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For successful chimeric antigen receptor T (CAR-T) cell therapy, CAR-T cells must be manufactured without failure caused by suboptimal expansion. In order to determine risk factors for CAR-T cell manufacturing failure, we performed a nationwide cohort study in Japan and analysed patients with diffuse large B-cell lymphoma (DLBCL) who underwent tisagenlecleucel production. We compared clinical factors between 30 cases that failed (7.4%) with those that succeeded (n = 378). Among the failures, the proportion of patients previously treated with bendamustine (43.3% vs. 14.8%; p, CAR-T細胞製造を成功させるためのレシピ --アフェレーシス前の下ごしらえでの工夫--. 京都大学プレスリリース. 2023-04-27.

The standard cryoprotectant for human cellular products is dimethyl sulfoxide (DMSO), which is associated with hematopoietic cell infusion-related adverse events (HCI-AEs) in hematopoietic stem cell transplantation including peripheral blood stem cell (PBSC) transplantation (PBSCT). DMSO is often used with hydroxyethyl starch (HES), which reduces DMSO concentration while maintaining the postthaw cell recovery. The cryoprotectant medium CP-1 (Kyokuto Pharmaceutical Industrial) is widely used in Japan. After mixture of a product with CP-1, DMSO and HES concentrations are 5% and 6%, respectively. However, the safety profile of CP-1 in association with HCI-AEs has not been investigated.To compare CP-1 with other cryoprotectants, we conducted a subgroup analysis of PBSCT recipients in a prospective surveillance study for HCI-AEs. Moreover, we validated the toxicity of CP-1 in 90 rats following various dose administration.The PBSC products cryopreserved with CP-1 (CP-1 group) and those with other cryoprotectants, mainly 10% DMSO (non-CP-1 group), were infused into 418 and 58 recipients, respectively. The rate of ≥grade 2 HCI-AEs was higher in the CP-1 group, but that of overall or ≥grade 3 HCI-AEs was not significantly different, compared to the non-CP-1 group. Similarly, after propensity score matching, ≥grade 2 HCI-AEs were more frequent in the CP-1 group, but the ≥grade 3 HCI-AE rate did not differ significantly between the groups. No significant toxicity was detected regardless of the CP-1 dose in the 90 rats.Infusion of a CP-1-containing PBSC product is feasible with the respect of HCI-AEs.

Chimeric antigen receptor (CAR) T-cell therapy has revolutionized the approach to patients with relapsed or refractory diffuse large B-cell lymphoma (r/r DLBCL). This study retrospectively analyzed patients treated with commercially available tisagenlecleucel at our hospital and evaluated its safety and effectiveness. Of the 21 patients evaluated, any grade and grade ≥3 cytokine release syndrome (CRS) occurred in 85.7% and 9.5% of the patients, respectively. A total of 66.7% received tocilizumab and 28.6% received glucocorticoids for the treatment of CRS. The complete response (CR) rate at 3 months was 61.9% (95% confidence interval [CI] 38.4-81.9). After a median follow-up of 6.3 months following CAR-T infusion, the progression-free survival (PFS) and overall survival rates at 6 months were 53.1% (95%CI 28.3-72.7) and 69.2% (95%CI 43.7-84.9), respectively. Severe cytopenia and hypogammaglobulinemia occurred frequently following CAR-T infusion. Eight patients (38.1%) had comorbidities that would have made them ineligible for leukapheresis in the JULIET trial. However, the presence of comorbidities at the time of leukapheresis had no significant effect on the rates of CR, PFS, and adverse events. Tisagenlecleucel for r/r DLBCL in the real-world setting showed high efficacy and manageable safety profile comparable with the pivotal trial.

Background Hematopoietic cell infusion-related adverse events (HCI-AEs) in hematopoietic stem cell transplantations (HSCTs) have been largely attributed to toxicity of dimethyl sulfoxide (DMSO) for cryopreservation, but HSC products also contain various cells and plasma components. Our recent prospective study of 1125 HSCT recipients revealed the highest overall HCI-AE rate in bone marrow transplantation (BMT) using fresh/noncryopreserved products, although products of peripheral blood stem cell transplantation and cord blood transplantation (CBT) are generally cryopreserved with DMSO containing smaller plasma volumes. We aimed to clarify if product volume and component effects are more substantial in small recipients including children. Study design and methods We performed subgroup analysis on 219 recipients of 45 kg or less body weight (whole small recipients), including 90 children (pediatric recipients), from the original cohort (general recipients). Results Whereas overall HCI-AE rates did not differ among hematopoietic stem cell sources in the general recipients, bradycardia most often occurred after CBT in whole small recipients. Conversely, whole small and general recipients shared the same trend of having the highest rate of hypertension in BMT. The overall HCI-AE rate was higher in allogeneic HSCT compared with autologous HSCT. Notably, pediatric recipients showed a 10-fold higher incidence of nausea and vomiting in allogeneic HSCT compared with autologous HSCT, suggesting a possible role of allogeneic antigens. Multivariate analysis identified a relatively large infusion volume per body weight as a significant factor correlating with HCI-AE in whole small recipients. Conclusions We should be aware of product volume and specific HCI-AEs such as nausea and vomiting in small patients including children.

Gene rearrangements of MLL/KMT2A or RUNX1 are the major cause of therapy-related leukemia. Moreover, MLL rearrangements are the major cause of infant leukemia, and RUNX1 rearrangements are frequently detected in cord blood. These genes are sensitive to topoisomerase II inhibitors, and various genes have been identified as potential fusion partners. However, fetal exposure to these inhibitors is rare. Therefore, we postulated that even a proliferation signal itself might induce gene rearrangements in hematopoietic stem cells. To test this hypothesis, we detected gene rearrangements in etoposide-treated or non-treated CD34+ cells cultured with cytokines using inverse PCR. In the etoposide-treated cells, variable-sized rearrangement bands were detected in the RUNX1 and MLL genes at 3 hours of culture, which decreased after 7 days. However, more rearrangement bands were detected in the non-treated cells at 7 days of culture. Such gene rearrangements were also detected in peripheral blood stem cells mobilized by cytokines for transplantation. However, none of these rearranged genes encoded the leukemogenic oncogene, and the cells with rearrangements did not expand. These findings suggest that MLL and RUNX1 rearrangements, which occur with very low frequency in normal hematopoietic progenitor cells, may be induced under cytokine stimulation. Most of the cells with gene rearrangements are likely eliminated, except for leukemia-associated gene rearrangements, resulting in the low prevalence of leukemia development.

Most of the diseases for which apheresis therapy is indicated are intractable and rare, and each patient has a different background and treatment course prior to apheresis therapy initiation. Therefore, it is difficult to conduct large-scale randomized controlled trials to secure high-quality evidence. Under such circumstances, the American Society for Apheresis (ASFA) issued its guidelines in 2007, which were repeatedly revised until the latest edition in 2019. The ASFA guidelines are comprehensive. However, in the United States, a centrifugal separation method is mainly used for apheresis, whereas the mainstream procedure in Japan is the membrane separation method. The target diseases and their backgrounds are different from those in Japan. Due to these differences, the direct adoption of the ASFA guidelines in Japanese practice creates various problems. One of the features of apheresis in Japan is the development of treatment methods using hollow-fiber devices such as double filtration plasmapheresis (DFPP) and selective plasma exchange and adsorption-type devices such as polymyxin B-immobilized endotoxin adsorption columns. Specialists in emergency medicine, hematology, collagen diseases/rheumatology, respiratory medicine, cardiovascular medicine, gastroenterology, neurology, nephrology, and dermatology who are familiar with apheresis therapy gathered for this guideline, which covers 86 diseases. In addition, since apheresis therapy involves not only physicians but also clinical engineers, nurses, dieticians, and many other medical professionals, this guideline was prepared in the form of a worksheet so that it can be easily understood at the bedside. Moreover, to the clinical purposes, this guideline is designed to summarize apheresis therapy in Japan and to disseminate and further develop Japanese apheresis technology to the world. As diagnostic and therapeutic techniques are constantly advancing, the guidelines need to be revised every few years. In order to ensure the high quality of apheresis therapy in Japan, both the Japanese Society for Apheresis Registry and the guidelines will be inseparable.

Outcomes after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in nonremission acute myeloid leukemia (AML) are dismal [2-year overall survival (OS): 20–30%]. Though several risk classifications have been used, some factors are unavailable until the start of conditioning or transplantation. We analyzed prognostic gene mutations by targeted next-generation sequencing to identify predisposing factors for predicting OS at 1 month before transplantation. We enrolled 120 patients with nonremission AML who underwent first allo-HSCT between 2005 and 2018. Mutations were found in 98 patients; frequently mutated genes were FLT3-ITD, TP53, RUNX1, and WT1. TP53 mutation was detected in 21 patients and was the only predictor of poor OS. Multivariate analysis using Cox regression hazard model revealed primary AML, monosomal karyotype (MK), and TP53 mutation as independent factors for predicting poor OS. Based on these, patients were stratified into three groups. The low-risk group included patients with prior myeloid disorder without MK (n = 26). Among the rest, patients with TP53 mutation were assigned to the high-risk group (n = 19) and the rest into the intermediate-risk group (n = 75). Two-year OS in low-, intermediate-, and high-risk groups differed significantly (50.0%, 24.9%, and 0%, respectively). This suggests that the indication of allo-HSCT should be carefully judged for high-risk patients.

Adverse events (AEs) associated with blood transfusions, including component-specific red cell, platelet, and plasma products, have been extensively surveyed. In contrast, surveillance of AEs associated with hematopoietic stem cell (HSC) products in HSC transplantation (HSCT) has been less rigorous, even though HSC products include a diversity of immature and mature hematopoietic cells, substantial plasma, and dimethyl sulfoxide (DMSO) in the case of cryopreserved HSC products. HSC infusion-related AEs have been attributed to DMSO toxicity, but AEs associated with the infusion of noncryopreserved HSC products are not uncommon. To quantify the frequencies, types, and risk factors of HSC infusion-related AEs, we implemented national surveillance for AEs observed within 24 hours after infusion. Herein we report on 1125 HSCTs, including 570 peripheral blood stem cell transplantations (PBSCTs) (290 autologous [auto-] and 280 allogeneic [allo-]), 332 allo-bone marrow transplantations (allo-BMTs) and 223 allo-cord blood transplantations (allo-CBTs). Unexpectedly, incidences of grade ≥ 2 AEs were most frequent in allo-BMTs (37.7%) with no DMSO in any product compared with auto-/allo-PBSCTs (20.9%, P .001) and allo-CBTs (19.3%, P .001) typically cryopreserved with DMSO. Hypertension was most often noted in BMTs, whereas nausea/vomiting, fever, and allergic reactions were most frequent in allo-PBSCTs. In a multivariate analysis, a history of transfusion reactions was a risk factor for overall AEs in all HSCTs (odds ratio [OR] = 1.459, P = .045). For grade ≥ 2 AEs in allo-HSCTs, a history of transfusion reactions (OR = 1.551, P = .044) for overall AEs, and high infusion volume (OR = 7.544, P = .005) and allo-PBSCTs (versus BMTs, OR = 9.948, P = .002) for allergic reactions were identified as risk factors. These findings suggest that some factors unrelated to DMSO, such as allo-antigens, contribute to HSC infusion-related AEs. As severe AEs, a total of 117 grade ≥ 3 AEs were reported in 1125 HSCTs, including life-threatening complications in 3 (0.3%) HSCTs: 1 allo-CBT (anaphylaxis) and 2 allo-PBSCTs (hypoxia, kidney injury) with cryopreserved product. Our data show that HSC infusion risks vary by product, can be severe, and should be monitored with the same rigor as modern transfusion hemovigilance programs.

Chimeric antigen receptor (CAR) T-cell therapy is effective in treating relapsed and refractory B-cell non-Hodgkin lymphoma. However, because of the mortality risk associated with immune effector cell-associated neurotoxicity syndrome and pseudoprogression, patients with central nervous system (CNS) involvement are less likely to receive CAR T-cell therapy.We report a case of a 61-year-old, male patient with intravascular large B-cell lymphoma who suffered a CNS relapse after standard chemotherapy.A diagnosis of intravascular large B-cell lymphoma with CNS involvement was made.We treated the patient using CAR T-cell therapy following a conditioning regimen consisting of thiotepa and busulfan and autologous stem cell transplantation. Although he experienced grade 1 cytokine release syndrome, no other serious adverse events, such as immune effector cell-associated neurotoxicity syndrome or pseudoprogression, were observed.The patient achieved complete remission after the CAR T-cell infusion.CAR T-cell therapy following autologous stem cell transplantation is a viable option for relapsed/refractory lymphoma with CNS infiltration. Further clinical studies are warranted to verify its safety and efficacy.

Background: Additional cytogenetic abnormalities (ACA), the most frequent form of cytogenetic changes, are considered as a result of genetic instability and clonal evolution of leukemia cells. Recently, we described that ACA at the first relapse was associated with the significantly lower second complete remission (CR2) rate and poor survival in adult acute myeloid leukemia (AML) patients (Hematol Oncol. 2018;36:252-257). However, the prognostic impact of ACA after allogeneic stem cell transplant (allo-SCT) has not been elucidated in adult AML patients. Patients and methods: Of the 145 adult AML patients who underwent the first allo-SCT in CR2 between 1997 and 2019, 98 patients whose cytogenetic abnormality data both at diagnosis and at the first relapse were available were included in this study. Cytogenetic changes between at diagnosis and the first relapse were classified into four groups: (1) no change, (2) ACA was acquired at the first relapse, (3) cytogenetic abnormalities observed at diagnosis were reduced or had disappeared at the first relapse, and (4) cytogenetic abnormalities observed at diagnosis were reduced or had disappeared, and completely different ACA was acquired at the first relapse. In this study, groups 2 and 4 were defined as ACA acquisition. Overall survival (OS) was defined as the interval from the date of transplant to the date of death. Fisher's exact test was used to compare binary variables. The cumulative incidence (CI) of non-relapse mortality (NRM) and relapse were evaluated with Gray's test, considering relapse and NRM as a competing risk, respectively. OS was estimated with the Kaplan-Meier method and compared using the log-rank test. Factors associated with at least borderline significance (p < 0.20) in univariate analyses were subjected to multivariate analysis. The Cox and Fine-Gray proportional hazard model were used for multivariate analysis of prognostic and risk factors, respectively. Values of p < 0.05 were considered to indicate statistical significance. Results: Of the 98 patients included in this study, 57 were male, and 41 were female. The median age at transplant was 45 years (range, 17-71 years). The median duration of CR1 was 12.4 months (range, 1.3-70.3 months) and cytogenetic risk groups were good, intermediate, and poor in 26 (27%), 70 (71%), and two patients (2%), respectively. Donor types were related, unrelated, and cord blood in 23 (24%), 59 (60%), and 16 patients (16%), respectively, and 86 (87%) and 61 patients (62%) were conditioned with myeloablative and total body irradiation-containing regimens, respectively. According to the definition described above, 20 patients (20%) acquired ACA at the first relapse. There was no significant difference in baseline characteristics and transplant procedures between patients with and without ACA acquisition. The OS rates were not significantly different between two groups (55% vs. 72% at three years after transplant; p = 0.28). The CI of relapse was significantly higher in patients with ACA acquisition than those without ACA acquisition (59% vs. 15%; p < 0.01), while the CI of NRM were not significantly different between two groups (5% vs. 19%; p = 0.17). Multivariate analysis for OS revealed that age over 50 years (hazard ratio [HR] = 2.4; p < 0.01), but not ACA acquisition, was identified as an independent prognostic factor. ACA acquisition (HR = 4.7; p < 0.01) was extracted as an independent risk factor of relapse, while use of reduced intensity conditioning regimens (HR = 3.1; p = 0.03) and more than or equal to 1 of performance status at transplant (HR = 2.7; p = 0.04) showed independent risks of NRM. The similar OS rates between two groups might resulted from an offset of the lower relapse rate with the higher NRM rate in patients without ACA acquisition despite not reaching statistical significance. This increasing NRM rates in those without ACA acquisition was potentially associated with use of reduced intensity conditioning regimens in larger proportion (0% vs. 15%; p = 0.12). Conclusion: These findings suggested that ACA acquisition at the first relapse was associated with a higher risk of relapse even after allo-SCT in CR2 in adult AML patients. As AML cells with ACA acquisition was resistant to not only chemotherapy but also graft-versus-leukemia effect, innovative therapeutic strategy is warranted. Disclosures Handa: Janssen: Honoraria; Kyowa Kirin: Research Funding; Takeda: Honoraria, Research Funding; BMS: Honoraria; Chugai: Research Funding; Ono: Honoraria; Sanofi: Honoraria, Research Funding; Abbvie: Honoraria; MSD: Research Funding; Shionogi: Research Funding; Celgene: Honoraria, Research Funding; Daiichi Sankyo: Research Funding.

Gene rearrangements of MLL/KMT2A or RUNX1 are the major cause of therapy‐related leukemia. Moreover, MLL rearrangements are the major cause of infant leukemia, and RUNX1 rearrangements are frequently detected in cord blood. These genes are sensitive to topoisomerase II inhibitors, and various genes have been identified as potential fusion partners. However, fetal exposure to these inhibitors is rare. Therefore, we postulated that even a proliferation signal itself might induce gene rearrangements in hematopoietic stem cells. To test this hypothesis, we detected gene rearrangements in etoposide‐treated or non–treated CD34+ cells cultured with cytokines using inverse PCR. In the etoposide‐treated cells, variable‐sized rearrangement bands were detected in the RUNX1 and MLL genes at 3 hours of culture, which decreased after 7 days. However, more rearrangement bands were detected in the non–treated cells at 7 days of culture. Such gene rearrangements were also detected in peripheral blood stem cells mobilized by cytokines for transplantation. However, none of these rearranged genes encoded the leukemogenic oncogene, and the cells with rearrangements did not expand. These findings suggest that MLL and RUNX1 rearrangements, which occur with very low frequency in normal hematopoietic progenitor cells, may be induced under cytokine stimulation. Most of the cells with gene rearrangements are likely eliminated, except for leukemia‐associated gene rearrangements, resulting in the low prevalence of leukemia development., MLL and RUNX1 rearrangements, which occur with very low frequency in normal hematopoietic progenitor cells, may be induced under cytokine stimulation. Most of the cells with gene rearrangements are likely eliminated, except for leukemia‐associated gene rearrangements, resulting in the low prevalence of leukemia development.

A 51-year-old man underwent allogeneic bone marrow transplantation (BMT) for recurrent acute myeloid leukemia. Although the patient developed slight edema, pleural effusion, and cardiac effusion 6 months after BMT, his clinical condition improved with furosemide treatment. The patient was transfused with red blood cells for the management of anemia 8 months after BMT. He developed acute respiratory failure with pulmonary alveolar hemorrhage 80 min after the transfusion. He was diagnosed with transfusion-associated circulatory overload (TACO) due to the presence of acute pulmonary congestion and depressed left ventricular systolic function. Reduced circulatory load due to sufficient furosemide led to ventilator weaning 3 days later. Other causes of pulmonary alveolar hemorrhage were excluded, and the patient's condition improved by cardiac failure treatment only. This clinical course indicated that pulmonary alveolar hemorrhage would breakdown the blood vessels due to acute pulmonary congestion. Chemotherapy and prolonged anemia are high risks for cardiac failure in patients with hematological malignancies. Therefore, the possibility of cardiac failure is considered when patients with hematological malignancies have fluid retention, such as cardiac enlargement, edema, and pleural effusion. Moreover, the body fluids should be monitored before and after blood transfusion.

Recent studies have shown that CD25 expression at the time of diagnosis of acute myeloid leukemia (AML) may be associated with an unfavorable outcome. We focus on patients with AML without complete remission (CR) and examine the clinical correlation between surface CD25 expression at the time of transplant and subsequent transplant outcomes. We observed a significant difference in overall survival (OS), disease-free survival (DFS) and cumulative incidence of relapse (CIR) between CD25 positive (+) (n = 22) and negative (-) groups (n = 44) (2-year OS; CD25 (+) group: 5% vs. CD25 (-) group: 40%, p

Increased levels of normal B cell precursors, termed hematogones (HGs), are observed in regenerating bone marrow after chemotherapy or hematopoietic stem cell transplantation (HSCT). Recent reports suggest that emergence of HGs is associated with better outcomes following allogeneic HSCT (allo-HSCT). We reviewed the emergence of HGs and the clinical features of 192 patients after allo-BMT. Patients undergoing allo-BMT from related donors were more likely to develop HGs at day 30 compared to unrelated donors. Furthermore, patients undergoing allo-BMT from HLA-mismatched donors were less likely to develop HGs at day 30. The emergence of HGs at day 30 was an independent prognostic factor among patients who underwent BMT. We found no difference in the relapse rate between HG-positive (+) and HG-negative (-) patients undergoing BMT. HG (-) patients had high non-relapse mortality, grade II to IV acute graft-versus-host-disease (GVHD), fungal infection, and lower IgG levels compared to HG (+) patients. The emergence of HGs at day 30 among patients undergoing BMT may be a very useful indicator of subsequent survival outcomes or acute GVHD in common clinical practice.

BACKGROUND The plasma fraction of blood components has an essential role in the etiology of allergic transfusion reactions (ATRs). The difference of incidences of ATRs between fresh-frozen plasma (FFP) and platelet concentrates (PCs), in which plasma is the main component, is not clearly understood. This study compares the frequency of ATRs to FFP versus PCs on both first and subsequent (nonfirst) transfusions and considers the factors influencing the risk of ATRs. STUDY DESIGN AND METHODS Five hospitals agreed to systematically collect and share 2 years of data (January 2010 through December 2011). This was a retrospective observational analysis of data including the number of transfusion episodes and ATRs for FFP and PCs on first-transfusion patients (without transfusion history) and previously transfused patients. RESULTS The incidence of ATRs to PCs (2.51%) was significantly higher than to FFP (1.68%) on subsequent transfusions (p

Although peripheral blood stem cell (PBSC) transplantations in an unrelated transplant setting have been performed since 2010, prior harvesting and cryopreservation of PBSCs from unrelated donors has not been approved in Japan. There are no restrictions with regard to related donors. Therefore, in April 2015, we conducted a nationwide survey and obtained written answers from 123 transplant hospitals throughout Japan. Our survey revealed that as much as 81.3% of transplant hospitals routinely perform prior harvesting and cryopreservation of PBSCs from related donors and that both cell processing and quality management of cryopreserved products have been appropriately conducted in line with domestic guidelines, although post-thaw quality control and storage period setting require further improvements. Moreover, informed consent obtained from both patients and donors with regard to cryopreservation of PBSCs was not always sufficient in some hospitals. We found that the average number of unused or discarded cryopreserved PBSCs is 1.09 per hospital, and the overall nonuse or discard rates of cryopreserved PBSCs were estimated to be as low as 2.67%.

Background Myelodysplastic syndromes (MDS), commonly seen in elderly patients, represent a heterogeneous group of clonal hematopoietic stem cell disorders caused by the accumulation of gene mutations. By contrast, congenital bone marrow failure syndromes and genetic predispositions associated with MDS are known in pediatric patients. However, little is known about the pathogenesis of MDS in adolescent and young adult (AYA) patients. Previous reports showed the patients with MDS aged under 40 or 41.5 years at allo-HSCT were associated with good survival compared to those among the older population (N Engl J Med. 2017;376:536-547, Blood. 2017;129:2347-2358). However, AYA-MDS is rare, and its clinical features and genetic abnormalities have not been analyzed enough. It is suspected that the clinical and genetic features of AYA-MDS patients might be different from those of elderly patients or pediatric patients. Therefore, we investigated the gene abnormalities of AYA-MDS patients and aimed to elucidate the genetic characteristics associated with the good outcome of allogeneic hematopoietic stem cell transplantation (allo-HSCT). We analyzed the patients younger than 50 years of age in order to reduce the variation of patient-related factors. Methods We analyzed the outcomes of all consecutive patients aged under 50 years who were diagnosed with MDS or acute myeloid leukemia evolving from MDS in our hospital between January 2005 and July 2018. The study was approved by the institutional review board, and patients gave written informed consent for the study, according to the Declaration of Helsinki. Cytogenetic analysis and genomic DNA extraction were carried out using diagnostic bone marrow samples. We performed targeted next-generation sequencing to identify mutations in 68 driver genes using AmpliSeq for Illumina Myeloid Panel and On-Demand Panel on the MiniSeq system (Illumina). Gene variants were detected by in-house analysis pipeline. Overall survival (OS) was analyzed for all patients, and the Kaplan-Meier survival curve was used to assess OS using the log-rank test. Additionally, the cumulative incidence of relapse (CIR) was analyzed for patients who underwent allo-HSCT. Gray's test was used to evaluate the CIR. Results A total of 85 patients with MDS aged under 50 years (U40 between 15 and 39 years old: N=37, 40s between 40 and 49 years old: N=48) were analyzed. The median follow-up time of survivors was 2,041 days (range 176-5,085). There were no significant differences in patient characteristics between U40 and 40s. The 3-year OS of U40 were superior to 40s (79.9% vs. 58.1%, P=0.018), especially lower risk IPSS categories (3-year OS, 95.5% vs. 50.8%, P=0.002). In total, 69 of 85 patients (U40: N=31, 40s: N=38) had undergone allo-HSCT. U40 patients had lower percentage of bone marrow blasts at just before HSCT than 40s patients (over 10%, 12.9% vs. 36.8%, P=0.048), and better 3-year OS from HSCT in lower-IPSS (88.8% vs. 53.8%, P=0.024); but not in higher-IPSS (45.0% vs. 43.2%, P=0.834). In this cohort, at least one driver mutation was detected in 61% of allo-HSCT recipients. Frequently mutated genes (more than 10%) were ASXL1 and RUNX1; however, both of the genes did not have significant impact on the outcomes. While, only one patient in 40s had TP53 mutation. We detected 0.8 (range 0-3) and 1.8 (range 0-6) mutations at average in U40 and 40s, respectively (P=0.06). The proportions of the patients without any gene mutations were 52% in U40 and 30% in 40s. Transplanted patients with 0 or 1 mutation showed lower relapse rate than those with 2 or more mutations (3-year CIR, 23.3% vs. 45.2%, P=0.049). Conclusions The clinical outcomes of U40 patients with MDS were favorable than those in the 40s, especially in lower disease risk. The number of driver mutations in U40 tended to be lower than that in 40s. MDS in adult is regarded as a stem-cell aging disease with gene mutations; however, MDS-associated mutations were not detected in the half of U40. Moreover, TP53 mutation that is associated with extremely poor posttransplant survival was not detected in U40 patients. MDS patients with less than 2 mutations showed lower relapse rate, which maybe indicate genetic mutations have a great impact on transplant outcomes between 15 and 49 years old. Disclosures No relevant conflicts of interest to declare.

Background: Granulocyte-colony stimulating factor (G-CSF) is widely used to mobilize peripheral blood stem cells (PBSCs) in healthy donors. A few reports have shown that some healthy donors developed acute respiratory distress syndrome or capillary leak syndrome after more than several rounds of G-CSF administration or leukapheresis. Case Report: We report the case of a healthy donor for allogeneic stem cell transplantation who developed severe hypoxemia 1 h after only the first administration of G-CSF. The donor was administered 10 μg/kg G-CSF (lenograstim) subcutaneously for PBSC mobilization. 1 h after the first administration of G-CSF, the donor suddenly presented with dry cough and dyspnea. The oxygen saturation by pulse oximetry (SpO2) in the room air was 88%. An electrocardiogram and chest radiography revealed no abnormalities. We excluded other causes of severe hypoxemia and diagnosed the donor with hypoxemia due to G-CSF administration, which was subsequently terminated. The donor was administered 2 l/min oxygen via a nasal cannula and 100 mg hydrocortisone intravenously. He subsequently recovered, and SpO2 in the room air returned to 98% 10 h after hypoxemia. Conclusion: These respiratory symptoms might be related to anaphylactoid or hypersensitivity reaction. The donors should be observed for at least 1 h after the first administration of G-CSF.

Background Although the outcome after allogeneic hematopoietic stem cell transplantation (Allo-HSCT) for the patients with acute myeloid leukemia (AML) in complete remission has been improved, the prognosis of patients with active disease is still dismal, generally with 20 - 30% of overall survival (OS) at 2 years. Prognostic value of gene mutations detected by the next generation sequencing (NGS) for this extremely poor group remains to be evaluated. Methods A total of 120 patients with AML not in hematological remission who received the first allo-HSCT at our institute between April 2005 and December 2017 were enrolled. For each patient, genomic DNA was extracted from the frozen bone marrow sample harboring leukemic blasts which was preserved at the nearest available date before the initiation of conditioning regimen. Sequencing was performed using TruSight Myeloid Sequencing Panel® on the MiniSeq system (Illumina). Gene variants were detected by in-house analysis pipeline. Charts were retrospectively reviewed on survival, relapse, and non-relapse mortality (NRM). The Kaplan-Meier method was used to assess OS using the log-rank test. Univariate and multivariate analysis were performed to identify potential prognostic factors. The Cox proportional hazards method was used for the multivariate analysis to assess OS. Gray's test and the Fine-Gray test were used to assess the cumulative incidence of relapse (CIR). Competing risks were relapse and NRM. Results Median follow-up of survivors was 1345 days (235 - 4888 days). Median age at transplant was 51 (range 21 - 71). Grafts were from bone marrow (n = 67, 55.8%), peripheral blood (n=42, 35.0%) and cord blood (n=11, 9.2%). Refined disease risk index (Blood. 2014;123:3664-71) scored high (n=61, 51.3%) and very high (n=58, 48.7%). OS at 2 years of the whole cohort was 27.3% (95% confidence interval [CI], 19.4% - 35.7%). There was no significant difference in OS between patients in primary induction failure and in relapse (OS at 2 years: 26.5% [n=50] vs 28.7% [n=70], p= 0.293). NGS analysis revealed TP53 loss-of-function mutation in 23 (19.2%) patients. Among all detected gene mutations, TP53 mutation was the most powerful predictor of poor OS after allo-HSCT (OS at 2 years: 13.5% vs 30.5% for TP53+ [n=23] vs TP53- [n=97], p= 0.0184). Consistent with previous reports, monosomal karyotype (MK, J Clin Ocnol 2010:26;4791-7) was significantly associated with positive TP53 mutation (13.3% of non-MK vs 37.9% of MK, p=0.006). Of note, all the patients (n=11) positive for both prognostic factors died within 1 year after allo-HSCT, whereas OS of the patients without either factor (n=78) was 33.6% at 2 years. Multivariate analysis on OS revealed MK, TP53 mutation, de novo AML (no prior history of MDS), ECOG performance status score 2 or more, C reactive protein 1.0 mg/dL or more, peripheral blood blast frequency of 1% or more at the initiation of conditioning regimen were independent prognostic factors for poor OS. Among them, to determine the prognostic factors critical for deciding the indication of allo-HSCT, we chose pre-transplant factors which were available around one months before transplant. From this point of view, multivariate analysis revealed independent prognostic factors for poor OS after allo-HSCT including MK (Hazard ratio [HR] 2.05; 95% confidence interval [CI] 1.30 - 3.25, p=0.00217), TP53 mutation (HR 1.72; 95% CI 1.04 - 2.86, p=0.035), and de novo AML (HR 1.67; 95% CI 1.04 - 2.86, p=0.036). Because the HR of these three factors were comparable, a score of 1 was assigned to each factor. OS at 2 years was 49.7%, 26.5% and 13.1% for patients with low (score 0, n=23), intermediate (score 1, n=63) and high (score 2 or 3, n=34) risk, respectively (p Conclusions NGS-analysis revealed the importance of TP53 mutation on transplant outcome of AML in non-remission. The combined score with the information on MK, de novo AML, and TP53 mutation stratified the patients into three groups, including low risk with 50% survival rate, and high risk with shorter survival after allo-HSCT for whom the other strategy might be optimal. Our results would require further validation in larger cohorts. Disclosures Harada: Celgene: Research Funding.

Adverse events (AEs) associated with blood component transfusion have been widely surveyed. In contrast, surveillance of AEs associated with hematopoietic stem cell (HSC) infusion in HSC transplant (HSCT),including bone marrow transplant (BMT), peripheral blood stem cell transplant (PBSCT), and cord blood transplant (CBT),has been less rigorous, even though HSC products contain cells of diverse maturity and viability,plasma with various antigens, cytokines and antibodies, and dimethyl sulfoxide (DMSO) in the case of cryopreserved products. In fact, HSC infusion is associated with several AEs, e.g., allergic reactions, flushing, hypo- or hypertension, and respiratory distress, which have been attributed to toxicity of dead cells and DMSO (Otrock et al, Transfusion, 2017). However, our recent prospective surveillance revealed that HSC infusion-related AEs often occurred in each HSC type and the overall rates of AEs were greater in allo-BMT with no DMSO, compared with auto-PBSCT, allo-PBSCT, and allo-CBT typically cryopreserved with DMSO. Hypertension was the most common AE in each HSC source, with the highest rate in BMT, while allergic reactions were the most frequent in allo-PBSCT. A multivariate analysis identified a history of transfusion reactions as a risk factor of HSC infusion-related AEs (Ikeda et al, Transfus Med Rev, 2018). These findings suggest that some DMSO-independent factor(s), such as plasma components, may contribute to HSC infusion-related AEs. Thus, we asked if HSC volume and component effects were more substantial in small recipients, and if age-related factors alter susceptibility to HSC infusion-related AEs in pediatric patients. So far, data on HSC infusion-related AEs in pediatric and low body-weight recipients are lacking. Here, to address these issues, we investigated AEs due to HSC infusions in 219 recipients of The rates of overall HSC infusion-related AEs were quite similar among HSC sources among low body-weight/pediatric recipients (Table 1) andexclusivepediatric recipients (P= .158 in auto-/allo-HSCT and .867 in allo-HSCT), in contrast to general recipientswhose rate of AEs was highest in BMT. In addition, bradycardia was more often reported in CBT compared with PBSCT and BMT (Table 1), especially in pediatric recipients (30.8% in CBT and 0% in others, P< .001). On the other hand, there were some similar trends between low body-weight/pediatric recipients and general recipients: PBSCT and CBT recipients, but not BMT recipients, complained of malodor, whereas the rate of hypertension was highest in BMT (Table 1). Next, we compared HSC infusion-related AEs between auto- and allo-HSCT using cryopreserved products (Table 2). This comparison showed higher overall rates of AEs in allo-HSCT compared with auto-HSCT, especially in pediatric recipients, suggesting that plasma componentsrather than DMSO contribute to HSC infusion-related AEs in low body-weight/pediatric recipients as well as in general recipients. Of note, pediatric recipients showed a 10-foldhigher incidence of nausea/vomiting in allo-HSCT versus auto-HSCT, instead of allergic reactions, for which the incidence was significantly higher in allo-HSCT than auto-HSCT in general recipients. Subsequently, we sought factors that correlate with HSC infusion-related AEs in allo-HSCT using multivariate analysis with logistic regression, which identified lymphoid neoplasms over myeloid neoplasms as a factor for overall AEs (OR 3.013, P= .026), while history of transfusion reactions did not reach statistical significance (OR 2.368, P= .066). Notably, in a multivariate analysis for any grade ≥2 AEs, there were some factors that did not correlate with general recipients but did with low body-weight/pediatric recipients, including complications before HSCT (OR 5.764, P= .019), without plasma or red blood cell removal for ABO mismatch (OR 3.815, P= .032), and >10 ml/kg infusion volume (OR 5.306, P= .027). In conclusion, our data quantifysome specific symptoms associated with HSC infusionin low body-weight and pediatric recipients. We should be mindful ofinfusion volume and preexisting complications when small recipients receive HSC infusion. Disclosures Fujiwara: Astellas: Consultancy; Kyowa-Hakko: Consultancy; Kirin: Consultancy; Chugai: Consultancy; Pfizer: Consultancy; Shire: Consultancy. Muroi:JCR: Speakers Bureau; Becton: Speakers Bureau; Dickinson and Company: Speakers Bureau; Japanese Red Cross Society: Speakers Bureau. Mori:Astella Pharma: Honoraria; Kyowa Hakko Kirin: Honoraria; Japan Blood Products Organization: Honoraria; MSD: Research Funding; Ono: Honoraria; Novartis Pharma: Honoraria; Eisai: Honoraria; Janssen: Honoraria; Asahi Kasei: Research Funding; Novartis Pharma: Research Funding; Celgene: Honoraria; Taisho Toyama Pharmaceutical Co: Honoraria; Shire Japan: Honoraria; Pfizer: Honoraria; CHUGAI: Honoraria; MSD: Honoraria; SHIONOGI: Honoraria. Nagai:Ono Pharmaceutical Co.Ltd.: Consultancy; Kaneka Corporation: Research Funding; Kawasumi Laboratories Inc.: Research Funding.

我々は，D抗原陰性者に対しD陰性の赤血球輸血を行なっていたにもかかわらず，抗D抗体価がクームス法で最高4,096倍と著しく上昇した症例を経験した．輸血に使用された血液製剤の遺伝子解析を行なったところ，Del型赤血球の特徴であるRHD遺伝子の1227番目がグアニンからアデニンに置換している製剤が含まれていたことが明らかとなった． したがって本症例はDel型赤血球の輸血により，D抗原の感作を受け，抗D抗体を産生したと考えられた．なお，輸血後明らかな溶血の所見は認められなかった． 通常のD陰性血の中にはDel型が含まれていることがあり，Del型赤血球輸血がD陰性者に抗D抗体を産生させる可能性があることは十分認識するべきであると思われた．

【背景・目的】赤血球濃厚液（RCC），血小板濃厚液（PC），新鮮凍結血漿（FFP）などの輸血用血液製剤（輸血用血液）は献血者の人体の一部であり，とりわけ有効利用が求められるが，一定量が有効期限切れで廃棄されているのが現状である．廃棄血を減少させるため有効期限内で別の患者に転用する努力が行われているが，一病院の中では限界がある．そこで，われわれは病院間で輸血用血液を転用し，有効利用する方法（病院間有効利用）を検討し，実施したので報告する． 【方法】東京都が運営する7病院が本研究に参加した．まず，7病院において有効期限切れが原因で廃棄となる輸血用血液の量を調査した（平成17年1～7月）．その後，平成19年9～12月の間に各病院で有効期限切れが切迫している輸血用血液の情報をインターネットメールで毎日，定時に発信し，使用できる病院があれば，その病院に搬送し輸血に用いた．搬送にあたっては血液製剤搬送用温度安定剤を用い，温度を管理しながら搬送した．搬送後の品質を管理するため，温度と外観，搬送時間などを評価し記録に残した． 【結果】平成19年9～12月の間に，RCC 18本，PC 1本，FFP 4本の輸血用血液が病院間で有効利用された．その期間のRCCの廃棄率は1.06％で，H19年度の病院間有効利用を行わなかった期間の廃棄率1.78％と比較し明らかに低かった． 【結論】輸血用血液の廃棄量を減少させるために期限切れの前に他の病院で利用することは有効であり，今後，多くの病院間で試みる意義があると思われた．

The development of NK cells from hematopoietic stem cells is thought to be dependent on IL-15. In this study, we demonstrate that stimulation of human cord blood CD34+ cells by a Notch ligand, Delta4, along with IL-7, stem cell factor, and Fms-like tyrosine kinase 3 ligand, but no IL-15, in a stroma-free culture induced the generation of cells with characteristics of functional NK cells, including CD56 and CD161 Ag expression, IFN-γ secretion, and cytotoxic activity against K562 and Jurkat cells. Addition of γ-secretase inhibitor and anti-human Notch1 Ab to the culture medium almost completely blocked NK cell emergence. Addition of anti-human IL-15-neutralizing Ab did not affect NK cell development in these culture conditions. The presence of IL-15, however, augmented cytotoxicity and was required for a more mature NK cell phenotype. CD56+ cells generated by culture with IL-15, but without Notch stimulation, were negative for CD7 and cytoplasmic CD3, whereas CD56+ cells generated by culture with both Delta4 and IL-15 were CD7+ and cytoplasmic CD3+ from the beginning and therefore more similar to in vivo human NK cell progenitors. Together, these results suggest that Notch signaling is important for the physiologic development of NK cells at differentiation stages beyond those previously postulated.

背景·目的: わが国ではアルブミン製剤の使用量が多く，輸入に依存しているが，その使用実態に関しては必ずしも明らかではない．当院はアルブミン製剤を輸血部門で管理し，使用前に厚生労働省の適応基準に準拠することを確認しながら使用してきており，使用量が少ない．当院におけるアルブミン製剤の使用状況の特徴を明らかにするため，全国の総合病院7施設と比較した． 対象·方法: 当院および協力の得られた全国の7総合病院における入院患者で，平成17年4月の1カ月間にアルブミン製剤が使用された全症例を対象とし，患者背景およびアルブミン製剤の使用状況を調査した． 結果: 症例数およびアルブミン製剤使用本数は，当院およびその他7病院で，それぞれ，56例，180本，および444例，2,512本であった．アルブミン製剤の使用理由は，当院で循環動態の維持，7病院では低アルブミン血症が最も多かった．投与前の患者血清アルブミン値は，当院2.3±0.5g/dl，7病院2.8±0.7g/dl（p＜0.000001），1回のアルブミン使用量は当院23.5±15.3g，7病院44.7±59.3g（p＜0.000001），アルブミン投与1カ月後の死亡率は，当院16.1%，7病院14.6%（P=0.77）であった．全体的に見て投与前のアルブミン値が低いほど予後が悪い傾向を認めた． 考察: 当院のアルブミン製剤の使用量は7病院に比較して大幅に少なかった．これは使用前に輸血部門で適応を評価することによると思われた．また，低アルブミン血症は予後不良の指標になるが，アルブミン製剤の投与によっても予後は改善できない可能性が考えられた．

We describe herein two cases of delayed hematological recovery (DHR) following autologous peripheral blood stem cell transplantation (auto-PBSCT) using cells harvested during second molecular remission after treatment with arsenic trioxide (As(2)O(3)). Current therapeutic strategies with As(2)O(3) plus auto-PBSCT might be hampered by potential mechanisms of DHR. Our observations highlight the need for study of the real effects of As(2)O(3) on the kinetics of normal hematopoietic engraftment following auto-PBSCT.

血液製剤の細菌汚染を予防することは輸血の安全性を確保するために重要である. 今回, 我々は細菌検出システムBDSおよびその改良型機種のeBDSの性能を評価するために, 日本赤十字社から供給された200製剤のPCを測定した. BDSおよびeBDSは細菌の消費する酸素を測定することで細菌増殖を検出するシステムである.BDSで測定したPC 100製剤のうち1製剤に細菌汚染があると判定され, eBDSで測定したPC 100製剤はすべて陰性であった. 陽性となったPCの一部を, BacT/ALERTにより1週間培養後, 測定したが菌は検出されず, 偽陽性と判断された. 結局, 今回の検討では200製剤のPCはすべて細菌汚染はないと判断された. また, BDSおよびeBDSともに検体採取から培養開始にかかる時間は2～3分であった.eBDSは大量の検体を迅速に処理でき, 結果は24時間で判定できるので, 血小板製剤の細菌汚染のスクリーニング検査として有用性が高いものと思われた.

It has been recently reported that many Caucasians with IgA deficiency carry the HLA-A1, B8, DR3 haplotype. This haplotype may be widely associated with the pathogenesis of immune deficiency. To date, however, there has been no study of HLA typing of IgA deficiency in Japanese.We performed HLA-A, B, and DR typing in nine healthy blood donors who had been identified to be IgA-deficient by systematic screening in Saitama Prefecture, Japan. Results showed that these IgA-deficient blood donors had HLA antigens generally found in the Japanese population and no HLA-A1, B8 or DR3 antigens were observed. While the gene frequency of B54 antigen was significantly higher in this group than in the Japanese population, no significant differences for other antigens were identified. It is suspected that the characteristics of IgA deficiency in Japanese are different from those in Caucasians.

We assessed the performance of a leukocyte reduction filter, WBF2, which has been developed for preparation of blood products with pre-storage leukocyte reduction, and analyzed residual leukocyte subsets in the filtered whole blood.Whole blood was collected from six healthy volunteer donors and filtered 4 hours after collection. Leukocyte subsets of unfiltered and filtered whole blood were analyzed by flow cytometry, and absolute cell numbers of each leukocyte subset were determined.Total leukocyte counts per bag were 1.75±0.34×109 for the unfiltered blood and 2.23±1.37×104 for the filtered blood. Average log10 leukocyte reduction rate was 4.83. In the filtered whole blood, the number of granulocytes, CD4+ T lymphocytes, CD8+ T lymphocytes and NK cells was 8.67±7.66×103, 11.6±36.5×103, 4.05±0.80×103 and 1.66±1.35×103, respectively. No monocytes or B lymphocytes were detected in the whole blood after filtration.These results show that leukocyte reduction rate using the WBF2 filter varies according to leukocyte subset; in particular, monocytes and B lymphocytes are reduced more effectively by this filter.

Approximately 50% of adult patients with acute myeloid leukemia (AML) do not show clonal chromosome aberrations at diagnosis, and although this group shows an intermediate prognosis, a minority of ...

The plasma fraction of blood components has an essential role in the etiology of allergic transfusion reactions (ATRs). The difference of incidences of ATRs between fresh-frozen plasma (FFP) and platelet concentrates (PCs), in which plasma is the main component, is not clearly understood. This study compares the frequency of ATRs to FFP versus PCs on both first and subsequent (nonfirst) transfusions and considers the factors influencing the risk of ATRs.Five hospitals agreed to systematically collect and share 2 years of data (January 2010 through December 2011). This was a retrospective observational analysis of data including the number of transfusion episodes and ATRs for FFP and PCs on first-transfusion patients (without transfusion history) and previously transfused patients.The incidence of ATRs to PCs (2.51%) was significantly higher than to FFP (1.68%) on subsequent transfusions (p0.001). On the other hand, there were no significant differences in the incidences of ATRs to FFP (2.67%) and PCs (2.82%) on first transfusions. This discrepancy was most pronounced among males: FFP versus PCs on first transfusions, 2.02% versus 2.60% (p = 0.30); and on subsequent transfusions, 1.58% versus 2.46% (p = 0.0007). Among females, FFP versus PCs on first transfusions was 3.59% versus 3.13% (p = 0.61) and on subsequent transfusions was 1.87% versus 2.61% (p = 0.029).Repeated exposure rather than the total volume of transfused components may influence the incidence of ATRs.

Background and Objectives This study compares the frequency of adverse transfusion reactions (ATRs) after first transfusions with the frequency of ATRs for subsequent (non-first) transfusions. Materials and Methods Five hospitals agreed to systematically collect and share 2 years of data. This was a retrospective observational analysis of data including the number of transfusion episodes and ATRs for red blood cells (RBCs), fresh frozen plasma (FFP) and platelet concentrates (PCs) given to first-time transfusion recipients and to those previously transfused. Results First transfusion ATRs to RBCs, FFP and PCs were 1·08%, 2·84% and 3·34%, respectively. These are higher than ATR incidences to RBCs (0·69%), FFP (1·91%) and PCs (2·75%) on subsequent transfusions. Specifically, first transfusion incidences of febrile non-haemolytic transfusion reactions (FNHTRs) to RBCs (0·43%) and allergic reactions to FFP (2·51%) were higher than on subsequent transfusions (RBCs: 0·23%, FFP: 1·65%). Conclusion There are risks of ATRs on the first transfusion as well as transfusions of patients with transfusion history.

The case of a 34-year-old man with relapsing Ph+ acute lymphoblastic leukemia (ALL), which occurred five months after allogeneic bone marrow transplantation, is described. He was originally treated with aggressive chemotherapy, which put him in hematological remission, and he subsequently received donor leukocyte infusion (DLI) form the original donor. To assess the efficacy of this adoptive immunotherapy, we monitored minor-BCR/ABL (m-BCR/ABL) mRNA levels using the recently established real-time quantitative RT-PCR (RQ-PCR) assay. The results were compared with those obtained using conventional qualitative RT-PCR assays run in parallel. RQ-PCR, but not RT-PCR-based, minimum residual disease (MRD) detection showed a good correlation with the rapid changes documented during the post-DLI clinical course. Currently, six months after DLI, the patient continues to be in remission, which is consistent with the undetectable levels of m-BCR/ABL mRNA in the leukemic clone using RQ-PCR found in this study. Thus, monitoring of m-bcr/abl transcripts using RQ-PCR provides more useful information on a clinical assessment of MRD.

Recently, a completely automated procedure, the AutoPBSC program for the COBE Spectra cell separator, has been developed for peripheral blood stem cell harvest (PBSCH). We compared the performance of the AutoPBSC program with the standard mononuclear cell (MNC) program in the same patients and in a donor. Peripheral blood stem cells (PBSC) were collected from 3 patients or a donor alternately using the MNC program and the AutoPBSC program in a course of PBSC mobilization. Equal blood volume was processed from each patient (200 ml/kg) and a donor (150 ml/kg). We used a harvest volume of 3 ml and a chase volume of 7 ml in all AutoPBSC procedures. The procedure duration was almost equivalent for both programs. The volume of products was significantly lower in the AutoPBSC program (71 +/- 13 ml) than in the MNC program (183 +/- 30 ml). MNC yields were fewer, and total nucleated cell (TNC) and MNC collection efficiency was less for the AutoPBSC program compared to the MNC program. The CD34+ cell collection efficiency was less for the AutoPBSC program (26.5 +/- 13.7%, compared with 77.7 +/- 60.6%; p > 0.05). The contamination of platelets and red cells was significantly less in the AutoPBSC program than in the MNC program. In conclusion, we consider that the collection efficiency in the new program should be improved by modification of parameters because there exist great advantages to automated procedures.