and Graft Engineering protocol. This study was approved by Institutional Review Board, and informed consent was obtained from all patients. There was a very good correlation between the numbers of XN-HPCs and CD34+ cells (R2=0.952) in all samples, at a wide range of CD34+ cell concentrations (range; 0.3–12830.5 cells /μL) (Fig. 1). The correlation was unaffected by WBC counts, use of EDTA as an anticoagulant, sample type, or timing of collections. The XN-HPC concentration in the 3L-apheresis products (3L-HPCs) correlated well with CD34+ cell concentration in the final products (R2=0.948). The estimated total number of XN-HPCs in the final products, which was calculated from the 3L-HPC concentration or pre-apheresis HPC concentration in the peripheral blood (PB-HPCs), also correlated well with the total number of CD34+ cells in the final products (R2=0.918 or 0.950, respectively), suggesting that the final amount of collected CD34+ cells could be predicted from the total number of HPCs in the final products, as well as from pre-apheresis PB-HPCs and from the intermediate products during apheresis (3L-HPCs). The change in PB-HPCs closely resembled that of CD34+ cells during the bone marrow recovery phase after chemotherapy (Fig. 2), also suggesting that XN-HPC might be a good indicator for the optimal timing of PBSC collection. In conclusion, XN-HPC could be a surrogate for CD34+ cells in PBSCT, and further investigation of their usefulness and clinical applications are warranted. Disclosures: Tanosaki: Sysmex Corporation: They provided hematology analyzers, a flow cytometer and reagents.