Table of Content

25 December 2022, Volume 42 Issue 6

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Laboratory Capability Verification and Accreditation

A Retrospective Study on Proficiency Testing Samples in the Field of Laboratory Animal Testing from 2013 to 2022

Han QIAO, Jie WEI, Feifei WANG, Hong WANG, Jin XING, Rui FU, Jing XIAO, Meng ZHAO, Bingfei YUE, Xinhua XIANG

2022, 42(6):  483-489.  DOI: 10.12300/j.issn.1674-5817.2022.139

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Objective To analyze the status of proficiency testing samples in the field of laboratory animal testing, and to evaluate the role of the testing samples in the laboratory animal laboratory proficiency testing program. Methods A retrospective study was conducted to compare the 30 proficiency testing plans and reports in the field of laboratory animal organized by National Institute for Food and Drug Control (NIFDC) from 2013 to 2022. The type, quantity, preparation method, homogeneity and stability of the proficiency testing samples, as well as the satisfaction of the participating units to the samples were analyzed. Then, the supply of new samples, the addition of quantitative projects, the improvement of stability research and the attention to customer needs were discussed. Results The 30 proficiency testing programs included 10 items in the direction of bacteria, 10 items in the direction of virus and 10 items in the direction of genetics in the field of experimental animals. A total of 80 non-nucleic acid samples (including serums, feces, tracheal flushing fluid and proteins) and 4 nucleic acid samples were provided, all of which were qualitative samples. Compared with nucleic acid samples, non-nucleic acid samples always required large numbers of animals for sample preparation. The pass rate of homogeneity verification of the sample was 100%, but there is no quantitative study at present. Stability verification can be conducted for the short-term storage, transportation, freeze-thaw, different concentrations and terminal stability of different samples, but it lacks long-term stability verification. Conclusion In the past ten years, the proficiency testing samples have basically met the basic requirements of the proficiency testing plan of laboratory animal testing area. However, the provision of nucleic acid samples should be reasonably increased; quantitative research samples and projects should be added; the influence of proficiency testing in the field of laboratory animal testing should be continuously expanded and maintain high-quality services.

Laboratory Proficiency Testing and Accreditation

Analysis of Laboratory Proficiency Testing Results on the Detection of Porcine Parvovirus Nucleic Acid

Xiaobo LI, Ji WANG, Hong WANG, Shujing WANG, Shasha WANG, Xiao QIN, Wei LI, Bingfei YUE, Rui FU

2022, 42(6):  490-497.  DOI: 10.12300/j.issn.1674-5817.2022.137

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Objective To understand the testing level of the relevant laboratories and standardize the detection of porcine parvovirus (PPV) nucleic acid by implementing laboratory proficiency testing plan of the PPV nucleic acid testing. Methods From January to August 2022, NIFDC organized the laboratory proficiency testing plan for PPV nucleic acid detection (plan number: NIFDC-PT-364). According to relevant CNAS standards, inactivated PPV was added into the serum of PPV negative pigs to prepare proficiency testing samples. After passing the homogeneity and stability tests, the samples should be distributed to the participating laboratories. Each laboratory was distributed 3 samples and was required to conduct qualitative detection of PPV nucleic acid, and submit the results and relevant original records within the specified time. Finally, the results submitted by each laboratory were summarized and analyzed. Results A total of 9 laboratories registered to participate in this proficiency testing, and all of them fed back the results within the specified time. Among them, the results of 8 laboratories met the expectations, accounting for 88.9%. Three samples from one laboratory were all tested positive for PPV, which was not consistent with the expected results, and it was judged as unsatisfactory. Most laboratories adopted real-time fluorescent PCR detection method, and used domestic reagents for nucleic acid extraction, while the PCR amplification reagents were mainly imported reagents. The use of different brands of reagents didn't have impact on the detection results. There were some problems in some laboratories, such as unclear diagram of experimental results and incomplete information of original records. Conclusion Most of the participating laboratories are capable of PPV nucleic acid detection, but the detection details need to be improved.

Result Analysis of Proficiency testing for Four Respiratory Pathogens in Laboratory Animals

Yufang FENG, Hong WANG, Rui FU, Xueqing ZHANG, Qiang GAO, Bingfei YUE, Jin XING

2022, 42(6):  498-504.  DOI: 10.12300/j.issn.1674-5817.2022.140

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Objective To assess the ability of the inspection institutions of laboratory animals and to improve their detection levels through the implementation of the proficiency testing for the detection ability of Bordetella bronchiseptica, Pasteurella pneumotropica, Pasteurella multocida and Corynebacterium kutscheri in laboratory animals. Methods According to the proficiency testing scheme approved by China National Accreditation Service for Conformity Assessment (CNAS), the two projects, NIFDC-PT-309 and NIFDC-PT-363 in 2021 and 2022 were set up, respectively. The samples of respiratory tract pathogens in laboratory animal were prepared by freeze-drying at low temperature. The samples were randomly grouped and numbered through the CNAS platform and issued to participating units after passing the homogeneity and stability tests. A work instruction was attached with each sample set. The inspection reports and original records were required to be submitted within the specified time. If the feedback results were consistent with the sample setting, the results were deemed as satisfactory, and if the feedback results were inconsistent or not submitted on time, the results were deemed as unsatisfactory. Results There were 29 laboratories joined to NIFDC-PT-309, 27 laboratories (93.1%) achieved satisfactory test results. There were 30 laboratories joined to NIFDC-PT-363, the satisfaction rate is 100%. Conclusion Laboratory animal quality inspection institutions have the ability to detect respiratory pathogens, which gets steadily improved through the ability verification activities.

Evaluation Report on the Proficiency Testing of SNP Detection in Laboratory Mice Nucleic Acid Samples

Jie WEI, Xinyan ZHANG, Hong WANG, Lan ZHAO, Wei LIU, Huan LI, Rui FU, Han QIAO, Meng ZHAO, Xinhua XIANG, Bingfei YUE

2022, 42(6):  505-510.  DOI: 10.12300/j.issn.1674-5817.2022.138

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Objective To test the single nucleotide polymorphism (SNP) detection technology level of mice genetic quality control in laboratories, promote the application of nucleic acid samples and SNP detection technology by implementing a nationwide proficiency testing program. Methods In 2022, National Institutes for Food and Drug Control implemented domestic laboratory proficiency testing named“NIFDC-PT-365 Laboratory Proficiency Testing Program of SNP Marker Detection Ablity in Laboratory Mice DNA Samples”. According to the requirements of the program, each participating laboratory received 2 blind samples of DNA from BALB/c mice and C57BL/6 mice with random numbers, standard operation procedure and 2 pairs of primers for rs3023177 and rs3023382 SNP loci. The participants should submit the results and original records before the deadline. If the mutation site amplification and sequencing results were consistent with the preseted results, it was considered as satisfactory; otherwise, it was considered as unsatisfactory. Meanwhile, PCR amplification system, sequencing direction and alignment methods of participating laboratories were compared to analyze as the relevant factors which may affect the SNP detection results of nucleic acid standard samples. Results The results of 10 participating laboratories were consistent with the preset. They all obtained satisfactory results. Differences in amplification volume (20 μL, 25 μL, 30 μL), Taq enzyme system (single component and PCR MIX), sequencing company, sequencing direction and alignment software had no influence on the accuracy of the results. Conclusion The participating laboratories have high detection ability in SNP detection method of mice nucleic acid samples. SNP detection technology is simple and feasible in mouse genetic quality control and is worth popularizing.

Analysis on the Standardized Expression of the Accredited Ability Scope for Hematology, Blood Biochemistry and Urinalysis in Laboratory Animal Clinical Testing

Jian WANG, Jin LU, Lingyun TAO, Hongkun FU, Cheng GAO, Baojin WU, Yao LI, Yufeng TAO

2022, 42(6):  511-517.  DOI: 10.12300/j.issn.1674-5817.2022.172

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To improve the application of hematology and urine testing of laboratory animals, and the consistency and effectiveness of the on-site review in China, there is a need for further standardization of the expression of testing ability in this field. In addition, it promotes better understanding and acceptance of the China National Accreditation Service for Conformity Assessment (CNAS) recognized results by relevant parties. In this paper, the expression of the accredited competence scope in the domestic and international institutes of laboratory animal hematology, blood biochemistry, and urine analysis fields were analyzed and compared. This study provides the CNAS with recommendations to standardize the expression of accreditation ability. In addition, it provides a detailed theoretical reference for further improving the process of standardizing testing of laboratory animals in China.

Discussion on the Scope Expression of CNAS Laboratory Accreditation in Veterinary Pathology Testing Field

Zhiqiang YU, Jingjing WANG, Hongkun FU, Cuie WANG, Yufeng TAO

2022, 42(6):  518-525.  DOI: 10.12300/j.issn.1674-5817.2022.177

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At present, due to some differences in the scope expression of veterinary pathology testing accreditation ability, the laboratory accreditation of China National Accreditation Service for Conformity Assessment (CNAS) is not standardized and uniform in the process of application of expanded items in the field of veterinary pathology testing and the process of assessors' evaluation, which makes many laboratories engaged in laboratory animal pathology testing and scientific research fail to obtain relevant qualifications.This article discussed the problems of scope expression in the field of CNAS veterinary pathology accreditation, and compared the expression with other veterinary pathology laboratories in other countries including the United Kingdom, the United States, Canada, New Zealand, and Singapore. The scope expression in medical pathology laboratories accredited by CNAS and some other ISO/IEC 17025 accreditation organizations were also compared. Combined with the accreditation practice of veterinary pathology laboratories in China, this paper put forward some suggestions on the scope expression in veterinary pathology testing field, so as to further unify and standardize the scope expression in veterinary pathology testing field in China, and improve the standardization of accreditation system in China.

Discussion on Expression of Laboratory Accreditation Scope in the Field of Toxicology Testing

Xiangmei LIU, Zhongchun MA, Hongkun FU, Feng GAO, Yufeng TAO

2022, 42(6):  526-530.  DOI: 10.12300/j.issn.1674-5817.2022.143

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With the series of regulations issued by the State Administration for Market Regulation entitled "Release, Regulation, and Service" and the rapid development of the biomedical health industry, the demand for toxicology testing is increasing, and the number of testing institutions is also growing. Statements of accreditation capacity scope of toxicology laboratories must also be more specialized and international. In this paper, the project classification, detection objects, items/parameters, statements of accreditation and scope of the Chinese regulatory toxicology testing field were described. The accreditation capacity scope of 4 mainstream international accreditation institutions in the field of toxicology testing, in New Zealand, the United States, Japan, and the United Kingdom, was organized and analyzed. Results showed that China, New Zealand and other countries mentioned above used the same basic expression regarding the scope of accreditation capacity in the field of toxicology testing; these expressions covered detection objects, detection methods/technologies, detection standards and field codes, but their classification principles for codes varied slightly. In order to standardize and uniformly express the scope of accreditation capacity in this field and improve the professional level of laboratories, this study puts forth suggestions on detection objects, items/parameters, field codes, names(method)and numbers(including year numbers) of detection standards, and the scope of limitations.

Animal Models of Human Diseases

Establishment of a Vaginal Atrophy Rat Model and its Application in Pharmacodynamic Evaluation

Liya YANG, Tao SONG, Jialin HE, Yiming GUO, Mingkang QI, Hanbi WANG, Huiping WANG

2022, 42(6):  531-540.  DOI: 10.12300/j.issn.1674-5817.2022.014

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Objective To establish an animal model suitable for screening vaginal preparations, administering the corresponding drugs, and evaluating biological indicators for monitoring drug treatment effects. Methods Bilateral ovariectomy (OVX) was used to establish the disease model of rat. The estrous cycle, uterine wet weight, and vaginal tissue morphology were observed from day 15 to day 21 after OVX to determine the optimal time for successful vaginal atrophy model after the OVX operation. Sprague-Dawley female rats were randomly divided into the following groups: normal group, sham group, model group, drug group 1, drug group 2, and solvent control group (n=10). Drug group 1, drug group 2, and solvent control group were treated with promestriene, Colpotrofin?, and solvent control for 14 days, respectively. Three days after the last administration, the body weight and uterus wet weight of the rats were measured, the histological morphology of the vagina was analyzed by HE staining, and estrogen receptors-α (ERα) expression was detected by immunohistochemistry. Results Twenty-one days after OVX, the vaginal atrophy model was established, which was suitable for the follow-up experiments. The change of estrous-cycle wasn't observed from day 17 to 21 after OVX. On 21 days after OVX, the vaginal epithelium thickness reduced (P<0.05), vaginal folds reduced, no squamous epithelium was observed, body weight increased (P<0.05), and uterine wet weight decreased (P<0.05). In drug group 1 and drug group 2, the symptoms of vaginal atrophy improved. Compared with the model group, the body weight of rats in drug group 1 and 2 decreased (P<0.05), the vaginal histomorphology improved, the expression of ERα protein in vaginal tissue up-regulated (P<0.05), and the ratio of uterine wet weight to body weight increased (P<0.01), but the endometrial thickness was not significantly thickened (P>0.05). Conclusion The rat OVX model is suitable for screening vaginal preparations. Vaginal histomorphology, endometrial thickness, and uterine/body weight ratio can be used as indicators to monitor drug efficacy.

In Vivo Colonization Test of Two CRISPR-Engineered Escherichiacoli in Mice

Chenyin REN, Siqi GAO, Hao LI, Biao TANG, Hua YANG, Yuehuan LIU

2022, 42(6):  541-550.  DOI: 10.12300/j.issn.1674-5817.2022.088

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Objective The colonization ability and efficiency of two Escherichia coli ( E. coli)-engineered strains, Nissle1917 and BW25113, in the intestine were evaluated in mice, we aimed to screen out strains for subsequent research on clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system-engineered bacteria to eliminate drug-resistant bacteria via high intestinal colonization efficiency. Methods Seventy ICR mice (18–20 g), half male and half female, were randomly divided into 7 treatment groups by gender, with 10 mice in each group (6 for experiment and 4 for control). The experimental group was gavaged with 2×10 ¹⁰ of the engineered strains at a final volume of 200 μL, and the control group was gavaged with an equal volume of PBS. At 1, 3, 6, 12, 24, 48, and 72 hours after gavage, the mesenteric lymph nodes, stomach, ileocecal and colonic tissues, and intestinal contents of the mice were removed. The two E. coli strains were detected using plate inoculation, fluorescence microscopy, and PCR amplification to compare their in vivo colonization ability and efficiency. Results At 1, 3, 6, 12, 24, and 48 hours after gavage, both E. coli strains had colonized in the stomach, ileocecal and colonic tissues as detected using the three methods, and no leakage of E. coli fluid from the lymph nodes was observed; at 72 hours, only Nissle1917 colonized in the ileocecal and colonic tissues, comparing the colonization efficiency of the two E. coli strains, that of Nissle1917 was 100% and that of BW25113 was 0 at 72 hours. Conclusion Nissle1917 has a higher colonization efficiency than BW25113 and can colonize in the mucosal surface of ileocecal and colonic tissues for a long time, suggesting that it can be used as a carrier for the CRISPR system to prevent and control drug resistance gene transmission.

Effect of the Traditional Chinese Medicine Shuganjieyu Formula on Constipation Type Irritable Bowel Syndrome and Brain-gut Axis in Rats

Bin WU, Xu WANG, Dongxu FU, Yujun ZHU, Jinlu HUANG, Shunxing ZHU

2022, 42(6):  551-559.  DOI: 10.12300/j.issn.1674-5817.2022.135

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Objective To observe the intervention effect of Shuganjieyu Formula on the rat model of constipation irritable bowel syndrome (IBS-C) and its effects on 5- hydroxytryptamine (5-HT), corticotropin releasing hormone (CRH) and adrenocorticotropic hormone (ACTH), and to explore their mechanism of regulating brain-gut axis function. Methods Totally fourty rats were randomly divided into four groups with 10 rats in each group, including normal control group, model group, low (5 mg/kg) and high (20 mg/kg) doses of Shuganjieyu Formula groups. Except for the normal control group, the other three groups of rats were gavaged with ice water for preparing the IBS-C model. Subsequently, the low and high doses groups of rats were treated with Shuganjieyu Formula for 2 weeks via oral gavage. The body weight of rats, total particle number and water content of fecal in the four groups were observed for 28 days. At last, serum of inferior vena cava and colon were collected from rats after anesthesia of 3% pentobarbital sodium. The morphological changes of colon tissue and the count of mast cells (MC) were observed with light microscope. Immunohistochemistry was used to detect the positive expression of 5-HT in the colon of rats in each group. The serum levels of 5-HT, CRH and ACTH were detected by ELISA. Results Compared with the normal control group, the body weight of rats in IBS-C model group increased slowly, the number of fecal grains and the fecal water content decreased (P<0.05); the number of MC cells, the positive expression of 5-HT in colon tissue increased significantly in IBS-C group (P<0.05); the content of 5-HT in serum increased significantly, while the content of ACTH decreased significantly in IBS-C group (P<0.05). Colonic morphology showed no abnormal pathological changes in IBS-C model group. Compared with the model group, the body weight, fecal granule number and fecal water content of rats in each dose group of traditional Chinese medicine increased, but the differences were not statistically significant. The colon tissue structure of rats in each dose group of traditional Chinese medicine was normal. Compared with the model group, the number of MC cells and the positive expression of 5-HT in each dose group of traditional Chinese medicine decreased significantly (P<0.05), the content of 5-HT in serum decreased significantly, while the content of ACTH increased significantly (P<0.05). The dose-effect relationship between the low and high doses groups of Shuganjieyu Formula was not statistically significant. Conclusion The traditional Chinese medicine Shuganjieyu Formula can effectively improve the dysfunction of brain-gut axis in IBS-C model rats, and has a good therapeutic effect on IBS-C.

Research Progress in Rat Models of Intrauterine Adhesion

Shaofeng HUANG, Zhong LIN, Xuehong ZHU, Shuchen LIU, Li BIN

2022, 42(6):  560-565.  DOI: 10.12300/j.issn.1674-5817.2022.025

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Intact endometrium and proper endometrial thickness are the necessary conditions for normal uterine reproductive function. Intrauterine adhesion (IUA) can cause recurrent abortion and infertility, which seriously affects the normal reproductive function of patients. At present, the treatment methods for IUA include surgery elimination and use of estrogen, but there are problems such as poor effectiveness, high rate of recurrence, and still with low pregnancy rate. The specific pathogenesis and developmental mechanism of IUA and the repair mechanism of damaged endometrium are still unclear. Establishing an appropriate animal model is conducive to the research of human IUA in all aspects. Here, we introduced the common rat models of IUA in recent years, including physical and mechanical damage method, chemical damage method, biological damage method and combined damage method, and described its application from the aspects of modeling rate, stability, and pathogenesis of IUA, and discussed the observation indexes to evaluate the success of modeling, in order to provide useful reference for selecting appropriate modeling methods. Generally, the combined model of repeated endometrial damage and infection of pregnant rats may be more suitable for the clinical IUA caused by multiple abortions and infections after curettage.

Laboratory Animal Welfare and Ethics

A Brief Interpretation of CCAC Guide on Humane Endpoints of Animals: 2022 Edition

Xiaying LI, Yusheng WEI, Yonglu TIAN, Wanyong PANG

2022, 42(6):  566-571.  DOI: 10.12300/j.issn.1674-5817.2022.077

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The 2022 Canadian Council On Animal Care (CCAC) guidelines: Identification of Scientific Endpoints, Human Intervention Points, and Cumulative Endpoints (CCAC Guide) supplements existing laboratory animal humane endpoint theory according to the latest available literature. This article summarized the main content of the 2022 CCAC Guide, and elaborated and analyzed the determination, implementation and supervision of the scientific endpoints, humane intervention points, and cumulative endpoints of animal experiments, in order to provide useful reference and information.

Establishment and Interpretation of Group Standard for Laboratory Animal—Guideline for the Welfare of SPF Chicken

Haibo YU, Jie ZHOU, Jianhong LI, Changwen LI, Caixia Gao, Changyou XIA

2022, 42(6):  572-576.  DOI: 10.12300/j.issn.1674-5817.2022.095

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Specific pathogen free (SPF) chickens are the main laboratory poultry used in biomedicine and animal husbandry. SPF chickens are used for scientific research, production and testing of biological products for humans, livestock and poultry， so they are indispensable laboratory animals and raw materials. This paper introduced the background, purpose, scope, content, and significance of the group standard Laboratory Animal—Guideline for the Welfare of SPF Chickens. This standard provides the necessary welfare for SPF chickens in institutions, animal facilities, animal environments, daily management, experimental operations, and transportation. The implementation of the standard is aimed at regulating the management of SPF chickens, improving the quality of SPF chickens, and providing better services for research and production. It also promotes scientific development, protect human health, and bring the SPF chicken laboratory animal industry in line with international standards.

Laboratory Animal Management

The Current Status of Laboratory Animal License Administration in Guangdong Province

Huiping LI, Chujun LIANG, Weibo ZHAO

2022, 42(6):  577-582.  DOI: 10.12300/j.issn.1674-5817.2022.107

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This study examined and summarized the operations and achievements of laboratory animal license administration in Guangdong Province and presents preliminary suggestions based on the deficiencies in the current administration. The study utilized laboratory animal licensing data of Guangdong Province obtained from the National Laboratory Animal License Inquiry and Administration System, and uses a combination of statistical analysis, literature review, and comparative research. Various aspects of the workings of the laboratory animal license administration in Guangdong Province were analyzed, including license issuance, regional distribution, animal species, organization types, facility scale, and development trend of laboratory animal license administration in Guangdong Province to identify areas of improvements. The following measures were recommended to the Guangdong Province: increasing social awareness about the importance of using laboratory animals, promoting the publicity and implementation of laws and regulations on laboratory animals, strengthening the strategic resource reserve of experimental animals, improving the management system of aquatic laboratory animals, and optimizing information management. Through such measures, we aimed to provide reference for the administration of laboratory animals in other regions.