Long-term external quality assessment program for CD4+ T-lymphocyte enumeration in Thailand

The accuracy and reliability of CD4+ T-lymphocyte enumeration are crucial for HIV healthcare management. Thailand’s CD4 external quality assessment (EQA) program was implemented in 2003. This program aims to improve the quality of CD4 testing by providing the quality control material, remedial action, and technical training. In this article, the overall longitudinal performance of the participating laboratories from 2003 to 2015 is reported. The EQA blood samples were sent to the participating laboratories in Thailand and other Southeast Asian (SEA) countries. The participants were requested to enumerate CD3+ and CD4+ T-lymphocytes. The trimmed mean, standard deviation (SD), coefficient of variation (CV), and standard deviation index (SDI) of both fraction and number were calculated from the returned data. Our result indicated a continuously increasing number of participants. The response rate of 72 trials was 98 %. The outlier rates for the fraction and number of CD4+ T-lymphocytes were 3.3 % and 3.4 %, respectively. The average CV of the fraction and number of CD4+ T-lymphocytes was 7.1 % and 9.4 %, respectively. In addition, the analysis of our current EQA trial no. 072 revealed that more than 98 % of participating laboratories had SDI values between ?2 and +2 for both fraction and number of CD3+ and CD4+ T-lymphocytes. In conclusion, the implementation of the CD4 EQA program in Thailand and other SEA countries has led to a reduction in the variability of the results of the CD4 count between laboratories and a continuous improvement in laboratory performance.     

External quality assessment of point-of-care CD4 testing in Thailand and Southeast Asia

Accreditation and Quality Assurance - In recent years, the use of point-of-care (POC) CD4 testing technologies has increased dramatically in Thailand and other Southeast Asian (SEA) countries....     

Toward standardization of quality assessment in laboratory medicine by using the same matrix samples for both internal and external quality assessments

The main purpose of quality assurance procedures in clinical laboratories is to ensure that test results are appropriate to maintain excellence in the diagnosis, monitoring, and treatment of disease. However, in current practice, no standardized procedure or frequency for the evaluation of methods exists, particularly in external quality assessment. Furthermore, different quality control materials are typically used for internal and external quality assessment. To overcome these discrepancies, we used samples with the same matrix for both internal and external quality assessments of a group test performed in our laboratory. We then calculated total error using real bias (target value obtained by reference method) and the imprecision of each test and compared our results with the total error allowable, derived from biological variation data. We suggest that the strategy of using the same matrix samples for both internal and external quality assessment is cost-effective, can be readily used by staff, and will facilitate the standardization of quality control in clinical laboratories.     

Surveys on the accreditation of providers of proficiency testing and external quality assessment schemes

Two surveys among providers of proficiency testing (PT) and external quality assessment (EQA) schemes were carried out during 2004 and 2005. The main objectives were to explore the current status of accreditation/certification and collect the providers’ views. Information based on the response from 160 providers in 32 countries reveals a strong tendency towards accreditation of PT/EQA. It is shown that this type of accreditation is based on several combinations of normative documents, hence illustrating a lack of harmonisation of national accreditation bodies. The surveys also show that schemes are operated under considerably different conditions and that providers’ competence may or may not be underpinned by other certification and/or accreditation. This paper elaborates on a number of issues related to PT/EQA accreditation, including customers’ views, normative documents, providers’ experience from the accreditation process, views expressed by international organisations, and effects of accreditation on participation fees, quality and availability.     

Developments in external quality assessment for clinical microbiology laboratories

There has been considerable development in external quality assessment programs for clinical microbiology laboratories. Over the last 5 years samples have progressively become more clinically realistic and relevant. Programs are tending to produce more samples that look and act like real samples. As programs change to a broader range of challenge targets, the observed rate of errors is substantially greater for pre-analytic and post-analytic challenges than with analytic phase challenges. Rather than focusing only on analytic phase procedures, schemes should target to a greater extent pre-analytic and post-analytic aspects of the laboratory cycle.Fourth EURACHEM/CITAC/EQUALM International Workshop on Proficiency Testing in Analytical Chemistry, Microbiology and Laboratory Medicine, UK, February 2003.     

Application of ISO 13528 robust statistical method for external quality assessment of blood glucose measurements in China

External quality assessment (EQA) including extensive participants often result in abnormal distribution. Robust statistical methods are insensitive to slight deviation for a given probability model and can estimate the population parameters utilizing robust algorithm. The aim of this study was to evaluate the blood glucose testing performance of EQA in China using ISO 13528 robust statistical method. A total of 4571 participants measured two lots of materials at different concentrations and submitted the results through the Clinet-EQA reporting system V1.5. Data were collected and analyzed by Clinet-EQA evaluating system and SPSS 13.0. Participants were classified by method principles they adopted. Acceptability of relative differences between each result and robust mean of the group was calculated using biological variation desirable quality specification for allowable total error. The kernel density plots and Youden Plot were also applied to show the distribution of z-scores. The robust means of the two lots were 6.31 mmol/L and 15.71 mmol/L, and the robust standard deviations were 0.23 mmol/L and 0.5 mmol/L, respectively. Acceptability of relative differences was more than 90 % among all groups. About 91.5% and 89.7 % of the participants out of the two lots fulfilled the condition of |z| ≤ 2. In addition, 87.2 % participants fell inside the confidence ellipse for probability levels of 95 % in Youden Plot. According to the statistical results, we can conclude that most participants are qualified for performing blood glucose test and ISO 13528 robust statistical method is appropriate for EQA result analysis.     

Instability of mercury in specimens of human urine for external quality assessment

An under-recovery of inorganic mercury added to urine and a wide range of results is observed in quality assessment schemes (EQAS) for trace elements. Furthermore, the under-recoveries are inconsistent suggesting features associated with the urine matrix may make the mercury unavailable for measurement. To investigate the instability of mercury in urine the following experiments were set up: (1) a sample of Hg²⁺ in water with various ‘stabilizers’ added was sent to UK external quality assessment scheme participants. (2) Urine was collected from volunteers who also completed a 3-day food diary. Hg, Ca, Mg, Se, uric acid, phosphate, creatinine, reducing substances and protein were measured. Inorganic mercury was spiked into the urine, stabilizers were added and the mercury determined following storage. The results confirmed under-recovery of mercury in association with the urine matrix. Further investigations of how urinary components affect the measurement of mercury are necessary.     

Lyophilized human whole blood for internal and external quality assurance of lead in blood assays

Summary Lyophilized human whole blood control material containing lead was prepared for internal and external quality assurance to evaluate and improve the analytical performance of lead.The samples were prepared in four different concentrations from outdated human whole blood stabilized with glucose and a citrate/phosphate buffer and provided under clean room conditions to avoid contamination. The lyophilized samples are easy to reconstitute with water. The materials were evaluated according to a statistical model.The lead concentrations in the specimens are close to blood lead levels usually following environmental and industrial exposure, particular in occupational health to the control of lead exposure at work regulations. The materials are available to the commercial as well as the scientific community.The Danish External Quality Assessment Scheme (DEQAS) for lead in blood is intended to complement the internal quality control for the Danish laboratories assaying lead in blood. During 1990 the scheme will be tested in an international external assessment scheme.     

Reference materials and analytical standards to stimulate improved laboratory performance: Experience from the external quality assessment scheme for trace elements in biological samples

The performance of a large number of laboratories measuring trace elements in biological fluids has been monitored over many years by examination of their results in the Guildford external quality assessment scheme. Specific experiences of the UK trace elements reference laboratories have been used to stimulate improvements in performance of other participants in the scheme. The key features of these initiatives were: specially prepared reference materials, used as internal quality control specimens within a common procedure, contributed to accuracy control; proposed standards of satisfactory and unacceptable analytical performance associated with a new system for scoring; regular non-threatening open discussion of performance with interested colleagues. The impact of these features is illustrated with reference to measurements of Al and Zn in serum and Pb in whole blood.     

A human mutant CD4 molecule resistant to HIV-1 binding restores helper T-lymphocyte functions in murine CD4-deficient mice

CD4 is a cell surface glycoprotein that acts as a co-receptor for the T cell antigen receptor by binding to a non-polymorphic portion of MHC molecules. CD4 also functions as a receptor for human immunodeficiency virus type-I (HIV-1) because the viral envelope glycoprotein gp120 binds to CD4 with a high affinity. We have previously demonstrated that introduction of mutations into CD4 abolished the binding of gp120 and prevented HIV-1 from entering cells and spreading. However, whether introduction of such mutations into CD4 causes decreased binding to MHC and loss of function is yet to be determined. We generated transgenic mouse lines by injecting a mutant human CD4 (muthCD4) gene under a murine CD4 enhancer/promoter to ensure tissue and stage specific expression. To exclude the influence of endogenous murine CD4, transgenic mice were crossed with murine CD4-targeted mice to produce muthCD4 transgenic mice lacking endogenous CD4 (muthCD4TG/KO mice). In these mice, T lymphocytes expressing muthCD4 expanded and matured in the thymus and were present in the spleen and lymph nodes. They also activated B cells to mount an antibody response to a T-dependent antigen. The results from this study suggest that a human variant of CD4 modified to be resistant to HIV-1 binding can rescue the signaling for T cell development in the thymus in vivo, having helper T cell functions. Thus, further characterization of muthCD4 molecules should open the way to new HIV treatment modalities.     

Product angular distribution for the H + CD4 --> HD + CD3 reaction

Using an analytical potential energy surface previously developed by our group, namely PES-2002, we analyzed the gas-phase reaction between a hydrogen atom and perdeuterated methane. We studied the effect of quasiclassical trajectory (QCT) and reduced dimensionality quantum-scattering (QM) calculations, with their respective limitations, on CD3 product angular distributions in the collision energy range 16.1-46.1 kcal x mol(-1). It was found that at low collision energy, 16.1 kcal x mol(-1), both the QCT and QM calculations yielded forward scattered CD3 products, i.e., a rebound mechanism. However, at high energies only the QM calculations on the PES-2002 surface reproduced the angular scattering found experimentally.     

Maintenance of CD8+ T-cell anergy by CD4+CD25+ regulatory T cells in chronic graft-versus-host disease

In a murine model of systemic lupus erythematosus (SLE)-like chronic graft-versus-host disease (cGVHD), donor CD8+ T cells rapidly fall into anergy to host cells, while donor CD4+ T cells hyperactivate B cells and break B-cell tolerance to self-Ags in the recipient mouse. The functional recovery of donor CD8+ T cells can result in the conversion of cGVHD to acute GVHD (aGVHD), indicating that donor CD8+ T-cell anergy is a restriction factor in the development of cGVHD. In this report, we present evidence that donor CD4+CD25+ regulatory T cells (Treg cells) are critical in maintaining the donor CD8+ T-cell anergy and thus suppressing the development of aGVHD in mice that are naturally prone to cGVHD. Our results provide a novel insight into the role of Treg cells in determining cGVHD versus aGVHD.     

Developing external quality assessment programmes for primary health: care level in resource limited countries

Laboratory services are an essential component of health care delivery in tropical countries and play a vital role in improving the accuracy of clinical diagnosis and the investigation of disease outbreaks. In developing countries, laboratories face numerous constraints to providing quality services, including poor selection of techniques, difficulties in equipment availability and maintenance, and shortages of supplies, staffing and supervision. Currently in the eastern African countries (Kenya, Tanzania and Uganda), internal quality control procedures are inconsistently carried out in most laboratories. National External Quality Assessment Schemes (EQAS) have been established in all countries addressing a limited number of tests, but are constrained by difficulties of sustainability and poor coverage. The Laboratory Programme of the African Medical and Research Foundation (AMREF) has been operating a simple EQAS for primary heath care laboratories since 1993. Tests addressed are those carried out in primary health care laboratories in eastern Africa. A total of 81 laboratories from 5 countries in the eastern African region have participated in the scheme since 1993 and 9 distributions were submitted since the start of the scheme. No laboratory participated in all distributions; 24 (30%) laboratories participated in 4 or more distributions. Of these, the hospital laboratories in Kenya and Tanzania showed improved average mean scores between the first two and subsequent distributions. The educational benefit of participation in the scheme is emphasised. There was an overall low rate of participation of laboratories (35%) once the scheme was expanded to include laboratories outside direct AMREF project activities. Contributing factors include shortages of staff and lack of time in busy rural laboratories, together with difficulties in communication; however, the voluntary nature and lack of appreciation of the benefits of participation may also play a role. To increase participation in the scheme and to address the quality of laboratory services throughout the region, AMREF is currently developing a Regional EQAS in collaboration with the Ministries of Health of Kenya, Tanzania and Uganda, in affiliation with the World Health Organisation (WHO). The approaches used to establish reference values for haemoglobin samples are discussed. The scheme has also been utilised to examine the performance of different techniques for haemoglobin estimation, demonstrating the inaccuracy of the visual comparator methods. Received: 13 April 2002 Accepted: 5 July 2002     

The multivariate coefficient of variation for comparing serum protein electrophoresis techniques in external quality assessment schemes

External quality assessment (EQA) schemes are national or transnational programmes designed to control the analytical performance of clinical laboratories and to maintain inter-laboratory variability within acceptable limits. In such EQA programmes, participants are usually grouped by the type of assay technique/equipment they use. The coefficient of variation (CV) is a simple tool for comparing the inter-laboratory reproducibility of such techniques: the lower the CV, the better the analytical performance. Serum protein electrophoresis, a laboratory test profile consisting of five fractions (albumin, α ₁, α ₂, β and γ globulins) summing up to 100% of total proteins, can also be assayed in different ways depending on the media or the analytical principle. We propose a multivariate CV for comparing the performance of electrophoretic techniques in EQA, thus extending the univariate CV concept. First, the compositional nature of electrophoretic data requires a one-to-one transformation from the five-dimensional to the four-dimensional space. Next, robust estimations of the mean and the covariance matrix are needed to avoid the effect of outliers. The new approach is illustrated on electrophoretic datasets from the French and Belgian national EQA programmes.     

Evidence for vibrational excitation in H+ + CH4, CD4 collisions by means of a surface-hopping mechanism

The energy-loss spectra for H⁺ scattered at small angles from CH₄ and CD₄ display an unusual bimodal character and extremely large average excitation energies. We propose a potential energy surface-hopping mechanism to account for the novel features of this system.     

Mode correlation of product pairs in the reaction OH+CD4-->HOD+CD3

The hydrogen abstraction reaction from methane by a hydroxyl radical produces two polyatomic molecules. Each product has several vibrational modes that characterize distinct, concerted motions of the constituent atoms of the molecule. This communication describes the first measurement that maps out the coincident information on how the mode of excitation of one product varies with that of the other co-product. Such information on mode correlation of product pairs is particularly appealing in that it provides intuitively a glimpse of the reaction paths by which the chemical transformation occurs.     

AIDS病毒与CD^＋4靶细胞检测方法的研究

本文对ＡＩＤＳ病毒原及其靶细胞检测方法的研究进展了较系统的评述。深入分析了ｇｐ１２０，ＣＤ４受体分子与小肽的相互作用。并给出了抗ＡＩＤＳ病毒药物分析 方法的最新研究方向。     

人外周血CD4+IL-21+记忆T细胞的特征

目的:探讨人外周血中白细胞介索21(IL-21)的产生细胞及其特征.方法:分离人外周血单个核细胞(PBMC),分为不刺激或anti-CD3(OKT3)、OKT3+anti-CD28、PMA+ionomycin刺激四个组,流式细胞术(FCM)检测产生IL-21的细胞亚群.PMA+ionomycin刺激PBMC、纯化CD4+、CD4+CD45RA-、CD4+CD45RA+细胞、脐带血单个核细胞(CB-MC),FCM分析产生IL-21细胞的表型特征和IL-21与Th1、Th2、Th17和Th22细胞因子之间的关系.结果:与OKT3、OKT3+anti-CD28相比,PMA+ionomycin能诱导最高量的IL-21产生.产生IL-21的主要细胞为CD4+T细胞,少数CD8+T细胞.CD4+IL-21+T细胞表达CD45RO,不表达CD45RA,其中部分细胞表达CCR6、CCR7或CXCR5.CD4+CD45RA-细胞表达IL-21远高于CIM+CD45RA+细胞.进一步研究表明,PBMC产生IL-21,而CBMC不产生.此外,大约24%的CD4+IL-21+细胞表达IFN-γ,小于10%CD4+IL21+细胞表达IL-4、IL-17或IL-22.结论:人PBMC在多克隆刺激的条件下,可以诱导IL-21的产生.产生IL-21的主要细胞亚群具有记忆CD4+T细胞的表型.其中一部分CD4+IL-21+T细胞的表型独立于Th1、Th2、Th17和Th22细胞亚群.