Laboratory Practice Guidelines Research Articles

Toxicity of a novel antifungal agent (ATB1651 gel) in Yucatan minipigs (Sus scrofa) following 4weeks of daily dermal administration.

ATB1651 gel is an antifungal drug candidate that enhances antifungal activity through substitution of several aryl rings, alkyl chains, and methyl groups. To ensure safety of use of ATB1651 gel, assessment of its potentially toxic side effects is necessary. In this study, we examined the repeated-dose toxicity of ATB1651 gel to Yucatan minipigs (Sus scrofa) in accordance with the Good Laboratory Practice guidelines. Five doses of ATB1651 gel (0%, 0.2%, 0.5%, 1.0%, 3.0%) were administered dermally to the left and right flanks of 38 minipigs daily for 4weeks. Mortality, clinical symptoms, dermal scores, body weights, and physiological, biochemical, pathological, and toxicokinetic analyses were performed after the treatment period. No systemic toxicological damage was observed in either male or female minipigs regardless of dose; however, dermal application of ATB1651 gel caused some skin alterations at the application sites. Specifically, erythema and eschar formation, edema, and scabs or raise spots were observed at the application site(s) in males in the 3.0% ATB1651 gel treatment group and in females at ATB1651 gel concentrations ≥ 1.0%, with dermal scores ranging from grade 1 to 2. Additionally, histopathological assay indicated infiltration of different types of inflammatory cells and the presence of pustule/crust at the application site(s) in both males and females at ATB1651 gel concentrations ≥ 0.5%. However, these changes were reversible after a 2-week recovery period and were considered a local irritation effect of ATB1651 gel. The no-observed-adverse-effect level of ATB1651 gel was 3.0% with regard to topical and systemic toxicity in both male and female minipigs. Collectively, our results imply that ATB1651 gel is a safe candidate for clinical development as an antifungal drug with a wide therapeutic window.

Targeting staphylococcal bone infections

A novel quinolone antibiotic was developed by Shiladitya Sengupta and colleagues using molecular-docking simulation, and one lead compound, VCD-077, was selected based on this strategy.1 Antimicrobial resistance is increasing, driven by widespread antibiotic overuse in medical and agricultural settings.2 Paradoxically, despite the urgent need to develop a new generation of antibiotics, their marketability is suppressed, partly because the new antibacterial drug may quickly develop new resistance and the development of new antibiotics is sluggish.3, 4 The ideal next-generation antibiotic should demonstrate excellent activity against drug-resistant bacteria, good biosafety, flexible drug delivery, and the ability to retard the development of resistance. In this regard, recently researchers have focused on developing novel antibiotics by improving drug performance, availability, targeted enrichment, and reducing systemic toxicity. Reporting in Nature Biomedical Engineering, Shiladitya Sengupta and colleagues now show that VCD-077 screened by molecular-docking simulations exhibits excellent activity against drug-resistant bacteria and its biofilms, reduces the development of bacterial resistance and is compatible with bone cement for local delivery and treatment of staphylococcal bone infections in rats (Figure 1).1 Bone infections are rising rapidly due to accidental fractures and the global aging population.5 Sengupta and co-authors designed a series of quinolone antibiotics using molecular-docking simulation. They selected VCD-077 from 17 candidate antibiotics, which can effectively against clinical drug-resistant staphylococcal and retard the development of resistance. The minimal inhibitory concentration (MIC) of ciprofloxacin, Staphylococcus aureus sensitive antibiotics, increased stepwise to 64.0 μg/ml by the 20th passage to S. aureus. While S. aureus were still sensitive to VCD-077 even at the 28th passage. Specifically, VCD-077 is potent against S. aureus and Staphylococcus epidermidis biofilms because it is a weak acid with a single pKa of 5.8, that is, it still maintains antibacterial activity at pH 5.5–7.4. In contrast, gentamicin lost part of its activity at acid pH 5.5. Therefore, VCD-077 has a unique advantage in the prevailing weakly acidic infected bone environment. Notably, VCD-077 is physicochemically compatible with polymethylmethacrylate bone cement, exhibits desired pharmacokinetics without loss of mechanical properties of bone cement. VCD-077-impregnated bone cement showed more powerful anti-infective activity compared with either FDA-approved rifampin or gentamicin-impregnated bone cement. Overall, the authors show that VCD-077-impregnated bone cement could be used as rational next-generation drugs or coatings to treat bone infections. The high efficacy of Sengupta and co-authors' VCD-077-impregnated bone cement suggests that flexible local administration can be achieved at ideal safe concentration, and is effective against resistant strains, as well as delaying the development of new resistance. However, before VCD-077-impregnated bone cement can be considered for the treatment of clinical bone infection, three main challenges were compelled to be discussed. First, the authors used rodents in this experiment, which may be differences between the efficacy of antibiotics-impregnated bone cement in rodents and human bones. Studies using larger animal infection models (such as pigs and monkeys) would employ to corroborate the impressive antimicrobial activity in rat models. Second, significant biosafety assessment under good laboratory practice guidelines should be performed. And third, new antibiotic development should focus on specific screening molecules and combinations with emerging nanoparticle-based delivery systems that can target both resistant and persistent bacterial populations.6 Developing novel antibacterial agent delivery systems to improve the bioavailability of antibiotics is a promising strategy for increasing the lifespan of newly developed antibiotics. Integration of such novel antibiotics and combinations into nanoparticle-based delivery systems or coating implants might transform the landscape of infectious diseases. Drafting the article: Zhang Xiangchun. Revising the manuscript: Xulin Hu and Hongping Chen. All authors have read and approved the final manuscript This research was supported by the National Key Research and Development Program of China (2021YFD1601102), Central Public-interest Scientific Institution Basal Research Fund (No. Y2022QC24), and Innovative Program of Chinese Academy of Agricultural Sciences (CAAS-ASTIP-2021-TRI, CAAS-ZDRW202011). The authors declare no conflict of interest. Not applicable. Not applicable.

Safety evaluation of the genotoxicity and subchronic toxicity of standardized Bacopa extract (Bacognize®) from Bacopa monnieri

Bacopa monnieri is an important medicinal plant widely used in various food systems and gaining interest these days for its health benefits such as boosting brain function and improving quality of life. The objective of the present study was to examine the safety of Bacognize®, a standardized botanical extract obtained from the whole herb Bacopa monnieri (L.) Wettst., in subchronic toxicity and genotoxicity studies conducted in compliance with Good Laboratory Practice (GLP) and test guidelines established by the Organization for Economic Cooperation and Development (OECD). In the subchronic toxicity study, treatment with Bacognize® did not result in any toxicologically significant treatment-related changes in clinical observations, and in the clinical pathology as studied by hematology, serum chemistry, urinalysis, and terminal necropsy. Treatment-related adverse effects were not observed in ophthalmic examinations, body weights, body weight gains, feed consumption, and organ weights. The results of genotoxicity studies as assessed by gene mutations in Salmonella typhimurium, in vitro chromosome aberrations and in vivo micronucleus test did not reveal any genotoxicity of Bacognize ®. The no-observed-adverse-effect level (NOAEL) for Bacognize® was established based on the subchronic study and was determined as at least 1000 mg/kg bw/day. These results indicate that Bacognize® does not cause significant adverse effects and suggest its tolerability up to 1000 mg/kg (highest dose tested) for the daily administration of 90 days in rats.

Antimicrobial sensitivity pattern of children with cystic fibrosis in Bangladesh: a lesson from a specialized Sishu (Children) Hospital

BackgroundInfection control in cystic fibrosis (CF) patients plays a crucial role in improving the survival of patients with CF. Antimicrobial sensitivity patterns in these patient groups in our country are currently lacking. Therefore, the purpose of the study was to evaluate the microbiological cultures and antimicrobial susceptibility pattern of pediatric CF patients.MethodA total of 50 respiratory samples were prospectively collected from the period between February 2021 and October 2021. Sputum and oropharyngeal swabs were processed for culture and microbiological testing. Sample collection and evaluation were performed according to the Good Laboratory Practice guidelines (GLP). Informed written consent was ensured before participation. Statistical analysis was performed with SPSS v 26.ResultThe median age of the children was 30 months (6–120) months, with a male predominance (66% vs 34%). Single and two organisms were isolated in 72% (n = 36) and 12% (n = 6) of cases, respectively. During the study period, 36% of the patients harbored Pseudomonas aeruginosa, 18% harbored Klebsiella pneumoniae, and both Staphylococcus aureus and Escherichia coli were detected in 16% of cases. Levofloxacin was found to be the most active antibiotic agent with 100% susceptibility. In contrast, nearly all isolates were resistant to amoxicillin, erythromycin and rifampicin.ConclusionLevofloxacin is the most effective agent to treat CF patients. Active surveillance of the resistance pattern should always continue to be promoted.

Tumorigenicity Assessment of Human Cancer Cell Lines Xenografted on Immunodeficient Mice as Positive Controls of Tumorigenicity Testing.

Recent advances in human pluripotent stem cell (hPSC)-derived cell therapies and genome editing technologies such as CRISPR/Cas9 make regenerative medicines promising for curing diseases previously thought to be incurable. However, the possibility of off-target effects during genome editing and the nature of hPSCs, which can differentiate into any cell type and infinitely proliferate, inevitably raises concerns about tumorigenicity. Tumorigenicity acts as a major obstacle to the application of hPSC-derived and gene therapy products in clinical practice. Thus, regulatory authorities demand mandatory tumorigenicity testing as a key pre-clinical safety step for the products. In the tumorigenicity testing, regulatory guidelines request to include human cancer cell line injected positive control group (PC) animals, which must form tumors. As the validity of the whole test is determined by the tumor-forming rates (typically above 90%) of PC animals, establishing the stable tumorigenic condition of PC animals is critical for successful testing. We conducted several studies to establish the proper positive control conditions, including dose, administration routes, and the selection of cell lines, in compliance with Good Laboratory Practice (GLP) regulations and/or guidelines, which are essential for pre-clinical safety tests of therapeutic materials. We expect that our findings provide insights and practical information to create a successful tumorigenicity test and its guidelines.

O-123 FMEA analysis of an automatic integration of time-lapse incubators into electronic medical records using CHLOE(Fairtility) shows risk reduction through automation of data capture and processing

Abstract Study question Can integrations and automatic data processing between time-lapse incubators and EMRs reduce the risks associated with manual moving of data from time-lapse incubators to EMRs Summary answer Redesigning data workflow using CHLOE(Fairtility) decreased risk occurrence and increased risk detection possibilities associated with embryo classification and selection to freeze, biopsy, transfer and discard. What is known already Decisions are made from information derived from time-lapse incubators. Clinically, embryologists decide which embryos (and when) are suitable for transfer, cryopreservation, biopsy, or discarding based on data derived from time-lapse incubators, manually annotated and summarised into the electronic medical record (EMR) where further information useful for embryo selection is stored. Manual movement of data from time-lapse incubators to EMRs is time-consuming, administrative, reduces the granularity of the data available and incurs risk of human-error inaccuracies. These challenges limit the possibilities of how this data can be used to optimise clinical decisions, improve the patient experience and proactively detect operational anomalies. Study design, size, duration Failure mode effects analysis (FMEA) analysis was carried out on the workflow integration into a large (>5000 cycles per annum) IVF centre following ESHRE guidelines for laboratory and time-lapse practice (ESHRE,2015,2020), comparing before and after the introduction of CHLOE(Fairtility). The FMEA analysis evaluated the possible data capture, processing and associated clinical decision risks from embryos entering to leaving the time-lapse incubator. The Risk Priority Number (RPN=likelihoodxseverityxdetection of incidence) was calculated for each failure mode (Rienzi,2015). Participants/materials, setting, methods Through authenticated REST API calls according to the OpenAPI standard, CHLOE(Fairtility) linked the treatment unique identifier from the EMR(LIVO, inhouse developed) to the time-lapse incubator, automatically processed the time-lapse data, captured quantitative and qualitative information (such as morphokinetic time points, PNs, cleavage and blastocyst morphological grades, unusual embryo developmental anomalies and prediction scores for blastulation and implantation) and automatically loaded into the EMR. Main results and the role of chance Before CHLOE(Fairtility), 8 process phases were identified, with 81 associated failure modes, among which 45 risks were given a moderate RPN [RPN>15, i.e. data entry error into the EMR; image feature detection missed (i.e. 2PNs, Inner Cell Mass, incorrectly diagnosing fragments as cells and vice versa; incorrectly diagnosing vacuoles as a PNs, asynchronous PNs missed),with consequences including inaccurate KPI monitoring (n = 20, RPN=4); reduced patient experience and increased stress (n = 18, RPN range 3-16); wrong embryo being selected (n = 42, RPN range 8-36). Wrong embryo selection had three possible consequences: viable embryo discarded leading to a reduction in efficacy of treatment; viable embryo not prioritised for transfer causing reduced chance of pregnancy, or increased time to pregnancy, increasing cost and emotional burden); euploid embryo not prioritised for biopsy, increasing cost. Overall, RPN ranged from 3 to 36. After CHLOE(Fairtility), 51 failure modes were eliminated completely, including quantitative and qualitative morphokinetic annotations, entering data into the EMR for daily embryo grades, and embryo fate decisions. A further 22 failure modes had reduced RPN, including blastocyst morphological grading, number of PNs, identification of unusual embryo cleavages; with 30 low RPNs and 6 moderate RPNs. Implementation of CHLOE(Fairtility) reduced the highest RPN from 36 to 16. Limitations, reasons for caution FMEA is a proactive method to identify potential incidents in order to develop strategies to mitigate risks, forming part of a framework for responsible innovation. The likelihood of incidences were estimated based on a PUBMED literature review, personal experience and the experience of colleagues. Wider implications of the findings CHLOE(Fairtility) has the potential to eliminate risks that exist when manually moving data from time-lapse incubators to EMRs: time-consuming, administrative, reduced data granularity and human-error-based inaccuracies. CHLOE(Fairtility) optimises clinical decisions, providing an opportunity for personalised patient care, improved patient engagement, and the potential to detect operational non-conformities before impacting clinically. Trial registration number NA

Laboratory Test Utilization Practices in Hamad Medical Corporation; Role of Laboratory Supervisors and Clinicians in Improper Test Utilization; a Descriptive Pilot Study.

BackgroundThe use of diagnostic laboratory tests is increasing worldwide. Improper test utilization (ITU) is a common problem for all healthcare systems as it costs substantial expenses for the health systems and impacts optimal patient care.PurposeThe present small-scale survey aims to highlight the current practice of ITU among the labs and physicians, and investigate the actions of diagnostic laboratories towards ITU, and identify the reasons affecting test ordering decisions among physicians.MethodsA cross sectional study based on two different surveys was developed and distributed from March 2017 to April 2017 to laboratory supervisors and physicians (clinicians) at Hamad Medical Corporation (HMC), Qatar. Fourteen laboratory supervisors and eighty-nine physicians were surveyed about improper test utilization practices. The overall results are descriptive data.ResultsThe overall proportion of improperly utilized tests detected by the laboratory supervisors were 50.0%, 35.7%, and 14.3% for overused, misused, and underused lab tests, respectively. Among the physicians, 91% used the electronic ordering template to select the appropriate tests. Moreover, 78.7% of the physicians used the clinical guidelines, while 73% were not employing the laboratory handbook. Furthermore, 95.5%% of the physicians preferred to get feedback about inappropriate tests, while 51.1% were not receiving any, and 40.9% were rarely receiving. Finally, 67.4% were unaware of the tests’ costs among surveyed physicians, and 63.6% showed a willingness to reduce their orders if the cost was high and unnecessary.ConclusionThe physician’s and the laboratories’ communication were inadequate and not systematized, causing ITU practices. The improvement strategy should focus on the communication between clinical labs and physicians and enhance physician implementation to order appropriate lab tests. This could be achieved by conducting legitimate educational methodologies, continuous feedback reviews, ongoing audits, executing health information technology instruments, engaging laboratory practice guidelines, and applying demand management and testing algorithms.

Subchronic toxicity and genotoxicity studies of Hericium erinaceus β-glucan extract preparation

The medicinal effects of Hericium erinaceus have been long documented in scientific studies of Eastern traditional medicine. It is widely consumed, because of its nutritional qualities and perceived health benefits. Also, it is rich in β-glucans, which has been shown to have immunomodulating and antitumor effects. The objective of the present study was to investigate adverse effects, if any, of β-glucan extract preparation from H. erinaceus in subchronic toxicity and genotoxicity studies. The conduct of these studies was in compliance with Good Laboratory Practice (GLP) and test guidelines established by the Organization for Economic Cooperation and Development (OECD). In the subchronic toxicity study, Sprague Dawley rats (12/sex/group) were administered (gavage) H. erinaceus β-glucan extract preparation at dose levels of 0, 500, 1000 and 2000 mg/kg body weight (bw)/day for 90 days. Treatment with H. erinaceus β-glucan extract preparation did not result in any toxicologically significant treatment-related changes in clinical observations, ophthalmic examinations, body weights, body weight gains, feed consumption, and organ weights. Clinical pathology including hematology, serum chemistry, urinalysisand terminal necropsy (gross or histopathology findings) did not reveal any treatment-related adverse effects. The results of genotoxicity studies as evaluated by gene mutations in Salmonella typhimurium, in vitro chromosome aberrations and in vivo micronucleus test in mice did not reveal any genotoxicity of H. erinaceus β-glucan extract preparation. Based on the subchronic study, the no observed-adverse-effect level (NOAEL) for H. erinaceus β-glucan extract preparation was determined as 2000 mg/kg bw/day, the highest dose tested.

Investigation of Ifosfamide Toxicity Induces Common Upstream Regulator in Liver and Kidney.

Ifosfamide is an alkylating agent, a synthetic analogue of cyclophosphamide, used to treat various solid cancers. In this study, the toxicity of ifosfamide was evaluated using single-and multiple-dose intraperitoneal administration in rats under Good Laboratory Practice guidelines, and an additional microarray experiment was followed to support toxicological findings. A single dose of ifosfamide (50 mg/kg) did not induce any pathological changes. Meanwhile, severe renal toxicity was observed in the 7 and 28 days consecutively administered groups, with significant increases in blood urea nitrogen and creatinine levels. In the tox-list analysis, cholesterol synthesis-related genes were mostly affected in the liver and renal failure-related genes were affected in the kidney after ifosfamide administration. Moreover, interferon regulatory factor 7 was selected as the main upstream regulator that changed in both the liver and kidney, and was found to interact with other target genes, such as ubiquitin specific peptidase 18, radical S-adenosyl methionine domain containing 2, and interferon-stimulated gene 15, which was further confirmed by real-time RT-PCR analysis. In conclusion, we confirmed kidney-biased ifosfamide organ toxicity and identified identically altered genes in both the liver and kidney. Further comprehensive toxicogenomic studies are required to reveal the exact relationship between ifosfamide-induced genes and organ toxicity.

Study design criteria for regulatory-based drug control action: Drug discrimination

This “methods paper” focusses on one specific and limited aspect of drug safety evaluations required for all new drug entities that affect the central nervous system – the drug discrimination (DD) assay. We focus on three critical factors involved in experimental design and protocol development for the conduct of DD studies for abuse liability risk assessment that comply with the Good Laboratory Practice Guidelines (GLPs). The selection of 1) the reference drug(s) choice, 2) training dose selection, and 3) the selected route-of-administration will determine the applicability of the data to meet the regulatory expectations of the 8-factors determinative of schedule control recommendations. The study conduct and resulting data submission to the FDA are intended for drug scheduling review by the Controlled Substances Staff in the Center for Drug Evaluation and Research (CDER) at the US Food & Drug Administration (FDA). These animal studies are required to meet the statutory requirements of the Controlled Substances Act of 1970. The abuse liability study is conducted during Phase II and III of human clinical trials. Procedural or method-based errors this late in drug development can result in a significant economic and business threat to the program.

Application and optimization of reference change values for Delta Checks in clinical laboratory.

BackgroundDelta check is a patient‐based QC tool for detecting errors by comparing current and previous test results of patient. Reference change value (RCV) is adopted in guidelines as method for delta check, but the performance is not verified. We applied RCV‐based delta check method to patients' data and modified for application.Materials and methodsReference change value were calculated using results of internal QC materials and biological variation data. Test results of 17 analytes in inpatients, outpatients, and health examination recipients were collected. The detection rates of currently used delta check method and those of RCV‐based method were compared, and the methods were modified.ResultsReference change value‐based method had higher detection rates compared to conventional method. Applied modifications reduced detection rates. Removing the pairs of results within reference interval reduced detection rates (0.42% ~ 10.92%). When RCV was divided by time interval, the detection rates were similar to prior rates in outpatients (0.19% ~ 1.34%). Using RCV multiplied by twice the upper limit of reference value as cutoff reduced the detection rate (0.07% ~ 1.58%).ConclusionsReference change value is a robust criterion for delta check and included in clinical laboratory practice guideline. However, RCV‐based method generates high detection rates which increase workload. It needs modification for use in clinical laboratories.

Pengembangan Panduan Praktikum Laboratorium Virtual Berbasis Keterampilan Proses Sains Untuk Meningkatkan Pemahaman Konsep Siswa

ABSTRACTEfforts to improve understanding of concepts in students can be seen from their academic abilities. Understanding concepts is a term used to mean the extent to which students are able to understand the basic concepts of a material provided by the teacher. One effort to improve the understanding of concepts in students is to do experimental learning models or practicum. Along with the advancement of time where technology can be used as a tool in learning, therefore the implementation of practicum can be done using a virtual laboratory. The use of virtual laboratories can run well and in accordance with procedures when using a virtual laboratory practicum guidelines. The making of practicum guidelines must not be separated from the scientific process skills because it has been regulated in Regulation of the Ministry of Education and Culture No. 22 of2016.The purpose of this study is to find out the development of practical process guidelines based on science process skills using virtual laboratories, to determine the impact of virtual laboratories on students' understanding of concepts, and to determine the strengths and weaknesses of virtual laboratories. After conducting field tests in SMA 1 Metro with a class XII IPA 4 as sample, it is known that the results of the feasibility of using a virtual laboratory practice guidelines based on science process skills, namely expert validation got a percentage of 84.6%, Teacher Response 91.25% and Student Response 88, 4% Based on the average percentage of the three assessments of 88.08%, it is in the very feasible category. As for understanding the concept an increase of 21.4 between the pretest and posttest scores. Based on the results of the study, the virtual laboratory practicum guidelines based on science process skills affect the learning process specifically to improve students' understanding of concepts.
 Keywords: Development of practicum guides, virtual laboratories, science process skills, conceptual understanding

Collection and Handling of Thoracic Small Biopsy and Cytology Specimens for Ancillary Studies Guideline from the College of American Pathologists (CAP): implications for the cytology community

The methods of collecting and handling thoracic small biopsy and cytology specimens can greatly impact downstream ancillary testing success, especially for non-small cell lung cancer (NSCLC). The College of American Pathologists (CAP) in collaboration with multiple other professional societies has recently developed an evidence-based laboratory practice guideline to provide clarity on how to best optimize these pre-analytic variables for small thoracic specimens. A total of 16 guideline statements were developed based on systematic literature review and expert panel consensus, covering topics ranging from optimal materials and techniques for fine-needle aspiration and small biopsy sampling to acquire sufficient amounts of material, to the use of rapid on-site evaluation to help appropriately triage, handle, and process these specimens. These guideline statements hold many implications for the practicing cytologist, though perhaps none more important than the recognition that the variety of cytology specimens we work with on a daily basis (including smears, cell blocks, and liquid-based cytology) can certainly be used for ancillary studies if supported by adequate validation studies. This commentary provides a brief overview of the newly developed CAP thoracic small biopsy and cytology specimen collection and handling guideline, as well as a discussion of how it may specifically impact the cytology community.

Association between Vaginal Micro-environment Disorder and Cervical Intraepithelial Neoplasia in a Community Based Population in China.

There are other factors that contribute to cervical carcinogenesis except HPV infection. This study aimed to investigate the association between vaginal micro-environment factors, including H2O2, vaginal PH value, vagina cleanness, β-glucuronidase, coagulase, neuraminidase and leukocyte esterase and cervical intraeipithelial neoplasia (CIN). In total 1019 participants, including 623 normal cervical (NC) women, 303 patients with low-grade cervical intraepithelial neoplasia (CIN1) and 93 patients with high-grade cervical intraepithelial neoplasia (CIN2/3), were enrolled into the study. HPV genotyping was detected by flow-through hybridization and gene chip. Vaginal H2O2, β-glucuronidase, coagulase, neuraminidase and leukocyte esterase were detected by Aerobic Vaginitis (AV) / Bacterial Vaginal Disease (BV) Five Joint Test Kit. Vaginal PH was measured on the glass slide after microscopy, using color strips with a PH range of 3.8-5.4. Vagina cleanness was determined according to the National Clinical Laboratory Practice Guideline. χ2test and Logistic regression were operated using SPSS 22.0 software. Our results showed that HPV16 infection rate and the abnormal rates of H2O2, PH, vagina cleanness, β-glucuronidase or neuraminidase increased gradually along with the severity of CIN (P<0.05). Abnormities of H2O2, cleanness, β-glucuronidase and neuraminidase were risk factors for CIN regardless of HPV16 infection, furthermore, abnormities of PH value, leukocyte esterase could also increase the risk of CIN in HPV16 positive group. In addition, women with abnormal vaginal micro-environment factors in HPV16 positive group had a significantly higher risk of developing CIN than HPV16 negative group. The results from generalized multifactor dimensionality reduction (GMDR) model showed that there was interaction effect with abnormities of vagina cleanness, H2O2, β-glucuronidase and neuraminidase on CIN2/3 in HPV16 negative group, while, there was interaction effect with abnormities of vagina cleanness, β-glucuronidase and neuraminidase on CIN1 and with abnormities of vagina cleanness, PH, H2O2, β-glucuronidase, neuraminidase and leukocyte esterase on CIN2/3 in HPV16 positive group. Our results suggested that vaginal micro-environment disorder could increase the risk of CIN, especially, the abnormality of H2O2, cleanness, β-glucuronidase and neuraminidase. There were interaction effects with abnormities of H2O2, vagina cleanness, β-glucuronidase and neuraminidase on CIN whether HPV16 was infected or not.

Implementing a quality management system using good clinical laboratory practice guidelines at KEMRI-CMR to support medical research.

Background: Good Clinical Laboratory Practice (GCLP) is a standard that helps ensure the quality and reliability of research data through principles of Good Laboratory Practice (GLP) and Good Clinical Practice (GCP). The implementation of GCLP includes careful documentation of procedures, competencies and safety measures. Implementation of GCLP is influenced by existing resources and quality systems, thus laboratories in low- and middle-income countries may face additional challenges. Methods: This paper describes implementation of GCLP at the Kenya Medical Research Institute-Center for Microbiology Research (KEMRI-CMR) as part of a quality system to support medical research. This study employed assessment, twinning (institutional mentorship) model, conducting relevant training workshops and Kaizen 5S approaches to implement an effective quality management system using GCLP standard. This was achieved through a collaboration between the KEMRI/Wellcome Trust Research Programme (KWTRP) and KEMRI-CMR. The aim was compliance and continuous monitoring to meet international GCLP standards in a way that could be replicated in other research organizations. Results: Following a baseline assessment in March 2017, training, mentorship and a cycle of quality audit and corrective action using a Kaizen 5S approach (sorting, setting in order, shining, standardizing and sustaining) was established. Laboratory personnel were trained in writing standard operating procedures and analytical plans, microbiological techniques, and good documentation practice. Mid-term and exit assessments demonstrated significant declines in non-conformances across all GCLP elements. KEMRI-CMR achieved GCLP accreditation in May 2018 by Qualogy Ltd (UK). Conclusions: Involving all the laboratory personnel in implementation of quality management system processes is critical to success. An institutional mentorship (twinning) approach shows potential for future collaborations between accredited and non-accredited organizations to accelerate the implementation of high-quality management systems and continuous improvement.

Rapid fluorescent focus inhibition test optimization and validation: Improved detection of neutralizing antibodies to rabies virus

The rabies rapid fluorescent focus inhibition test (RFFIT) is the most widely used cell-based assay for detecting and quantitating rabies virus neutralizing antibodies (RVNA) in human serum. However, it is a complex, labor intensive, and somewhat subjective manual assay, the performance of which may be affected by a number of factors including the quality of cells and virus, variability of assay reagents and the skill and expertise of analysts. This study sought to identify and evaluate conditions that may impact RFFIT performance and RVNA detection by evaluating assay parameters including: different serial dilution scheme of serum samples in a 96-well microplate using semi-automated pipetting systems, the range of dose of challenge virus standard (CVS-11) strain of rabies virus, the effect of complement (C′), the effect of cell seeding density and passage number, the effect of diethylaminoethyl (DEAE) dextran concentration on virus infectivity, and the assay incubation period prior to immunostaining. In addition the evaluation of counting fluorescent foci using a microscope versus using scanned images from a cell imaging reader was performed in an effort to ease the reading of slides and have permanent records of the raw data. The results from optimization of each parameter are presented along with subsequent assay validation in accordance with the International Conference on Harmonization (ICH) guidelines. The improved and optimized RFFIT accuracy, linearity and sensitivity was demonstrated by testing World Health Organization (WHO)-1 and WHO-2 Standard Rabies Immune Globulins (SRIGs) and complete assay development and validation was performed in compliance with Good Clinical Laboratory Practice (GCLP) guidelines.

Evaluating the Adoption of Laboratory Practice Guidelines.

To date, the College of American Pathologists (CAP) has developed 17 laboratory practice guidelines (LPGs) including updates. In 2013, the CAP was awarded a 5-year cooperative agreement grant from the United States Centers for Disease Control and Prevention to increase the effectiveness of LPGs. To assess the awareness and adoption of 2 CAP LPGs: immunohistochemical (IHC) assay validation and initial workup of acute leukemia. Baseline surveys for each LPG were conducted in 2010 and 2015, respectively. To measure the adoption of guideline recommendations and inform future updates, a follow-up study consisting of surveys, telephone interviews, and focus group sessions was conducted in laboratories that indicated they perform IHC testing. A follow-up study for the acute leukemia LPG is planned. For the IHC Validation LPG, a total of 1624 survey responses, 40 telephone interviews, and discussions with 5 focus group participants were analyzed. The response rate for the aforementioned 3 modalities was 46%, 13%, and 3%, respectively. All modalities indicated most respondents were aware of the LPG and had adopted most or all of its recommendations. Respondents expressed needs for continued communication, increased specificity, and more prescriptive recommendations when the guideline is updated. While data-driven development of evidence-based LPGs requires significant resources, active data collection to identify gaps and assess adoption contributes to improved laboratory testing practices in support of patient care. The CAP identified sustainable modalities to track metrics and developed multiple tools that should improve guideline development, adoption, and implementation. Of these modalities, written or electronic surveys were the most logistically feasible and had the highest response rate.

Analytical Validation of Variants to Aid in Genotype-Guided Therapy for Oncology

The Clinical Laboratory Improvement Amendments of 1988 require that pharmacogenetic genotyping methods need to be established according to technical standards and laboratory practice guidelines before testing can be offered to patients. Testing methods for variants in ABCB1, CBR3, COMT, CYP3A7, C8ORF34, FCGR2A, FCGR3A, HAS3, NT5C2, NUDT15, SBF2, SEMA3C, SLC16A5, SLC28A3, SOD2, TLR4, and TPMT were validated in a Clinical Laboratory Improvement Amendments-accredited laboratory. Because no known reference materials were available, existing DNA samples were used for the analytical validation studies. Pharmacogenetic testing methods developed here were shown to be accurate and 100% analytically sensitive and specific. Other Clinical Laboratory Improvement Amendments-accredited laboratories interested in offering pharmacogenetic testing for these genetic variants, related to genotype-guided therapy for oncology, could use these publicly available samples as reference materials when developing and validating new genetic tests or refining current assays.

Good Laboratory Practice in the Academic Setting: Fundamental Principles for Nonclinical Safety Assessment and GLP-Compliant Pathology Support When Developing Innovative Biomedical Products.

Development of new biomedical products necessitates nonclinical safety assessment in animals as a means of assessing potential risk to human patients. Pivotal nonclinical safety studies that support human clinical trials are performed according to Good Laboratory Practice (GLP) guidelines, which are designed to ensure that the study was conducted under carefully controlled conditions using standardized and validated procedures that will yield a reliable, reproducible, and traceable data set. The GLP guidelines established by different regulatory agencies address organizational structure, personnel responsibilities, personnel training practices, quality assurance (ensuring compliance), facilities, equipment, standard operating procedures, study documentation (record keeping), and record and sample retention. Academic institutions engaging in nonclinical safety assessment on-site have multiple options for implementing a GLP quality system. This article outlines the rationale supporting the use of a GLP-compliant or GLP-like quality system in academia and reviews key concepts needed to efficiently and effectively implement GLP in the academic setting. Emphasis is given to provision of GLP-compliant pathology support as (1) pathology data are an essential component of GLP nonclinical safety testing, (2) familiarity with pathology-related GLP procedures typically is gained first outside the academic setting, and (3) microscopic pathology diagnoses and interpretations require special accommodations to ensure that they are undertaken in a GLP-compliant fashion.

Implementing a quality management system using good clinical laboratory practice guidelines at KEMRI-CMR to support medical research

Background: Good Clinical Laboratory Practice (GCLP) is a standard that helps ensure the quality and reliability of research data through principles of Good Laboratory Practice (GLP) and Good Clinical Practice (GCP). The implementation of GCLP includes careful documentation of procedures, competencies and safety measures. Implementation of GCLP is influenced by existing resources and quality systems, thus laboratories in low- and middle-income countries may face additional challenges. Methods: This paper describes implementation of Good Clinical Laboratory Practice (GCLP) at the Kenya Medical Research Institute-Center for Microbiology Research (KEMRI-CMR) as part of a quality system to support medical research. This study employed assessment, twinning (institutional mentorship) model, conducting relevant training workshops and Kaizen 5S approaches to implement an effective quality management system using GCLP standard. This was achieved through a collaboration between the KEMRI/Wellcome Trust Research Programme (KWTRP) and KEMRI-CMR. The aim was compliance and continuous monitoring to meet international GCLP standards in a way that could be replicated in other research organizations. Results: Following a baseline assessment in March 2017, training, mentorship and a cycle of quality audit and corrective action using a Kaizen 5S approach (sorting, setting in order, shining, standardizing and sustaining) was established. Laboratory personnel were trained in writing standard operating procedures and analytical plans, microbiological techniques, and good documentation practice. Mid-term and exit assessments demonstrated significant declines in non-conformances across all GCLP elements. KEMRI-CMR achieved GCLP accreditation in May 2018 by Qualogy Ltd (UK). Conclusions: Involving all the laboratory personnel in implementation of quality management system processes is critical to success. An institutional mentorship (twinning) approach shows potential for future collaborations between accredited and non-accredited organizations to accelerate the implementation of high-quality management systems and continuous improvement.