The Center for HIV/AIDS Vaccine Immunology (CHAVI) multi-site quality assurance program for cryopreserved human peripheral blood mononuclear cells.
ABSTRACT: The Center for HIV/AIDS Vaccine Immunology (CHAVI) consortium was established to determine the host and virus factors associated with HIV transmission, infection and containment of virus replication, with the goal of advancing the development of an HIV protective vaccine. Studies to meet this goal required the use of cryopreserved Peripheral Blood Mononuclear Cell (PBMC) specimens, and therefore it was imperative that a quality assurance (QA) oversight program be developed to monitor PBMC samples obtained from study participants at multiple international sites. Nine site-affiliated laboratories in Africa and the USA collected and processed PBMCs, and cryopreserved PBMC were shipped to CHAVI repositories in Africa and the USA for long-term storage. A three-stage program was designed, based on Good Clinical Laboratory Practices (GCLP), to monitor PBMC integrity at each step of this process. The first stage evaluated the integrity of fresh PBMCs for initial viability, overall yield, and processing time at the site-affiliated laboratories (Stage 1); for the second stage, the repositories determined post-thaw viability and cell recovery of cryopreserved PBMC, received from the site-affiliated laboratories (Stage 2); the third stage assessed the long-term specimen storage at each repository (Stage 3). Overall, the CHAVI PBMC QA oversight program results highlight the relative importance of each of these stages to the ultimate goal of preserving specimen integrity from peripheral blood collection to long-term repository storage.
Project description:A large repository of cryopreserved peripheral blood mononuclear cells (PBMCs) samples was created to provide laboratories testing the specimens from human immunodeficiency virus-1 (HIV-1) vaccine clinical trials the material for assay development, optimization, and validation. One hundred thirty-one PBMC samples were collected using leukapheresis procedure between 2007 and 2013 by the Comprehensive T cell Vaccine Immune Monitoring Consortium core repository. The donors included 83 human immunodeficiency virus-1 (HIV-1) seronegative and 32 HIV-1 seropositive subjects. The samples were extensively characterized for the ability of T cell subsets to respond to recall viral antigens including cytomegalovirus, Epstein-Barr virus, influenza virus, and HIV-1 using Interferon-gamma (IFN-?) enzyme linked immunospot (ELISpot) and IFN-?/interleukin 2 (IL-2) intracellular cytokine staining (ICS) assays. A subset of samples was evaluated over time to determine the integrity of the cryopreserved samples in relation to recovery, viability, and functionality. The principal results of our study demonstrate that viable and functional cells were consistently recovered from the cryopreserved samples. Therefore, we determined that this repository of large size cryopreserved cellular samples constitutes a unique resource for laboratories that are involved in optimization and validation of assays to evaluate T, B, and NK cellular functions in the context of clinical trials.
Project description:<h4>Introduction</h4>The metabolomics quality assurance and quality control consortium (mQACC) evolved from the recognized need for a community-wide consensus on improving and systematizing quality assurance (QA) and quality control (QC) practices for untargeted metabolomics.<h4>Objectives</h4>In this work, we sought to identify and share the common and divergent QA and QC practices amongst mQACC members and collaborators who use liquid chromatography-mass spectrometry (LC-MS) in untargeted metabolomics.<h4>Methods</h4>All authors voluntarily participated in this collaborative research project by providing the details of and insights into the QA and QC practices used in their laboratories. This sharing was enabled via a six-page questionnaire composed of over 120 questions and comment fields which was developed as part of this work and has proved the basis for ongoing mQACC outreach.<h4>Results</h4>For QA, many laboratories reported documenting maintenance, calibration and tuning (82%); having established data storage and archival processes (71%); depositing data in public repositories (55%); having standard operating procedures (SOPs) in place for all laboratory processes (68%) and training staff on laboratory processes (55%). For QC, universal practices included using system suitability procedures (100%) and using a robust system of identification (Metabolomics Standards Initiative level 1 identification standards) for at least some of the detected compounds. Most laboratories used QC samples (>86%); used internal standards (91%); used a designated analytical acquisition template with randomized experimental samples (91%); and manually reviewed peak integration following data acquisition (86%). A minority of laboratories included technical replicates of experimental samples in their workflows (36%).<h4>Conclusions</h4>Although the 23 contributors were researchers with diverse and international backgrounds from academia, industry and government, they are not necessarily representative of the worldwide pool of practitioners due to the recruitment method for participants and its voluntary nature. However, both questionnaire and the findings presented here have already informed and led other data gathering efforts by mQACC at conferences and other outreach activities and will continue to evolve in order to guide discussions for recommendations of best practices within the community and to establish internationally agreed upon reporting standards. We very much welcome further feedback from readers of this article.
Project description:Advancements in multi-colour fluorescence activated cell sorting (FACS) panel warrant harmonized procedures to obtain comparable data between various laboratories. The intensifying clinical exploration of Natural Killer (NK) cell-based immunotherapy demands standardized and harmonized NK cell FACS panels and acquisition protocols. Eight colour FACS panels were designed to study human NK cell phenotype and function within peripheral blood mononuclear cells (PBMC). The panels were designed around fixed backbone markers and channels, covering antigens for non-NK lineage exclusion (CD3, TCR??, CD19, CD14, SYTOX® Blue) and NK cell selection (CD45, CD56, CD16), complemented with variable drop-in markers/channels to study NK cell phenotype (NKG2A, NKG2C, NKG2D and KIR2D) or NK cell function and activation (CD25, NKp44 and CD107a). Harmonized FACS set-up and data analysis for three different flow cytometers has been established, leading to highly comparable and reproducible data sets using the same PBMC reference samples (n?=?6). Further studies of NK cells in fresh or cryopreserved PBMC samples (n?=?12) confirmed that freezing and thawing of PBMC samples did not significantly affect NK phenotype or function. In conclusion, our data demonstrate that cryopreserved PBMC samples analysed by standardized FACS panels and harmonized analysis protocols will generate highly reliable data sets for multi-center clinical trials under validated conditions.
Project description:<h4>Background</h4>Cytokine flow cytometry (CFC) or intracellular cytokine staining (ICS) can quantitate antigen-specific T cell responses in settings such as experimental vaccination. Standardization of ICS among laboratories performing vaccine studies would provide a common platform by which to compare the immunogenicity of different vaccine candidates across multiple international organizations conducting clinical trials. As such, a study was carried out among several laboratories involved in HIV clinical trials, to define the inter-lab precision of ICS using various sample types, and using a common protocol for each experiment (see additional files online).<h4>Results</h4>Three sample types (activated, fixed, and frozen whole blood; fresh whole blood; and cryopreserved PBMC) were shipped to various sites, where ICS assays using cytomegalovirus (CMV) pp65 peptide mix or control antigens were performed in parallel in 96-well plates. For one experiment, antigens and antibody cocktails were lyophilised into 96-well plates to simplify and standardize the assay setup. Results ((CD4+)cytokine+ cells and (CD8+)cytokine+ cells) were determined by each site. Raw data were also sent to a central site for batch analysis with a dynamic gating template. Mean inter-laboratory coefficient of variation (C.V.) ranged from 17-44% depending upon the sample type and analysis method. Cryopreserved peripheral blood mononuclear cells (PBMC) yielded lower inter-lab C.V.'s than whole blood. Centralized analysis (using a dynamic gating template) reduced the inter-lab C.V. by 5-20%, depending upon the experiment. The inter-lab C.V. was lowest (18-24%) for samples with a mean of > 0.5% IFNgamma + T cells, and highest (57-82%) for samples with a mean of < 0.1% IFNgamma + cells.<h4>Conclusion</h4>ICS assays can be performed by multiple laboratories using a common protocol with good inter-laboratory precision, which improves as the frequency of responding cells increases. Cryopreserved PBMC may yield slightly more consistent results than shipped whole blood. Analysis, particularly gating, is a significant source of variability, and can be reduced by centralized analysis and/or use of a standardized dynamic gating template. Use of pre-aliquoted lyophilized reagents for stimulation and staining can provide further standardization to these assays.
Project description:BACKGROUND:The increasing number of clinical trials and their complexity make it challenging to detect and identify clinical quality issues timely. Despite extensive sponsor audit programs and monitoring activities, issues related to data integrity, safety, sponsor oversight and patient consent have recurring audit and inspection findings. Recent developments in data management and IT systems allow statistical modeling to provide insights to clinical Quality Assurance (QA) professionals to help mitigate some of the key clinical quality issues more holistically and efficiently. METHODS:We used findings from a curated data set from Roche/Genentech operational and quality assurance study data, covering a span of 8 years (2011-2018) and grouped them into 5 clinical impact factor categories, for which we modeled the risk with a logistic regression using hand crafted features. RESULTS:We were able to train 5 interpretable, cross-validated models with several distinguished risk factors, many of which confirmed field observations of our quality professionals. Our models were able to reliably predict a decrease in risk by 12-44%, with 2-8 coefficients each, despite a low signal-to-noise ratio in our data set. CONCLUSION:We proposed a modeling strategy that could provide insights to clinical QA professionals to help them mitigate key clinical quality issues (e.g., safety, consent, data integrity) in a more sustained data-driven way, thus turning the traditional reactive approach to a more proactive monitoring and alerting approach. Also, we are calling for cross-sponsors collaborations and data sharing to improve and further validate the use of statistical models in clinical QA.
Project description:Quality assurance and quality control (QA/QC) procedures are vital to good biorepository management. The National Eye Institute (NEI) core CLIA-certified laboratory of the eyeGENE(®) Network receives blood from individuals with inherited eye conditions and isolates DNA for clinical genetic diagnostic testing and research. Clinical genetic test results are returned to the affected individuals, making it imperative that sample integrity is preserved throughout laboratory processing. A clinically validated, short tandem repeat (STR)-based approach, termed Sample Confirmation Testing (SCT), was developed to ensure that no significant laboratory errors occurred during processing. SCT uses modified protocols from commercial kits to create and compare STR profiles for each participant's original blood and derived DNA. This QA/QC procedure has been performed on 47% of the more than 6000 participants in the eyeGENE Biorepository and has identified significant laboratory errors in 0.4% of samples tested. SCT improves the quality of the data returned to affected individuals and the data distributed to researchers using eyeGENE samples by ensuring the integrity of the samples and aiding in curation of the biorepository. This approach serves as a model for other repositories to improve sample quality and management procedures.
Project description:In recent years, the number and scope of outsourced activities in the pharmaceutical industry have increased heavily. In addition, also the type of outsourcing has changed significantly in that time. This raises the question of whether and how sponsors retain the capability to select and to control the contract research organizations (CROs) involved and what expertise still has to be present in the development department as well as other relevant departments to ensure adequate oversight, also in line with the expectations of regulators and health authorities. In order to answer these questions, a survey was conducted among the German vfa member companies. The survey describes the latest developments and experiences in outsourcing by 18 German vfa member companies. It concentrates on measures how to implement Quality Assurance (QA) when performing outsourced clinical studies. This study shows that the majority of companies apply a full-outsourcing, preferred-provider model of clinical trial services, with the clinical research department playing the major role in this process. A large amount of guiding documents, processes and tools are used to ensure an adequate oversight of the services performed by the CRO(s). Finally the guiding principles for all oversight processes should be transparent communication, a clearly established expectation for quality, a precise definition of accountability and responsibility while avoiding silo mentality, and a comprehensive documentation of the oversight's evidence. For globally acting and outsourcing sponsors, oversight processes need to be aligned with regards to local and global perspectives. This survey shows that the current implementation of oversight processes in the participating companies covers all relevant areas to ensure highest quality and integrity of the data produced by the outsourced clinical trial.
Project description:Previous attempts to maintain human spermatozoa without freezing were based on short-term storage in component-rich medium and led to fast decline in motility and increased incidence of chromosome breaks. Here we report a new method in which sperm are maintained without freezing in an electrolyte-free medium (EFM) composed of glucose and bovine serum albumin. Human sperm were stored in EFM or human tubal fluid medium (HTFM) or were cryopreserved, and their motility, viability, and DNA integrity were examined at different intervals. Cryopreservation led to significant decline in sperm motility and viability and induced DNA fragmentation. Sperm stored in EFM maintained motility and viability for up to 4 and 7 wk, respectively, much longer than sperm stored in HTFM (<2 and <4 wk, respectively). DNA integrity, assessed with comet assay, was also maintained significantly better in EFM than in HTFM. One-week storage in EFM yielded motility and viability similar to that of cryopreserved sperm, but DNA integrity was significantly higher, resembling that of fresh sperm. After several weeks of storage in EFM, sperm were able to activate oocytes, undergo chromatin remodeling, and form normal zygotic chromosomes after intracytoplasmic sperm injection. This study demonstrated that human spermatozoa can be stored in EFM without freezing for several weeks while maintaining motility, viability, and chromatin integrity and that 1-wk storage in EFM offers better protection of sperm DNA integrity than cryopreservation. Sperm storage in EFM may become a viable option for the physicians working in assisted reproduction technology clinics, which would avoid cryodamage.
Project description:The role of CD4(+) regulatory T cells (Tregs) and their subsets during HIV infection is controversial. Cryopreserved peripheral blood mononuclear cells (PBMC) are an important source for assessing number and function of Tregs. However, it is unknown if PBMC isolation and cryopreservation affect the expression of CD120b and CD39, markers that identify specific subsets of Tregs.HIV-uninfected (HIV-) and -infected (HIV+) men were randomly selected from the Multicenter AIDS Cohort Study (MACS). Percentages of CD120b(+) and CD39(+) Tregs measured by flow cytometry in whole blood and in corresponding fresh and cryopreserved PBMC were compared.Percentages of CD120b(+) Tregs were significantly lower in a) fresh PBMC relative to whole blood, and b) freshly thawed frozen PBMC relative to fresh PBMC when the recovery of viable cryopreserved cells was low. When present, low expression of CD120b in frozen PBMC was reversible by 4h of in vitro culture. In contrast, expression of CD39 on Tregs was not affected by isolation and/or cryopreservation of PBMC, or by relative recovery of cryopreserved PBMC. These findings were unaffected by the HIV status of the donor.The data suggest that percentages of CD120b(+) Tregs and CD39(+) Tregs can be validly measured in either whole blood or PBMC (fresh and frozen) in HIV- and HIV+ men. However, for measurement of CD120b(+) Tregs one type of sample should be used consistently within a given study, and thawed frozen cells may require in vitro culture if recovery of viable cells is low.
Project description:<h4>Background</h4>Rural/remote blood collection can cause delays in processing, reducing PBMC number, viability, cell composition and function. To mitigate these impacts, blood was stored at 4 °C prior to processing. Viable cell number, viability, immune phenotype, and Interferon-γ (IFN-γ) release were measured. Furthermore, the lowest protective volume of cryopreservation media and cell concentration was investigated.<h4>Methods</h4>Blood from 10 individuals was stored for up to 10 days. Flow cytometry and IFN-γ ELISPOT were used to measure immune phenotype and function on thawed PBMC. Additionally, PBMC were cryopreserved in volumes ranging from 500 µL to 25 µL and concentration from 10 × 10<sup>6</sup> cells/mL to 1.67 × 10<sup>6</sup> cells/mL.<h4>Results</h4>PBMC viability and viable cell number significantly reduced over time compared with samples processed immediately, except when stored for 24 h at RT. Monocytes and NK cells significantly reduced over time regardless of storage temperature. Samples with >24 h of RT storage had an increased proportion in Low-Density Neutrophils and T cells compared with samples stored at 4 °C. IFN-γ release was reduced after 24 h of storage, however not in samples stored at 4 °C for >24 h. The lowest protective volume identified was 150 µL with the lowest density of 6.67 × 10<sup>6</sup> cells/mL.<h4>Conclusion</h4>A sample delay of 24 h at RT does not impact the viability and total viable cell numbers. When long-term delays exist (>4 d) total viable cell number and cell viability losses are reduced in samples stored at 4 °C. Immune phenotype and function are slightly altered after 24 h of storage, further impacts of storage are reduced in samples stored at 4 °C.