Jang Soo Suh

Clinical impact of early absolute lymphocyte count after allogeneic stem cell transplantation

The role of repopulating lymphocytes after allogeneic stem cell transplantation (SCT) includes the prevention of serious infections and attacking residual tumour cells in the early post‐transplant phase. Therefore, the current study analysed the role of the absolute lymphocyte count (ALC) on day 21 after SCT in predicting transplant outcomes of 82 patients in terms of the risk of opportunistic infections and recurrence of original disease. The median dose of CD34+, CD3+ and mononuclear cells (MNC) infused was 6·41 × 106/kg, 1·96 × 108/kg and 6·81 × 108/kg respectively. The high ALC group (high ALC on day 21; ≥0·35 × 109/l) was associated with the use of peripheral blood stem cells, matched sibling donors and higher cell doses of MNC, CD3+ and CD4+ cells. The high ALC group also exhibited a better overall survival (56·3% vs. 17·7%) and disease‐free survival (50·1% vs. 15·9%) after 3 years and lower incidences of relapse (33·6% vs. 67·1%) and fungal infections (3·0% vs. 19·5%) after 1 year. The incidence of cytomegalovirus antigenaemia was lower in the high ALC group (47·7% vs. 73·7%). Accordingly, identifying the ALC on day 21 would appear to be a useful and simple measurement to predict those patients with a high risk of opportunistic infections and relapse after allogeneic SCT.

Multidrug resistance‐1 gene polymorphisms associated with treatment outcomes in de novo acute myeloid leukemia

Multidrug resistance‐1 (MDR‐1) gene single nucleotide polymorphisms (SNPs) have been identified as associated with the treatment outcomes of acute myeloid leukemia (AML) in Caucasians; yet, similar evidence is lacking for Asian populations. A total of 101 AML patients were enrolled in the current study. Two MDR1 SNPs (C3435T and G2677T/A) were analyzed with PCR/RFLP assay. As regards C3435T polymorphism, C/C genotype was significantly correlated with lower functional P‐glycoprotein (P‐gp) activity in leukemic blasts (7.5%) compared with C/T (10.7%) or T/T genotype (19.9%, p = 0.029). In genotypic analyses, C/C at −3435 (p = 0.05) and G/G at −2677 (p = 0.04) were strongly associated with a higher probability of complete remission (CR). In addition, the 3‐year event‐free survival (EFS) was higher in G/G genotype at −2677 (60.6%) than nonG/G (21.9%; p = 0.0241), in C/C at −3435 was higher than nonC/C genotype (p = 0.0139), and was higher in GC haplotype homozygote (58.2%) than nonGC homozygote (22.6%; p = 0.0427). In a multivariate analysis, the group without GC haplotype showed worse EFS (p = 0.030), with unfavorable cytogenetic risk (p = 0.008). However, no differences were noted in overall survival according to the MDR1 SNPs (p = 0.491 for C3435T and p = 0.955 for G2677T/A).

Different efficacy of mycophenolate mofetil as salvage treatment for acute and chronic GVHD after allogeneic stem cell transplant.

Abstract:  Objective:  The therapeutic options currently available for treating refractory graft‐vs.‐host disease (GVHD) are limited. Therefore, the present study evaluated the efficacy of mycophenolate mofetil (MMF) as a salvage treatment for acute and chronic GVHD in allograft patients.

Clinical implications of angiogenic factors in patients with acute or chronic leukemia: Hepatocyte growth factor levels have prognostic impact, especially in patients with acute myeloid leukemia

The present study evaluated the serum levels of known angiogenic factors and analysed their prognostic significance in patients with acute or chronic leukemia. Enzyme-linked immunosorbent assays (ELISAs) were performed to quantify the basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), tumor necrosis factor-alpha (TNF-alpha), angiogenin, and matrix metalloproteinase-9 (MMP-9) in stored samples obtained before treatment from patients with acute myeloid leukemia (AML; 30 patients), acute lymphoblastic leukemia (ALL; 10 patients), and chronic myelogenous leukemia (CML; 14 patients). The levels of VEGF, HGF, angiogenin, and MMP-9 were all significantly higher in patients with CML than in healthy individuals. The HGF levels were also higher in patients with AML than in healthy individuals, plus there was a significant correlation between the HGF level and the white blood cell count, monocyte count, and serum level of lactate dehydrogenase (LDH) in patients with AML. In a univariate analysis, age and HGF level were both found to be significant parameters predictive for an achievement of complete remission (CR) in patients with AML. Meanwhile, in a multivariate analysis using a logistic regression model, the HGF level was the only parameter strongly predictive for CR (P=0.047). The leukemia-free survival (LFS) rate for AML patients with a lower HGF concentration was better than that for AML patients with a higher HGF concentration (1 year LFS rates=75.0% vs. 37.5%, P=0.065). The HGF concentration was an independent prognostic factor for an achievement of CR, plus higher HGF concentrations were associated with a lower survival in patients with AML.

Slit-flow ektacytometry: Laser diffraction in a slit rheometer

Deformability of red blood cells (RBCs) is a determinant of blood flow resistance as RBCs pass through small capillaries of the microcirculation. Available techniques for measuring RBC deformability often require a washing process after each measurement, which is not optimal for day‐to‐day clinical use.

Transplantation with higher dose of natural killer cells associated with better outcomes in terms of non-relapse mortality and infectious events after allogeneic peripheral blood stem cell transplantation from HLA-matched sibling donors.

Abstract:  Background: Little is known about the role of the CD56+ natural killer (NK) cell dose on the outcome of allogeneic peripheral blood stem cell transplantation (PBSCT). Recently, higher dose of NK cells has been associated with a lower incidence of severe graft‐versus‐host disease (GVHD). The current study attempted to evaluate the effect of the NK cell dose on transplant outcomes in allogeneic PBSCT setting. Methods and materials: Sixty‐one cytokine mobilized PBSC recipients were analyzed according to the infused dose of CD34+ cells and NK cells in relation to overall survival (OS), non‐relapse mortality (NRM), GVHD, and infectious events. Results: The group received a higher dose of NK cells (≥5 × 107/kg) showed a lower incidence of NRM (P = 0.0186) and infectious events (P = 0.0107). In a multivariate analysis, a higher dose of NK cells was correlated to better transplant outcomes for NRM (P = 0.042) with CD34+ cell dose (P = 0.018), and for infectious events (P = 0.013) with CD34+ cell dose (P = 0.016). Higher NK cell infusion group also showed a faster immune recovery in serial measurements at days +90, +180, and +365. Conclusions: High dose of NK cells may play an important role in improving transplant outcomes, in terms of reducing NRM and infectious events together with CD34+ cells.

Flow cytometric detection of erythrocyte osmotic fragility

Hereditary spherocytosis (HS) is the most common cause of inherited chronic hemolysis. The various tests developed for HS screening have many shortcomings. The purpose of this study was to develop a new, simple, reliable test using flow cytometry.

Cryopreservation of amniotic fluid-derived stem cells using natural cryoprotectants and low concentrations of dimethylsulfoxide.

Amniotic fluid-derived stem cells (AFSCs) are a potential cell source for therapeutic applications. They can be easily mass produced, cryopreserved and shipped to clinics for immediate use. However, one major obstacle to the manufacturing of clinical grade stem cells is the need for current good manufacturing practices for cryopreservation, storage, and distribution of these cells. Most current cryopreservation methods used for stem cells include the potentially toxic cryoprotectant (CPA) dimethylsulfoxide (Me(2)SO) in the presence of animal serum proteins that prevent direct use of these cells in human therapeutic applications. To avoid any potential cryoprotectant related complications, it will be essential to develop non-toxic CPAs or reduce CPA concentration in the freezing media used. In this study, we assessed the use of disaccharides, antioxidants and caspase inhibitors for cryopreservation of AFSCs in combination with a reduced concentration of Me(2)SO. The thawed cells were tested for viability with MTT assays and a growth curve was created to measure population doubling time. In addition, we performed flow cytometry analysis for cell surface antigens, RT-PCR for mRNA expression of stem cell markers, and assays to determine the myogenic differentiation potential of the cells. A statistically significant (p<0.05) increase in post-thawed cell viability in solutions containing trehalose, catalase and (Z)VAD-fmk with 5% Me(2)SO was observed. The solutions containing trehalose and catalase with 5% or 2.5% (v/v) Me(2)SO produced results similar to those for the control (10% (v/v) Me(2)SO and 30% FBS) in terms of culture growth, expression of cell surface antigens and mRNA expression of stem cell markers in AFSCs cryopreserved for a minimum of 3 weeks. Thus, AFSCs can be cryopreserved with 1/4 the standard Me(2)SO concentration with the addition of disaccharides, antioxidants and caspase inhibitors. The use of Me(2)SO at low concentrations in cell freezing solutions may support the development of clinical trials of AFSCs.

Measurement of erythrocyte aggregation in a microchip stirring system by light transmission

In the analysis of red blood cell (RBC) aggregation using optical detection, various shearing methods have been used to disperse RBCs in confined geometries. This study investigated RBC aggregation measurement in a microchip-stirring system by analysis of light transmission. A stirring-aided disaggregation mechanism in a microchip, consisting of a flat-cylindrical test chamber (D=4 mm, H=0.3 mm) and a magnetic stirrer (d=0.14 mm, l=2.2 mm), was used to generate a given shear which was large enough to disperse RBC aggregates, but not large enough to cause any mechanical hemolysis of cells. After stirring for 10 s followed by an abrupt halt of the stirring, the intensity of the light transmitted through a microchip was measured with respect to time and analyzed. A comparative study was conducted with varying test chamber height and hematocrit. The AI and t1/2 as typical aggregation indices obtained by analysis of transmitted light, which showed a good reproducibility (coefficient of variation (CV)<2.8%, n=10), also were found to be nearly independent of the chamber dimensions (CV<3.4%). The present aggregometry also showed the similar results of aggregation indices with varying hematocrits compared to those obtained using a laser-assisted optical rotational cell analyzer (LORCA). The essential feature of the present design is the adoption of a disposable microchip requiring a minimum blood sample volume as small as 6 mul, which enables it to be used easily in a clinical setting.

A transient, microfluidic approach to the investigation of erythrocyte aggregation: The threshold shear-stress for erythrocyte disaggregation

Detailed analysis of red blood cells (RBCs) aggregation is often required in various clinical studies. Most conventional aggregation indices are dimensionless values and not available for comparison of across studies. Quite recently, we have developed microfluidic aggregometry that enables us to yield a critical shear-stress that are required to aggregate RBCs under the shearing hydrodynamic force. The present study investigated the relationships between the values of the critical shear-stress and conventional aggregation indices by comparing the critical shear-stress measured by the microfluidic aggregometry with the threshold shear-stress measured using a LORCA aggregometer. The results showed that the critical shear-stress did not vary with the hematocrit value while the threshold shear-rate decreased with the hematocrit value. The threshold shear-stress also showed the same hematocrit-independence as the critical shear-stress. These findings assist in rheologically validating the critical shear-stress, as defined in the microfluidic aggregometry, within the present range of hematocrit values.