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BACKGROUND We analyzed the 2019 external quality assessment (EQA) results to evaluate the analytical performance of maternal serum prenatal screening for Down Syndrome (DS) in the first trimester in China. METHODS In each round, five lyophilized EQA samples with detailed clinical information were distributed to participants and used to test the concentration of human chorionic gonadotropin free beta subunits (free β-HCG) and pregnancy-associated plasma protein-A (PAPP-A) to calculate the risk value of DS. The robust coefficient of variability (CV) was calculated or test results for free β-HCG and PAPP-A using analytes and major measurement platforms. For DS risk values, the robust CV was calculated using a professional calculation platform. Failed EQA results were analyzed using serum marker testing results and DS risk values. RESULTS EQA results were collected from 242 laboratories in round 1 and 239 laboratories in round 2. Total acceptable rates of testing results for free β-HCG and PAPP-A ranged from 95.04% to 97.91%. Overall acceptable rates of DS risk values ranged from 97.52% to 97.90%. The proportion of laboratories exhibiting satisfactory performance was higher in round 2 than in round 1 for serum marker testing results and DS risk values. The robust CV for risk values for each sample were significantly higher than those of serum markers. Three EQA result failure types were found, including result reporting errors, serum marker concentration testing errors, and DS risk calculation errors. CONCLUSIONS The analytical performance of maternal serum prenatal screening for DS in the first trimester in China can be improved further.

Objective To investigate the value of platelet count in evaluating the degree of liver fibrosis in patients with chronic hepatitis B (CHB). Methods A total of 158 CHB patients who underwent liver biopsy in our hospital were included, and the clinical characteristics of these patients were retrospectively analyzed. The diagnostic values of platelet count, aspartate aminotransferase‐to‐platelet ratio index (APRI), and the fibrosis index based on four factors (FIB‐4) for significant fibrosis (F ≥ 2) and early cirrhosis (F = 4) stages in CHB patients were assessed by the use of receiver operating characteristic (ROC) analysis. Results The median (F0: 221.0; F1: 210.0; F2: 188.0; F3: 171.0; and F4: 155.5) and mean rank (F0: 120.4; F1: 100.1; F2: 82.2; F3: 67.9; and F4: 49.5) of platelet count decreased along the aggravation of fibrosis (F0‐F4). The areas under the ROC curve for the platelet count in diagnosis of significant fibrosis stage was 0.70, which had no significant difference with FIB‐4 (0.73) and APRI (0.68) in diagnostic efficacy (P = .428). The areas under the ROC curve of platelet count in diagnosis of early cirrhosis were 0.72, which had no significant difference with FIB‐4 (0.76) and APRI (0.68) (P = .094). Conclusion The platelet count, as a simple and non‐invasive index, could evaluate the degree of liver fibrosis in CHB individuals. At the same time, the diagnostic efficiency of platelet count to evaluate the significant liver fibrosis and early cirrhosis is comparable to FIB‐4 and APRI.

Background Customer satisfaction is one of quality indicators which can be used to identify quality defects in the total process and remind clinical laboratories to take measures to improve the process. This study mainly reviewed and analyzed the general situation through a customer satisfaction survey in clinical laboratories in China and described the overall customer satisfaction of outpatients and inpatients. Methods Surveys in this study could be divided into two stages. The first stage was to investigate the general situation through a customer satisfaction survey in Chinese clinical laboratories. In the second stage, clinical laboratories carried out nationwide surveys on the satisfaction of outpatients and inpatients. Chi-square test and logistic regression analysis were applied to determine the relationship between the general characteristics of patients and patient satisfaction. Results The recovery rate in the first stage was 34.54%. In the second stage, we received 12,919 outpatient questionnaires and 11,818 inpatient questionnaires. Outpatients and inpatients had a common trend: satisfaction rates increased with age and decreased with frequency of visit. Patients who possessed a bachelor degree or above had a higher satisfaction rate than those who had only received a high school education or below. Conclusions There was a high level of patient satisfaction with clinical laboratory services, but aspects of dissatisfaction still existed, which provided opportunities for clinical laboratories in China to improve their service quality.

Background: Internal quality control (IQC) is essential for precision evaluation and continuous quality improvement. This study aims to investigate the IQC status of blood gas analysis (BGA) in clinical laboratories of China from 2014 to 2017. Methods: IQC information on BGA (including pH, pCO2, pO2, Na+, K+, Ca2+, Cl−) was submitted by external quality assessment (EQA) participant laboratories and collected through Clinet-EQA reporting system in March from 2014 to 2017. First, current CVs were compared among different years and measurement systems. Then, percentages of laboratories meeting five allowable imprecision specifications for each analyte were calculated, respectively. Finally, laboratories were divided into different groups based on control rules and frequency to compare their variation trend. Results: The current CVs of BGA were significantly decreasing from 2014 to 2017. pH and pCO2 got the highest pass rates when compared with the minimum imprecision specification, whereas pO2, Na+, K+, Ca2+, Cl− got the highest pass rates when 1/3 TEa imprecision specification applied. The pass rates of pH, pO2, Na+, K+, Ca2+, Cl− were significantly increasing during the 4 years. The comparisons of current CVs among different measurement systems showed that the precision performance of different analytes among different measurement systems had no regular distribution from 2014 to 2017. The analysis of IQC practice indicated great progress and improvement among different years. Conclusions: The imprecision performance of BGA has improved from 2014 to 2017, but the status of imprecision performance in China remains unsatisfying. Therefore, further investigation and continuous improvement measures should be taken.

Introduction Prenatal screening and diagnosis is important for the detection of birth defects and genetic diseases. The nationwide proficiency testing (PT) of maternal serum prenatal screening in second trimester in China has been launched since 2003 and partly reflected the performance of screening laboratories. This study analysed the 2015 PT results to examine the performance of clinical laboratories and different platforms. Materials and methods Fifteen lyophilized samples with different concentrations divided into three panels, were distributed to 613 clinical laboratories in 2015. Acceptable performance was defined as scores more than 80% of acceptable responses with the evaluation criterion of ± 30%. The robust coefficient of variation (CV) was also analysed. Chi-square (χ2) test was used to compare the acceptable performance while Kruskal-Wallis test and Mann-Whitney test were applied to compare the robust CV among analytes and mainstream platforms. Results Overall, 605, 61, 214, 416, 303 laboratories submitted effective results for alpha fetoprotein (AFP), total human chorionic gonadotropin (t-hCG), β-hCG, unconjugated estriol (uE3) and free β-hCG. The acceptable performances of AFP (μg/L), AFP (KIU/L), t-hCG, β-hCG, uE3, and free β-hCG were 98.45%, 99.24%, 95.58%, 98.72%, 94.50%, and 98.66%, respectively. The χ2 test indicated significant differences existed in the acceptable performances among different analytes and platforms for uE3. Kruskal-Wallis test and Mann-Whitney test suggested the robust CV differed significantly in different analytes and platforms. Conclusions The majority of results were acceptable. However, further effort is needed to achieve the standardization and harmonization among analytes and various platforms, particularly for uE3.

BACKGROUND The aim of this study was to analyze the 2018 external quality assessment (EQA) results of newborn screening by MS/MS of acylcarnitine by Chinese National Center for Clinical Laboratories and to determine the performance of clinical laboratories. METHODS Five dried blood spots were distributed to participants every round. Satisfactory performance was defined as scores more than 80 of acceptable results within the evaluation criterion. The robust coefficient of variability (RCV) of each sample was calculated by measurement systems. The chi-square (2) test was used to compare the correct recognition rates. RESULTS EQA results were collected from 150 laboratories for 15 different acylcarnitines between C0 - C18. The overall acceptable rates of the qualitative results were between 81.21% and 96.67%, and the proportion of acceptable quantitative results were between 78.38% and 93.24%. There were significant differences in the rates of acceptable quantitative results among different items and between the four methods. CONCLUSIONS Most of the participant laboratories had satisfactory performance for the quantitative results in this EQA scheme. But for qualitative assessment, a laboratory should re-evaluate and validate their reference intervals on a regular basis to improve the consistency of clinical assessment.

BACKGROUND To analyze the 2017 external quality assessment (EQA) results of newborn screening by MS/MS of amino acids by Chinese National Center for Clinical Laboratories, this study aimed to reflect the performance of clinical laboratories. METHODS Five dried blood spots were distributed to participants every round. Satisfactory performance was defined as scores more than 80 of acceptable results within the evaluation criterion. The robust coefficient of variability (RCV) of each sample was calculated by measurement systems. The chi-square () test was used to compare the correct recognition rates. RESULTS EQA results were collected from 150 laboratories for Ala, Val, Arg, Leu, Met, Phe, Tyr, Cit. The overall acceptable rates of the qualitative results were 87.42%, 92.72%, 73.33%, 94.04%, 92.72%, 94.70%, 92.72%, 94.04%, respectively, and the proportion of acceptable quantitative results were 76.51 %, 91.95%, 78.38 %, 92.62%, 93.29%, 93.29%, 94.63%, 91.28%, respectively. There were significant differences in the rates of acceptable quantitative results among different items and between four methods. CONCLUSIONS Most of the participant laboratories had satisfactory performance for the quantitative results in this EQA scheme. But for qualitative assessment, the laboratory should re-evaluate and validate their reference intervals on a regular basis to improve the consistency of clinical assessment.

Background This study observed and analyzed the internal quality control (IQC) of cystatin C (CysC) so that we can have overall knowledge about imprecision levels in Chinese medical laboratories. Methods Using the software developed by the National Center for Clinical Laboratories (NCCL), we can get the IQC information of CysC from 2014 to 2017. Then the proportion of laboratories meeting five quality specifications (pass rates) were calculated and the current CVs (coefficient of variation) were also compared among subgroups and years. Results We find that the current CVs between 2014 and 2017 show significant differences (p = 0.016) and the proportion of laboratories meeting the 1/3 TEa specification distributes randomly from 2014 to 2017 but all of them exceed 80 percent. When the optimum specification is applied, the pass rates all become very low and the distributions are wide spread (from 3.63% to 6.74%). Beckman, Roche, and Hitachi are mainstream analyzers, making up as much as 78% to 85% of all. We can see a significant difference of the pass rate between Beckman and Hitachi in 2014 (p = 0.005). The primary means of detecting CysC is particle-enhanced turbidimetric immunoassay (PETIA) which makes up 30.57% - 32.64% of detection methods. The good news is that the IQC practice has improved greatly from 2014 to 2017 in laboratories in China. Conclusions Medical laboratories have made some progress in IQC from 2014 to 2017, but it is still not satisfactory. As a result, there is a long way to go to improve detection quality of laboratories in China.

While emergency medical service (EMS) response time (ERT) is a major factor associated with the survival of patients with cardiovascular disease (CVD), relatively few studies have explored the factors associated with ERT. This study aimed to assess the current status of ERT and to identify the factors affecting ERT in patients with CVD in China. Between January 1, 2011 and December 31, 2015, EMS responses to CVD incidents in Guangzhou, China, were examined. The primary outcome was ERT, defined as the time from receipt of an emergency call to the arrival of paramedics on the scene. Factors associated with ERT were evaluated by multivariable logistic regression. A total of 44 383 CVD incidents were analysed. The median ERT was 12.58 min (interquartile range=9.98-15.67). Among the risk factors, distance (OR=13.73, 95% CI=11.76-16.04), level of hospital (OR=1.57, 95% CI=1.40-1.75), and site of the incident (OR=1.53, 95% CI=1.38-1.69) were the top three significant factors affecting the ERT. Our results suggest that greater attention should be given to factors affecting the ERT. It is essential to make continuous efforts to promote the development of effective interventions to reduce the response time.

Background This study aimed to investigate and analyze the status of internal quality control (IQC) for BNP and NT-proBNP from 2014 to 2017, so as to have an integral understanding of quality performance of measurement in clinical laboratories in China. Methods The 4-year IQC information for BNP and NT-proBNP of participant laboratories were collected through EQA reporting system. Percentages of laboratories meeting different quality requirements (pass rates) for current coefficient of variations (CVs) were calculated afterwards. Further analysis for current CVs and pass rates among different years and measurement systems were conducted. Finally, we analyzed and summarized IQC practice and its changes in 4 years. Results The current CVs for BNP and NT-proBNP have decreased significantly from 2014 to 2017, and pass rates both presented significant increasing trends. NT-proBNP had higher pass rates than BNP regardless of 1/3TEa or 1/4TEa specification was used. The main measurement systems for two analytes were different. For NT-proBNP, current CVs of Roche has decreased significantly among 4 years and were significantly lower than Radiometer and BioMerieux in 2015. Current CVs of Abbott also had decreasing tendency for BNP. Analysis of IQC practice indicated that control rules and IQC frequency had made great progress in 4 years. Conclusion The imprecision performance of measurement of BNP and NT-proBNP has improved with decreasing of current CVs and increasing of pass rates in 4 years. However, it still needs continual improvement. Clinical laboratories in China should take active actions to promote performance of BNP and NT-proBNP measurement.

Background This study aimed to evaluate the performance of HbA1c measurement systems quantitatively with sigma metric and promote the quality improvement. Methods 1066 laboratories enrolled external quality assessment (EQA) and reported their internal quality control (IQC) data for HbA1c in 2017 were included in this study. The bias and coefficient of variation were collected from EQA and IQC program designed by the National Center for Clinical Laboratory. The σ values was used to assess the performance of HbA1c, and combine Westgard Sigma Rules™ to select proper quality control rules in every laboratory. Results Totally, There were 388,190,185,100,59,55,89 participants used Bia-Rad HPLC, TOSOH G7/G8, ARKRAY HA8160/8180, HUIZHONG MQ-2000/2000PT, Roche, Primus HPLC and “Others” system, respectively. “HUIZHONG MQ-2000/2000PT” group had the smallest variation of bias. 56.19% (599/1066) of laboratories met bias criterion and 55.63% (593/1066) satisfied imprecision criterion. However, sigma metrics indicated that 80.21% (855/1066) of 1066 laboratories needed to improve their performance of HbA1c. Moreover, “TOSOH G7/G8” group had the highest constituent ration of σ ≥ 3. Conclusions The analytical quality of HbA1c in most laboratories need improving. Laboratories should pay more attention on the performance of HbA1c, and EQA organizers in China should improve evaluation criteria and push standardization work for HbA1c.

Introduction This survey was initiated to obtain knowledge on the current situation of internal quality control (IQC) practice for tumour markers (TMs) in China. Additionally, we tried to acquire the most appropriate quality specifications. Materials and methods This survey was a current status survey. The IQC information had been collected via online questionnaires. All of 1821 clinical laboratories which participated in the 2016 TMs external quality assessment (EQA) programme had been enrolled. The imprecision evaluation criteria were the minimal, desirable, and optimal allowable imprecisions based on biological variations, and 1/3 total allowable error (TEa) and 1/4 TEa. Results A total of 1628 laboratories answered the questionnaires (89%). The coefficients of variation (CVs) of the IQC of participant laboratories varied greatly from 1% (5th percentile) to 13% (95th percentile). More than 82% (82 - 91%) of participant laboratories two types of CVs met 1/3 TEa except for CA 19-9. The percentiles of current CVs were smaller than cumulative CVs. A number of 1240 laboratories (76%) reported their principles and systems used. The electrochemiluminescence was the most used principle (45%) and had the smallest CVs. Conclusions The performance of laboratories for TMs IQC has yet to be improved. On the basis of the obtained results, 1/3 TEa would be realistic and attainable quality specification for TMs IQC for clinical laboratories in China.

Introduction Specimen adequacy is a crucial preanalytical factor affecting accuracy and usefulness of test result. The aim of this study was to determine the frequency and reasons for rejected haematology specimens, preanalytical variables which may affect specimen quality, and consequences of rejection, and provide suggestions on monitoring quality indicators as to obtain a quality improvement. Materials and methods A cross-sectional survey was conducted and a questionnaire was sent to 1586 laboratories. Participants were asked to provide general information about institution and practices on specimen management and record rejections and reasons for rejection from 1st to 31st July. Results A total survey response rate was 56% (890/1586). Of 10,181,036 tubes received during the data collection period, 11,447 (0.11%) were rejected, and the sigma (σ) was 4.6. The main reason for unacceptable specimens was clotted specimen (57%). Rejected specimens were related to source department, container type, container material type, transportation method and phlebotomy personnel. The recollection of 84% of the rejected specimens was required. The median specimen processing delay in inpatient, outpatient and emergency department were 81.0 minutes, 57.0 minutes and 43.3 minutes, respectively. Conclusions Overall, rejection rate was a slightly lower than previously published data. In order to achieve a better quality in the preanalytical phase, haematology laboratories in China should pay more attention on training for phlebotomy and sample transportation, identify main reasons for clotted specimen and take effective measures. The platform in the study will be helpful for long-term monitoring, but simplification and modification should be introduced in the following investigation.

The aim of this study was to use Six Sigma(SM) (Motorola Trademark Holdings, Libertyville, IL) techniques to analyze the quality of point-of-care (POC) glucose testing measurements quantitatively and to provide suggestions for improvement.In total, 151 laboratories in China were included in this investigation in 2014. Bias and coefficient of variation were collected from an external quality assessment and an internal quality control program, respectively, for POC glucose testing organized by the National Center for Clinical Laboratories. The σ values and the Quality Goal Index were used to evaluate the performance of POC glucose meters.There were 27, 30, 57, and 37 participants in the groups using Optium Xceed™ (Abbott Diabetes Care, Alameda, CA), Accu-Chek(®) Performa (Roche, Basel, Switzerland), One Touch Ultra(®) (Abbott), and "other" meters, respectively. The median of the absolute value of percentage difference varied among different lots and different groups. Among all the groups, the Abbott One Touch Ultra group had the smallest median of absolute value of percentage difference except for lot 201411, whereas the "other" group had the largest median in all five lots. More than 85% of participate laboratories satisfied the total allowable error (TEa) requirement in International Organization for Standardization standard 15197:2013, and 85.43% (129/151) of laboratories obtained intralaboratory coefficient of variations less than 1/3TEa. However, Six Sigma techniques suggested that 41.72% (63/151) to 65.56% (99/151) of the laboratories needed to improve their POC glucose testing performance, in either precision, trueness, or both.Laboratories should pay more attention on the practice of POC glucose testing and take actions to improve their performance. Only in this way can POC glucose testing really function well in clinical practice.

BACKGROUND Free β subunit of human chorionic gonadotropin (free β-hCG) and pregnancy-associated plasma protein A (PAPP-A) are two important biomarkers in first-trimester prenatal screening. This study intended to reflect their analytical performance in clinical laboratories and main platforms of the 2015 External Quality Assessment (EQA) scheme for the first-trimester biochemical screening in China. METHODS Ten lyophilized EQA samples, divided into two sets and analyzed in two cycles 20151 and 20152, were distributed to each participant in 2015. Satisfactory performance was defined as scores more than 80 of acceptable results within the evaluation criterion of ± 30%. The robust coefficient of variability (CV) of each sample was calculated by analytes and mainstream platforms. The chi-square (2) test and the Mann-Whitney test were respectively used to compare the acceptable performance and the robust CV between two analytes, as well as two mainstream platforms. RESULTS A total of 142 laboratories were enrolled in this EQA scheme. For free β-hCG and PAPP-A, the number of effective results were 1330 and 1355, respectively, and the overall acceptable rates of results were 95.11% and 92.32%, respectively. For cycle 20151 and 20152, the acceptable rates were respectively 92.59% and 96.19% for free β-hCG and 89.78% and 94.78% for PAPP-A. The 2 test showed acceptable performance differed significantly between the two analytes, as well as two platforms for PAPP-A. Significant difference of the robust CV in different analytes and platforms was demonstrated by Mann-Whitney test. CONCLUSIONS Greater than 92% of results were acceptable. However, continuous improvement and further research of this EQA scheme is in process.

BACKGROUND This national survey was initiated to investigate the current status of quality control practice of prenatal screening by statistical analysis of the previous half year data of prenatal screening in 2015. METHODS Data were sent to all Chinese prenatal screening centers via the National Quality Assessment Scheme. This covered the software used, the risk cutoffs, monthly sample throughput, monthly median MoM of AFP, HCG, β-HCG, free β-HCG and uE3, monthly screening positive rates for trisomy 21, trisomy 18, and Open Neural Tube Defect (ONTD). RESULTS Screening protocols were versatile, 73.5% (133/181) used the two-marker model, 24.3% (44/181) used the three-marker model, and 2.2% used the four-marker model. Regarding the software used, 350 laboratories never updated the screening parameters, 89 laboratories had updated their median or parameter by manufacturers, and 24 laboratories had updated the parameters by themselves. Cutoffs differ between laboratories. 59.9% (275/459) use 1/270 as their cutoffs for trisomy 21. 66.2% (296/447) use 1/350 as their cutoff for trisomy 18. 96.5% (361/374) use cutoffs between 2.0 - 2.5 MoM for ONTD. Regarding the results of the monthly median MoM, the percentage of laboratories for which all six monthly median MoMs were within the target of 0.90 - 1.10 was 46.7% (155/332) for AFP, 20.0% (4/20) for hCG, 29.2% (28/96) for β-HCG, 15.7% (31/198) for free β-HCG, and 4.8% (11/228) for uE3. The percentage of laboratories for which all six monthly median MoMs were within the target of 0.95 - 1.05 was 14.2% (47/332) for AFP, 0% (0/20) for HCG, 4.3% (4/96) for β-HCG, 12.6% (31/198) for free β-HCG, and 4.8% (11/228) for uE3. Regarding the screening positive rate, there was a difference in the trisomy 21 positive rate in the same laboratory within the six month. CONCLUSIONS There were variations in the types of screening protocols, different kinds of soft platforms, randomness of choice or update of medians or other important parameters, and great difference in the awareness of quality control. Effective and practical quality control measures would help laboratories improve quality control.

Background To investigate the current status of IQC practice of neonatal screening in China. Methods The IQC data of Phe and TSH testing were collected. Coefficient of variations (CVs) and cumulative CVs of every month in 2014 were compared with the 1/3 and 1/4 TEa. Results Of the 219 laboratories, the return rates ranged from 71.23% to 89.95% of 12 months in 2014. The rates of CVs for 2 lots less than 1/3 TEa were 60.27% and 68.78% (Phe), 67.05% and 65.68% (TSH); less than 1/4 TEa were 32.10% and 35.99% (Phe), 32.35% and 27.55% (TSH); rates of cumulative CVs less than 1/3 TEa were 57.89% and 65.98% (Phe), 61.21% and 60.93% (TSH); less than 1/4 TEa were 29.61% and 30.74% (Phe), 27.48% and 22.41% (TSH). Conclusions The testing capabilities and performances of neonatal screening laboratories can be objectively evaluated by the quality specifications of acceptable imprecision.

Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is a key negative regulator of T cell activation and proliferation. Ipilimumab is a human monoclonal antibody that specifically blocks the binding of CTLA-4 to its ligand. To test the hypothesis that blockade of CTLA-4 by ipilimumab could augment graft-versus-malignancy (GVM) effects without a significant impact on graft-versus-host disease (GVHD), we conducted a phase I clinical trial of ipilimumab infusion in patients with relapsed malignancy following allogeneic hematopoietic stem cell transplantation (allo-HSCT). Here, we report the analysis of peripheral blood T lymphocyte reconstitution, T regulatory cell (Treg) expression, and T cell activation markers after a single dose of ipilimumab in 29 patients. Peripheral blood samples were collected from all patients before and after ipilimumab infusion. Lymphocyte immunophenotyes, including levels of CD4(+)CD25(high) cells and T cell activation markers, were analyzed in all cases. Levels of CD4(+)CD25(high)Foxp3(+) cells and intracellular CTLA-4 in CD4(+) T cells also were evaluated in the last 11 cases. We found lower baseline levels of CD4(+) and CD45RO(+) T cells in patients compared with normal controls. More than 50% of the patients had abnormally low lymphocyte counts (CD4 or/and CD8 T cells), and some had no circulating B lymphocytes. The percentages of both CD4(+)CD25(high) and CD4(+)CD25(high)Foxp3(+) T cells were significantly higher in patients before ipilimumab infusion than in healthy donors. Twenty of 29 patients exhibited an elevated level of CD4(+)CD25(low) activated T cells at baseline, compared with only 3 of 26 healthy donors. Both CD4(+) and CD8(+) T lymphocyte counts were significantly increased after ipilimumab infusion. There was no consistent change in absolute lymphocyte count or in the number of T cells expressing the activation marker CD69. However, increases in CD4(+)CD25(low) T cells were seen in 20 of 29 patients and increases in CD4(+)HLA-DR(+) T cells were seen in the last 10 patients in the first 60 days after ipilimumab infusion. Although the percentages of both CD4(+)CD25(high) and CD4(+)CD25(high)Foxp3(+) T cells decreased significantly during the observation period, the absolute cell counts did not change. Intracellular CTLA-4 expression in CD4(+)CD25(lo/-) T cells increased significantly after ipilimumab infusion. We conclude that CTLA-4 blockade by a single infusion of ipilimumab increased CD4(+) and CD4(+)HLA-DR(+) T lymphocyte counts and intracellular CTLA-4 expression at the highest dose level. There was no significant change in Treg cell numbers after ipilimumab infusion. These data demonstrate that significant changes in T cell populations occur on exposure to a single dose of ipilimumab. Further studies with multiple doses are needed to explore this phenomenon further and to correlate changes in lymphocyte subpopulations with clinical events.

The aim of the study was to promote the establishment and implementation of quality indicators (QIs) in clinical laboratories, catch up with the state of art, and provide preliminary quality specifications for established QIs.Clinical laboratories from different provinces in China were included in this QIs survey in 2015. All participants were asked to collect data related to QIs and complete QIs questionnaires. Defect percentages and sigma values were calculated for each QI. The 25th percentile, median, and the 75th percentile of defect percentages and TATs were calculated as optimum, desirable and minimum quality specifications. While 25th, median, and 75th of sigma values were calculated as minimum, desirable and optimum quality specifications, respectively.Five thousand seven hundred and fifty-three clinical laboratories from 28 provinces in China participated in this survey. Median defect percentages of pre-examination QIs varied largely from 0.01% (incorrect sample container) to 0.57% (blood culture contamination) with sigma values varied from 4.0σ to 5.1σ. Median defect percentages of examination phase QIs were all really high. The most common problem in examination phase was test uncovered by inter-laboratory comparison (86.67%). Defect percentages of critical values notification and timely critical values notification were all 0.00% (6.0σ). While the median of defect percentages of incorrect laboratory reports was only 0.01% (5.4σ).Improvements are needed in all phases of total testing process (TTP) in laboratories in China, especially in examination phase. More attention should be paid when microbiology specimens are collected and results are reported. Quality specifications can provide directions for laboratories to make effort for.

The aims of this article was to provide the quality control requirements of preanalytical variation for the determination of lead in samples of human origin, reduce the influence of preanalytical variation on the test results. According to the Clinical and Laboratory Standards Institute documents, control of preanalytical variation in trace element determinations, analytical procedures for the determination of lead in blood and urine and other references and guidelines, the methods of quality control of lead determination had been made, including: the factors needed to be considered before collection, preservation, transportation and other preanalytical factors, the abilities and considerations of laboratory staff, etc.

This study investigated whether TNF-α, Toll-like receptors (TLRs) 7/8 agonist resiquimod (R848), the TLR4 agonist lipopolysaccharide (LPS) and their combinations can enhance autologous AML-reactive T cell generation in an in vitro culture. AML peripheral blood or bone marrow mononuclear cells were cultured in medium supplemented with GM-CSF/IL-4 to induce dendritic cell (DC) differentiation of AML blasts (AML-DC). The impact of TNF-α, LPS, R848 and their combinations on AML-DC cultures was analyzed. Significantly enhanced CD80, CD40, CD83, CD54, HLA-DR and CD86 expression of AML cells was observed by addition of TNF-α, LPS, R848 alone or combinations. Induced CD80 expression of AML cells was significantly higher through the combination of TNF-α, LPS and R848 (T+L+R) than that by T alone. CTL induced from T+L+R, T+R, T+L, L+R and R, but not T, L alone stimulated cultures showed significantly higher IFN-γ release than the medium control in response to autologous AML cells. IFN-γ release by T+L+R was significantly higher than T or L alone, and T+R was significantly higher than T alone. CTL generated from T+L+R, T+L, T+R, L+R and L alone exerted significantly higher AML cell killing than medium control. AML cell killing by T+L+R and T+R was significantly higher than T or R alone. These results indicate that the combination of T+L+R induces a significantly enhanced antigen presentation effect of AML-DC. We speculate that the complementary effects of reagent combinations may better address the heterogeneity of responses to any single agent in AML cells from different patients.

To evaluate the external quality survey results of newborn hereditary deafness gene mutations to improve and enhance the testing quality of hereditary deafness gene mutations.Each of 30 voluntarily participating laboratories testing newborn hereditary deafness gene mutations received 15 lots of quality control blood spots in this survey at May 2013, including mitochondria DNA 12SrRNA 1555AG (201311-201315), SLC26A4 IVS7-2AG (201321-201325) and GJB2 235delC (201331-201335). The testing results, methods, equipments and reagent information were submitted for analyses of Clinet EQA, Microsoft Excel 2007 and SPSS 13.0. The rates of correction (number of correct results/total number of submitted results) were used for evaluating the performance of laboratories.Among them, 24 laboratories submitted the testing results of mitochondria DNA 12SrRNA 1555AG and the rate of submission was 80.0% (24/30). The rates of correction for each lot were 95.8% (23/24), 95.8% (23/24), 100% (24/24), 95.8% (23/24) and 95.8% (23/24) respectively and the overall rate was 96.7% (116/120). And 23 laboratories submitted the other two kinds of genetic test results, the rate of submission was 76.7% (23/30); the rates of correction for each lot of SLC26A4 IVS7-2AG genetic test were 95.7% (22/23), 95.7% (22/23), 100% (23/23), 95.7% (22/23) and 95.7% (22/23) respectively and the overall rate was 96.5% (111/115); the genetic test results of GJB2 235delC were all correct (23/23). In this survey, 2/3 laboratories employed fluorescent polymerase chain reaction (PCR) and the remainder microarray chips.The survey of newborn deafness gene mutation testing results is generally satisfactory. Only mitochondria DNA 12SrRNA and SLC26A4 have some errors. The laboratories of gene testing should improve their quality control system, correct mistakes during the test period without any delay and boost the rate of correction for newborn hereditary deafness gene testing.

Background: miR-153 has been found to be significantly decreased in non-small cell lung cancer (NSCLC) tissues; however, its clinical significance has not been investigated. Methods: The expression patterns of miR-153 in 137 pairs of human lung cancer tissues and adjacent normal lung tissues were analyzed using qRT-PCR. The relationships between miR-153 expression and clinicopathological parameters were examined by chi-square test. Kaplan-Meier method and the log-rank test were used to determine the difference in overall survival (OS) rates between two groups. Results: The expression of miR-153 was reduced significantly, compared with adjacent normal lung tissues (P

To investigate and analyze the reference interval application status of analytes in coagulation tests.Every laboratories attended 2012 external quality assessment program were received an internet questionnaire about the resource, grouping and ranges of analytes of coagulation test. By using Microsoft excel 2007 and SPSS 13.0 to analyze the submitted data and calculate the arithmetic averages, medians, maximum values, minimum values, and the 2.5% and 97.5% percentages after eliminating the outliers. The data of every analytes was divided into 4-5 subgroups by different kinds of reagents. Meanwhile, each subgroup was also conducted statistical analysis for further description.There were 403 laboratories submitting the investigation questionnaire. The top three resources of reference intervals were reagent manufacturer instruction (56.33%-57.32%), calculated by laboratories themselves (15.25%-21.34%), and national guide to clinical laboratory procedures (19.11%-23.00%). Most laboratories did not conduct gender grouping in PT, INR and Fbg. The reference intervals conducted gender grouping had little difference from the medians. The median of lower and upper limits of analytes of coagulation test were as of follows: PT, male (10.3, 14.4) s, female (10.8, 14.4) s, totally (10.0, 14.0) s; INR, male, female and totally (0.80, 1.20); APTT, male (25.0, 40.0) s, female (24.5, 40.0) s, totally (23.0, 38.5) s; Fbg, male, female and totally (2.0, 4.0) g/L. Some of lower and upper limits of subgroups of analytes of coagulation test had significant statistical differences between each other. The distributions of the 2.5% and 97.5% percentages of difference reagent manufacturers were not same as others.There were little differences between the arithmetic averages and medians of the lower and upper limits of PT, INR, APTT and Fbg, but the consistencies of the distributions of the 2.5% and 97.5% percentages were not very well. The resources of reference intervals had some major categories. We suggest establishing new reference intervals to apply most parts of China.

Background The nationwide external quality assessment (EQA) of tumor markers in China has been launched for years. The quality of the performance of Chinese clinical laboratories on tumor markers is partly reflected through analysis of EQA results. This report presents an 8-year EQA result of the six most common tumor markers from 2006 to 2013. Methods Ten freeze-dried EQA samples were distributed to participants every year. Satisfactory performance was defined as scores of more than 80% of acceptable responses with the evaluation criterion of ± 25%. The robust coefficient of variability (CV) between laboratories and percentage difference against the target value of each sample were also calculated by year. Results A total number of 1154 laboratories submitted results in 2013, which was more than threefold of 2006. The proportion of laboratories with satisfactory performance showed an overall rising trend over the years and was up to 95% for the second survey in 2013. The overall decrease of robust CV was observed for all analytes including alpha fetoprotein (AFP), carcinoembryonic antigen (CEA), total prostate specific antigen (t-PSA), cancer antigen 125 (CA 125), cancer antigen 15-3 (CA 15-3), and cancer antigen 19-9 (CA 19-9) except for CEA, which exhibited a rise followed by a flat trend. The percentage difference narrowed gradually and was less than 2% in 2013. Conclusions The 8-year EQA results showed a significant enhancement of degree of harmonization of tumor markers in China. However, standardization among various testing systems and improvement of harmonization has yet to be achieved.

Objective To analyze the 2014 results of neonatal screening external quality assessment (EQA) performed by the Chinese National Centre for Clinical Laboratories. Methods EQA test panels consisting of five dried blood spots (three panels for phenylalanine (Phe) and thyroid stimulating hormone (TSH), two for glucose-6-phosphate dehydrogenase (G6PD) and 17-alpha-hydroxy progesterone (17-OHP)) were distributed to laboratories and the results collected and evaluated. To compare the correct recognition rates, chi-square test was used. Results Test results were received from 170 laboratories for Phe, 176 for TSH, 65 for G6PD and 65 for 17-OHP. The total number of effective quantitative and qualitative results of Phe, TSH, G6PD and 17-OHP were 2520 and 2370, 2605 and 2450, 645 and 530, 645 and 645, respectively. The overall correct recognition rates for qualitative tests of Phe, TSH, G6PD and 17-OHP were 99.79 %, 99.67 %, 93.40 % and 99.84 %, and the proportion of acceptable quantitative results were 94.48 %, 98.31 %, 84.65 % and 99.84 %, respectively. There were significant differences in the rates of acceptable quantitative results of the two measurement systems for Phe, TSH and G6PD ( p2 test showed significant differences in correct recognition or acceptable rates among programmes ( pConclusion Most of the quantitative results were acceptable and the overall correct recognition rates in qualitative results approached 100%. Distributing more challenging samples and increasing the range of concentrations of EQA samples will improve standards in future assessments.

Stromal cell-derived factor 1 (SDF-1) is a chemokine that binds to the CXCR4 receptor. Its functions include acting as a chemotactic factor for hematopoietic stem and progenitor cells. We recently reported the synthesis of a small cyclized peptide analog (31 amino acids) of the terminal regions of SDF-1 that had biological function comparable to the native molecule (67 amino acids). In the present study, we investigated the effects of SDF-1 analogs (CTCE0021 and CTCE0214) in the chemotactic migration of peripheral blood hematopoietic cells (lineage-negative and CD34(+) cells). Enhanced chemotaxis of normal and G-CSF-mobilized hematopoietic cells was observed with both SDF-1 analogs in a dose-dependent manner. The increases were statistically significant (p < or = 0.016 by one-way ANOVA) at analog concentrations of 50 to 100 microg/mL. Colony-forming progenitor cells were not affected by exposure to the analogs up to 100 microg/mL. When different doses of the SDF-1 analog CTCE0214 were administered to mice, significant increases in circulating hematopoietic cells (identified by flow cytometry as lineage(low/-), Sca-1(+), and c-kit(+)) were observed after a single injection of 75 microg per animal. The effect was apparent at 4 hours and became significant at 24 hours. These results suggest that SDF-1 analogs can be considered for mobilization of hematopoietic stem cells.

Objectives. Acute myeloid leukemia (AML) cells express the cell surface antigen CD33 that can function as a downregulator of cell growth, mediating growth arrest and apoptosis. The protein kinase Syk is an essential element in several cascades coupling certain antigen receptors to cell responses. Recently we reported that CD33 recruits Syk for its signaling in AML cell lines. In this study, we further investigated the mechanism(s) of Syk engagement in CD33 signaling in primary AML samples. Methods. We investigated 25 primary AML samples for their proliferative response ( 3 H-thymidine incorporation) and biochemical changes (Western blot analysis) to anti-CD33 mAb treatment. Results. Proliferation studies demonstrated that 14 (56%) of AML samples were responsive (R) while 11 (44%) were nonresponsive (n-R) to inhibitory antibody activity. Seven of 25 AML samples (28%) expressed undetectable levels of Syk. However, cells from two of these patients expressed the ZAP-70 protein kinase. In Syk/ZAP-70 + samples, CD33 ligation inhibited proliferation in 70% of cases, while none of the Syk/ZAP-70 − samples was responsive. There were significant biochemical differences between responder and nonresponder AML populations. In responder samples, CD33 ligation induced phosphorylation of CD33 andSyk and formation of the CD33/Syk complex. In nonresponder samples, CD33 was not phosphorylated, and Syk was in complex with the SHP-1 protein phosphatase constitutively. Conclusions. Syk is an important component in the regulation of proliferation in AML cells. The differential response of AML cells to CD33 ligation is associated with the level of the Syk expression.

Evidence from allogeneic hematopoietic stem cell transplantation indicates a possible immune response against leukemia-associated antigens in patients with either acute myeloid leukemia (AML) or chronic myeloid leukemia (CML). However, autologous immune responses are less evident. We have developed a method using sequential modulation of growth factors (SMGF) to generate specific anti-AML T-cells from primary cultures of mononuclear cells (MNCs) from patients with AML. This culture method induces greater degrees of antigen presentation by inducing dendritic cell (DC) differentiation of AML in the presence of autologous lymphocytes, which are then expanded by interleukin (IL)-2 and costimulatory molecule ligation. MNCs consisting of 92.3% +/- 5.1% AML blasts and 3.4% +/- 3.2% CD3+ T-cells were obtained from AML patients (n = 12) and cultured in AIM-V medium with IL-4 and recombinant granulocyte-monocyte colony-stimulating factor. Recombinant IL-2 was added on day 8. On day 21, culture conditions were changed to anti-CD3/anti-CD28 monoclonal antibodies and IL-2. By day 42, 354 +/- 182-fold CD3+ T-cell expansion had occurred. Cytotoxic T-lymphocyte assays demonstrated that these T-cells caused significant lysis of autologous leukemia cells and AML cell lines, but not of cells of other lineages, in an HLA class I-dependent manner. Specific Vbeta subgroups (Vbeta3, -7, and -12a), possibly representing T-cell clones specific to AML-specific antigens, were expanded in the cultures of cells from 3 AML patients. SMGF can be used to induce and expand autologous T-cells with HLA class I-dependent antileukemia potential from the peripheral blood of AML patients. Adoptive transfer of these expanded T-cells to patients is a possible therapeutic approach for further study. Biol Blood Marrow Transplant 2002;8(10):557-68.

Background This study aimed to investigate the status of internal quality control (IQC) for cardiac biomarkers from 2011 to 2016 so that we can have overall knowledge of the precision level of measurements in China and set appropriate precision specifications. Methods Internal quality control data of cardiac biomarkers, including creatinine kinase MB (CK-MB) (μg/L), CK-MB(U/L), myoglobin (Mb), cardiac troponin I (cTnI), cardiac troponin T (cTnT), and homocysteines (HCY), were collected by a web-based external quality assessment (EQA) system. Percentages of laboratories meeting five precision quality specifications for current coefficient of variations (CVs) were calculated. Then, appropriate precision specifications were chosen for these six analytes. Finally, the CVs and IQC practice were further analyzed with different grouping methods. Results The current CVs remained nearly constant for 6 years. cTnT had the highest pass rates every year against five specifications, whereas HCY had the lowest pass rates. Overall, most analytes had a satisfactory performance (pass rates >80%), except for HCY, if one-third TEa or the minimum specification were employed. When the optimal specification was applied, the performance of most analytes was frustrating (pass rates

Background Internal quality control (IQC) plays a key role in the evaluation of precision performance in clinical laboratories. This report aims to present precision status of thyroid hormones immunoassays from 2011 to 2016 in China. Methods Through Clinet-EQA reporting system, IQC information of Triiodothyronine and Thyroxine in the form of free and total (FT3, TT3, FT4, TT4), as well as Thyroid Stimulating Hormone (TSH) were collected from participant laboratories submitting IQC data in February, 2011-2016. For each analyte, current CVs were compared among different years and measurement systems. Percentages of laboratories meeting five allowable imprecision specifications (pass rates) were also calculated. Analysis of IQC practice was conducted to constitute a complete report. Results Current CVs were decreasing significantly but pass rates increasing only for FT3 during 6 years. FT3, TT3, FT4, and TT4 had the highest pass rates comparing with 1/3TEa imprecision specification but TSH had this comparing with minimum imprecision specification derived from biological variation. Constituent ratios of four mainstream measurement systems changed insignificantly. In 2016, precision performance of Abbott and Roche systems were better than Beckman and Siemens systems for all analytes except FT3 had Siemens also better than Beckman. Analysis of IQC practice demonstrated wide variation and great progress in aspects of IQC rules and control frequency. Conclusion With change of IQC practice, only FT3 had precision performance improved in 6 years. However, precision status of five analytes in China was still unsatisfying. Ongoing investigation and improvement of IQC have yet to be achieved.

ObjectivesTo investigate the status of neonatal screening in China in 2013.MethodAll Chinese neonatal screening laboratories were asked to submit information about the number of newborns screened and for which diseases, the rate of acceptable blood spots, and the time between sample collection and delivery to laboratories and reporting.ResultsOf the 202 laboratories, 194 (96%) submitted data. In 2013, 73.6% (142/193) of laboratories testing for phenylketonuria (PKU), 72.7% (141/194) for congenital hypothyroidism (CH), 58.9% (43/73) for glucose-6-phosphate dehydrogenase (G6PD) deficiency and 53.1% (34/64) for congenital adrenal hyperplasia (CAH) screened more than 30,000 newborns. The incidences of PKU, CH, G6PD deficiency, and CAH were 1:12189, 1:2281, 1:44 and 1:6084, respectively. The estimated coverage of screening for PKU, CH, G6PD deficiency, and CAH were 86.3 ∼ 87.5%, 87.9 ∼ 89.1%, 24.0 ∼ 25.0% and 18.9% ∼ 19.9%, respectively. The rates of acceptable blood spots were ≥98% in 161 (87.0%) of 185 laboratories. More than half the laboratories could not achieve a time from collection to reporting of 5 days.ConclusionNeonatal screening has developed rapidly in China but the biggest challenge is still to increase coverage and expand the screening disease panel across the entire country. More government support is needed to make neonatal screening more efficient.

BACKGROUND: This study aims to investigate the imprecision of internal quality control of routine chemistry analytes so that we can have an overall knowledge of imprecision level of measurement in laboratories in China. METHODS: The internal quality control information including two kinds of coefficient of variations (CVs) of 22 analytes were collected via an on‐line questionnaire. Then, the percentages of laboratories meeting the quality requirement for each analyte were calculated according to five imprecision criteria. RESULTS: Among these 22 analytes, creatine kinase got the most satisfied result, the percentages of laboratories whose current CV met 1/3 total allowable error (1/3TEa), 1/4TEa, and the specifications based on biological variation were 99.3%, 95.9%, 99.6%, 99.9%, and 90.6%, respectively, while Na got the lowest satisfaction rate according to these specifications, which were 4.15%, 22.4%, 4.2%, 9.0%, and 1.3%, respectively. From an overall perspective, most of the 22 analytes got a satisfied result when applying the criteria defined by CLIA'88, while only a small amount of laboratories can meet the requirement when applying the specifications derived from biological variation. CONCLUSIONS: There is huge space for us to enhance and substantial effort is needed in improving and implementing quality management in China.

To analyze the results of prenatal karyotype of the external quality assessment program in 2013 in order to provide references and recommendations for improving the capability and performances of karyotype analysis of prenatal screening laboratories.Five lots of quality control cell photos were sent to 500 laboratories. The participants were asked to decide whether the photos have demonstrated any abnormal karyotype and determine the abnormal type. The results should be submitted before the deadline and compared with the standard results to evaluate the performances of the laboratory.One hundred forty three laboratories have returned their karyotype results for the survey. The standard answers were 7,XX,+18, 46,X,i(X)(q10), 46,XY,i(21)(q10) or 46,XY,+21,der(21;21)(q10;q10), 46,XY and 47,XY,+21 in sequential order, which were used to estimate the score of each participant. The pass rates for five lots were 97.9%, 97.2%, 95.8%, 100.0% and 97.9%, respectively. The total pass rate was 97.7%. The error rates were 2.1%, 2.8%, 4.2%, 0 and 2.1%, respectively. The total error rate was 2.3%.Some laboratories did not correctly identify the abnormal karyotypes, while some could not determine the right type of karyotype. The external quality assessment program of prenatal diagnosis of karyotype analysis should be conducted annually in order to improve the capability and performances of karyotype analysis of prenatal screening laboratories.

To analyze the sources and distribution decision limits on reference values of myocardial injury markers based on the feedback results of 150 laboratories which participated the 2012 external quality assessment (EQA) programs in China.Decision limits on myocardial injury markers from EQA programs were analyzed. Data from maternal and child care service center, children's hospital and corporations were excluded. Abnormal values and errors were eliminated. Mean, median, maximum value, minimum value, P2.5 and P97.5 were calculated. Sources of decision limits were summarized and data were filtered and only values obtained according to the reagent manufacturer's instructions as a reference source were used and grouped based on reagent characteristics.According to the surveys on reference interval sources, most of reference sources were derived from reagent manufacturer's instructions and the ratio of each item was: CK-MB (µg/L) 91.30%, Mb (µg/L) 88.44%, cTn-I (µg/L) 86.29%, cTn-T (µg/L) 92.00%, CK-MB (U/L) 70.65%. According to the surveys, the mean and median values of each test parameter were close to each other (CK-MB:3.74, 4.94 µg/L;Mb:76.09, 72.00 µg/L; CTn-I:0.06,0.09 µg/L;cTn-T:0.01, 0.02 µg/L;CK-MB:23.00, 25.00 U/L) . There were significant variations on P2.5 and P97.5 distribution ranges: CK-MB 2.48-7.09 µg/L, Mb 46.00-140.03 µg/L, cTn-I 0.01-1.68 µg/L, cTn-T 0.01-12.61 µg/L, CK-MB 6.00-30.60 U/L. According to the surveys on using various reagents, there were also significant variations on P2.5 and P97.5 reagent distributions.The decision limit of the mean and median for each test item is close to each other while the P2.5 and P97.5 distribution of each test item consistency is poor. There is therefore an urgent need to establish an uniform reference values for cardiac markers in China.

Background This study aimed to evaluate whether the quality performance of clinical laboratories in China has been greatly improved and whether Internal Quality Control (IQC) practice of HbA1c has also been changed since National Center for Clinical Laboratories (NCCL) of China organized laboratories to report IQC data for HbA1c in 2012. Methods Internal Quality Control information of 306 External Quality Assessment (EQA) participant laboratories which kept reporting IQC data in February from 2012 to 2016 were collected by Web-based EQA system. Then percentages of laboratories meeting four different imprecision specifications for current coefficient of variations (CVs) of HbA1c measurements were calculated. Finally, we comprehensively analyzed analytical systems and IQC practice of HbA1c measurements. Results The current CVs of HbA1c tests have decreased significantly from 2012 to 2016. And percentages of laboratories meeting four imprecision specifications for CVs all showed the increasing tendency year by year. As for analytical system, 52.1% (159/306) laboratories changed their systems with the change in principle of assay. And many laboratories began to use cation exchange high-performance liquid chromatography (CE-HPLC) instead of Immunoturbidimetry, because CE-HPLC owed a lower intra-laboratory CVs. The data of IQC practice, such as IQC rules and frequency, also showed significant variability among years with overall tendency of meeting requirements. Conclusion The imprecision performance of HbA1c tests has been improved in these 5 years with the change in IQC practice, but it is still disappointing in China. Therefore, laboratories should actively find existing problems and take action to promote performance of HbA1c measurements.

Objective To develop a novel method of generating multiple autologous acute myeloid leukemia (AML) reactive T-cell lines as a step toward adoptive immunotherapy for AML. Materials and Methods AML peripheral blood mononuclear cells (MNC), including >90% AML blasts and 1% to 3% T cells, were seeded in limiting dilution culture in which AML blasts were induced to undergo dendritic cell (DC) differentiation. T cells were primed and activated with the addition of a cytokine combination. Results Highly reactive anti-AML T-cell lines (both CD4 + and CD8 + ) were generated, selected, and expanded. The estimated average frequency of AML-reactive T cells or precursors was 6 ± 3/1,000,000 AML peripheral blood mononuclear cells (n = 11). Robust intracellular interferon-γ (IFN-γ) release from T-cell lines was demonstrated by flow cytometry after stimulation by autologous AML cells, but not an autologous B-lymphoblastoid cell line (LCL). These T-cell lines caused specific lysis of autologous AML cells, but not autologous LCL or allogeneic AML cells, and they depleted autologous AML colony-forming cells (CFC), but not normal CFC. Most CD4 + T-cell lines exerted strong proapoptotic effects on AML cells. AML cell apoptosis by CD4 + T-cell lines correlated with IFN-γ secretion. Conclusion This study demonstrates a methodology for generating large numbers of AML-reactive cytotoxic T cell lines (either class I or II restricted) that may be useful clinically in adoptive immunotherapy. This study also provides estimates of AML-reactive T-cell frequency in patients with AML.

To investigate the effects of curcumin (Cur) on p210(bcr/abl) level in K562 cells, and the relationship between these effects and the molecular chaperone functions of heat shock protein 90 (Hsp90).Flow cytometry and Western blot were used to examine the abundance of p210(bcr/abl), Hsp90, p23, Hsp70, and p60(Hop) in K562 cells treated with Cur. Reverse transcription polymerase chain reaction (RT-PCR) was used to determine the bcr-abl mRNA level in K562 cells treated with Cur. After co-immunoprecipitation of p210(bcr/abl) and its molecular chaperones, the immunoprecipitate was then subjected to Western blot analysis with anti-Hsp90, anti-Hsp70, anti-p23, and anti- p60(Hop)mAb.An exposure of K562 cells to Cur produced time-dependent down-regulation of p210(bcr/abl), the inhibition rate of p210(bcr/abl) in K562 cells determined by flow cytometry after treatment with Cur 27.2 micromol/L for 1 h, 6 h, 12 h and 24 h was 31.2%, 63.7%, 81.3% and 94.5%, respectively. In contrast, Cur had almost no influence on bcr-abl mRNA level. Treatment with Cur for 24 h reduced the association of p210(bcr/abl) with Hsp90/p23 complex, while increasing the association of p210(bcr/abl) with Hsp70/ p60(Hop) complex; however, the total protein abundance of Hsp90, p23, and p60(Hop) in K562 cells had no apparent change, while Hsp70 increased greatly.Down-regulation of p210(bcr/abl) by Cur involves dissociating the binding of p210(bcr/abl) with Hsp90/p23 complex. In contrast, the association of p210(bcr/abl) with Hsp70/ p60(Hop) complex increased.

Purpose: Gastrin-releasing peptide (GRP) is a growth factor for small cell lung cancer (SCLC). GRP belongs to the bombesin peptide family and has significant homology to bombesin. We constructed a bispecific molecule, OKT3xAntag2, by conjugating a monoclonal antibody OKT3 (anti-CD3) with a bombesin/GRP antagonist (Antag2) and evaluated cytotoxicity against SCLC cells. Experimental Design: We tested binding of the bispecific molecule to SCLC cell lines and T cells by flow cytometry, antibody-dependent cellular cytotoxicity (ADCC) of SCLC cells in vitro and in a murine SCLC xenograft model. We studied SCLC apoptosis and necrosis during ADCC and the activity and cleavage of caspase-3, caspase-9, and poly(ADP-ribose) polymerase (PARP). Results: The bispecific molecule functions as a cross-linker between T cells and SCLC cells, induces T cell activation, and mediates ADCC of SCLC cells; 40% to 80% growth inhibition of SCLC cells mediated by the bispecific molecule at low effector to target cell ratios was achieved. Activation of T cells by the bispecific molecule resulted in significant increases in IFNγ production and apoptosis and necrosis of SCLC cells associated with cleavage of PARP and caspase-3. Targeted immunotherapy with the bispecific molecule–armed human T cells significantly reduced SCLC tumor burdens in a mouse model. Conclusion: The bispecific molecule OKT3xAntag2 mediates growth inhibition and apoptosis of SCLC cells by activated T cells through activation and cleavage of caspase-3 and PARP in vitro and in vivo. Clinical trials of this bispecific molecule through adoptive transfer of ex vivo activated T cells in GRP receptor–positive tumors, such as SCLC, are warranted.

The therapeutic potential of the IgM complement-fixing murine monoclonal antibody (mAb) PM-81 (anti-CD15) against acute myeloid leukemia (AML was assessed in a SCID/hu leukemia model. Intraperitoneal (i.p.) injection of NB4 leukemia cells resulted in aggressive growth of leukemia cells in the peritoneal cavity of irradiated SCID/CB-17 mice. Flow cytometric analysis of human CD15, 33 and 45 expression, as well as cytologic examination, revealed that leukemia cells disseminated into the peripheral blood and multiple tissues of the mice. The approximately linear relationship between the injected leukemia cells and the subsequent leukemia cell proliferation provided a reliable model for monitoring the therapeutic effects of immunotherapy. Intraperitoneal injection of the mAb PM-81 markedly suppressed leukemia cell growth in this SCID/leukemia model. Most of the untreated mice died within 35–50 days of leukemia cell inoculation. Four weeks after inoculation of NB4 cells, five of nine mAb PM-81 treated mice had no solid tumor growth and six of nine had no detectable peritoneal exudate leukemia cells as determined by flow cytometry. In contrast, 100% of the mice in the untreated or control mAb groups were found to have both solid and peritoneal leukemia growth. In further experiments designed to evaluate the effects of therapy on survival, 50% ( 4 8 ) of PM-81 treated mice survived to 150 days, and had no detectable solid or suspension leukemia cells detectable at necropsy. In contrast, the median survival of untreated or negative control antibody-treated mice was 40 days (comparison to PM-81 treated; p = 0.006 and p = 0.03, respectively). The mechanism of leukemia cell suppression is not likely due to complement fixation since we could not demonstrate in vitro any cytotoxicity mediated by SCID mouse plasma. Further study is required to understand the mechanism of the antileukemia effect of PM-81 in this model.

Bispecific antibodies recognizing tumour-associated antigens and trigger molecules expressed on immune effector cells have been shown to redirect cytotoxicity of several types of peripheral blood cells against relevant tumour targets. Among various effector cells, natural killer (NK) cells appear to play a role in defence against leukaemia. Here we report the successful chemical conjugation of monoclonal antibodies to CD33 and CD16 to create a bispecific antibody (BsAb 251 x 3G8). This bispecific antibody is capable of augmenting the killing of otherwise resistant leukaemia cells by peripheral blood lymphocytes (PBL), purified resting NK (R-NK) cells, and activated NK (A-NK) cells. BsAb 251 x 3G8 may play a role in the therapy of acute myeloid leukaemia (AML) through redirecting the cytotoxic activity of endogenous or adoptively transferred NK cells.