ILAR Journal, 2021, Vol. 62, No. 3, 337–342 https://doi.org/10.1093/ilar/ilac003 Advance Access Publication Date: 24 March 2022 Article ICLAS LAQ Network for the Promotion of Animal Quality in Research Patri Vergara1,2,∗ , Gregory Ballard3 , Cynthia Besch-Williford4 , Nobuhito Hayashimoto5 , Cynthia Pekow6 , Ana Perez7 , Katja Schmidt8 , William Shek9 , Martin Toft10 and Atsushi Yoshiki11 1 Veterinary School, Universitat Autonoma de Barcelona, Barcelona, Spain, 2 ICLAS LAQ Network Coordinator, 3 The Jackson Laboratory, Bar Harbor, Maine, USA, 4 IDEXX BioAnalytics, Columbia, Missouri, USA, 5 Central Institute for Experimental Animals, Kawasaki, Japan, 6 University of Washington, Seatle, Washington, USA, 7 Humodigen, Burlingame, California, USA, 8 German Cancer Research Center (DKFZ), Heidelberg, Germany, 9 Charles River Laboratories (RADS), Wilmington, USA, 10 QM Diagnostics, Radboud University, Radboud, Netherlands and 11 RIKEN BioResource Research Center, Ibaraki Japan *Corresponding Author: Patri Vergara, Facultat de Veterinaria, Universitat Autonoma de Barcelona, 08193 Barcelona, Spain. E-mail: patri.vergara@uab.cat Abstract ICLAS Laboratory Animal Quality Network (LAQN) programs currently consist of the Performance Evaluation Program (PEP), which focuses on microbial monitoring by and for laboratory animal diagnostic laboratories, and the Genetic Reference Monitoring Program (GENRef), which provides assay-ready reference DNA for genetic testing of mouse strains. Since 2008, PEP has grown to become a truly international program with participating laboratories in 5 continents. Launched in 2016, GENRef currently distributes DNA from 12 common inbred mouse strains for use in genetic monitoring of locally inbred colonies as well as for genetic testing of stocks, particularly genetically engineered stocks, of uncertain origins. GENRef has the capacity to include additional strains as well as additional species. PEP and GENRef provide the reagents at cost, as a resource to the international scientific community, in the interest of improving research quality in an environment of growing concern for research quality, rigor, and reproducibility. Key words: animal models, diagnostic testing, genetic testing, mice, rats, reproducibility of results, quality control, virus nutrition, environment, genetics, and disease status. Quality BACKGROUND assurance refers to how a process is performed to assure quality. Since its inception in 1956, a core objective of the International Quality control refers to the quality management procedures Council for Laboratory Animal Science (ICLAS) has been to pro- that fulfil quality requirements (eg, of accrediting organiza- mote international harmonization in the quality of research and tions). Toward this end, ICLAS has contributed to and published of the animals involved in research. The goal of this harmoniza- guidance regarding laboratory animal nutrition1 and laboratory tion is to mitigate confounding variables in research involving animal nomenclature,2,3 and LAQN current activities promote animal subjects both to enhance the quality and reproducibility quality assurance in disease assessment and genetic monitoring of the science and to reduce the numbers of animals required of laboratory rodents. to achieve rigor and validity. Animal health and well-being and In 1969, in conjunction with the World Health Organization, the quality of research are influenced by many factors, including ICLAS established 2 areas where reference centers for laboratory Received: January 19, 2022. Revised: February 1, 2022. Accepted: February 2, 2022 © The Author(s) 2021. Published by Oxford University Press on behalf of the National Academies of Sciences, Engineering, and Medicine. All rights reserved. For permissions, please email: journals.permissions@oup.com This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https://creative commons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com 337 338 Vergara et al. animals were defined: Virus Reference Centers for Rodents and PEP’s aim was to improve health monitoring by providing Histocompatibility Reference Centers for Mice, followed later by well-characterized serum and microbiology samples to be used the addition of other laboratories for other species (ie, dogs and to assess the accuracy of testing by participating diagnostic non-human primates) and biomarkers. In 1983, these reference laboratories. Network “member laboratories” prepare standard- laboratories were replaced by the ICLAS Monitoring and Refer- ized sera and verified microbiological specimens and send them ence Centers Program. Under this program, the Central Institute as unknown samples for analysis to any diagnostic laboratory for Experimental Animals in Japan served as a monitoring center (“participating laboratory”) in the program. A comparison of the to promote breeding and maintenance of microbiologically and participant laboratory’s results with the verified contents of the genetically standardized laboratory animals to standardize and specimens, as detailed in a report (expected results) sent later harmonize research and testing procedures. by the LAQN, indicates the accuracy and/or sensitivity of the Several organizations have implemented the so-called “ring participating laboratory’s assays. test,” also called a ring trial or proficiency test, which is an inter- Key features of PEP eligibility and participation (for partici- laboratory test to evaluate the performance of testing laborato- pating laboratories) include the following: ries based on analyses of verified samples. These are established 1. The program is open to all diagnostic laboratories worldwide approaches to monitor the quality of analytical results, identify with no specific eligibility requirements; assays that need improvement, and serve as quality control 2. Diagnostic performance is self-assessed, and participating measures to demonstrate competency to accreditation bodies. laboratories are not asked to submit reports of their results Examples of other regional quality control initiatives included to ICLAS or any other agency; and the National Diagnostic Quality Assurance Program, based at 3. The program is self-financed; participating laboratories pay the Rockefeller University (New York, NY, USA), supported by in advance to cover the costs of sample production, shipment, the National Institutes of Health in the United States from and program administration. about 1978 to 1995, and the Quality Assurance Program of the Deutsches Krebsforschungszentrum, the inter-laboratory testing (ring-testing) program in Europe where the subscribing laborato- IMPLEMENTATION OF PEP ries received the same unknown sample for testing. However, the Following a successful development stage,5 PEP became fully objective of these programs was to reach a diagnostic consensus operational in 2008 with 2 production member laboratories, rather than to provide already confirmed samples to serve as Charles River USA (RADS) and RADIL (University of Missouri), reference specimens for laboratories.4 and 9 participating laboratories. Since 2008, the network has The ICLAS Monitoring and Reference Centers Program was incorporated 4 more production member laboratories (Central active until 2006 when it was replaced by the current Labora- Institute for Experimental Animals, Japan; QM Diagnostics, Rad- tory Animal Quality Network (LAQN), which focuses on provid- boud University, Netherlands; German Cancer Research Center, ing verified samples to research laboratories to independently Germany; Cerberus Sciences, Australia), and in 2010, the SIAL assure the accuracy of their in-house diagnostic tests on rodent laboratory at the Universitat Autonoma de Barcelona (Spain) genetics and infectious agents. was established as the specimen storage and distribution center, thanks to a grant from the Spanish Ministry of Science. LABORATORY ANIMAL QUALITY NETWORK In 2004, after discussions with internationally recognized scien- PEP PARTICIPANTS tists in the field of health monitoring, a decision was made to In its first 12 years, PEP has grown to become a truly international replace the ICLAS Monitoring and Reference Centers Program program with participating laboratories from 5 continents. Since with a new initiative more international in scope and able to 2008, more than 1000 specimens have been shipped worldwide, serve as a truly transnational reference in the field of high- and PEP participants have grown from 10 in 2008 to an average quality laboratory animal models. of 24 for the last 5 years of the program. In 2020, the combination These discussions led to the creation of the ICLAS Network program was the most popular (15), compared with serology for Promotion of Animal Quality in Research (LAQN) in 2006, only (5) and microbiology only (3). A total 34% of participating whose founding members were Patri Vergara and Cecilia Car- laboratories were based in Asia, 33% in Europe, 21% in the United bone (ICLAS Governing Board members), Bill Shek (RADS, Charles States, 8% in Oceania, and 4% in South America (Table 1). River, USA), Lela Riley (RADIL, University of Missouri), Werner Nicklas (German Cancer Research Center), Esther Schoonder- mark (Radboud University, the Netherlands), and Marge Strobel PEP PROCEDURE (The Jackson Laboratory, USA). The LAQN’s mission was to develop programs to help achieve Current PEP Programs ICLAS’s goal of improving the quality of research and the ani- Each laboratory participating in PEP can choose to join for serol- mals used in research in the areas of animal health and genetic ogy and/or microbiology programs and receives 10 or 20 samples monitoring. The programs developed under the umbrella of the of positive sera and/or microbiology according to the program LAQN were intended to have an educational component (orga- chosen (Table 2). nization of workshops and seminars) as well as to provide tools for the benefit of the laboratory animal community. PEP Specimen Production 1. Network member laboratories produce standardized serology PERFORMANCE EVALUATION PROGRAM and/or microbiology specimens. LAQN’s first initiative focused on microbial diagnostic testing 2. Specimen samples are sent to another network member for health monitoring and led to the creation of the ICLAS laboratory for confirmation, that is, to verify that the target Performance Evaluation Program (PEP) in 2007. agent(s) can be detected. If the target agent is not detected ILAR Journal, 2021, Vol. 62, No. 3 339 Table 1. PEP Participating Laboratories: 2008–2020 and the Type of Program Subscribed z Serology ‡ Microbiology only „ Combination  Not participating Participating Laboratories 08 09 10 11 12 13 14 15 16 17 18 19 20 001 EU „ „ „   „ „      002 UK „ „„ „ „ „    003 EU „ „ „ „ „ „„ „ „ „ „ „ „ 004 EU „ „ „ „ „ „„ „ „ „ „ „ „ 005 AU z „ „ „ „ „„ „ „ „ „ „ „ 006 US z z z z         007 US „ „          „ 008 AS „ „ „ „ „ „„ „ „ „ „ „ „ 009 AU „ „ „ „ „ „„ „ „ „ „ „ „ 010 EU z z z z  z ‡ 011 EU z „ „ „ „ „ ‡ ‡ ‡ ‡ ‡ ‡ ‡ 012 EU z z z z z z z z z z z z z 013 US „ „   „ „ „ ‡ ‡    014 AS z „ „ „ „ „  „ „ „ „ 015 AS z „ „ „ „ „ „ „ „ „ „ 016 US „ „ „ „ „ „ „ „ „ „ „ 017 US „ „ „ „ „ „ „ „ „ „ „ 019 AS „  „   „     018 SA „ „ „ „ „ „ „ „ „ 020 EU „ „ „ „ „ „ „ „ „ 021 EU „ z z z „ z z z z 022 AS „ z  „      023 US z z z z „ „ z z z 024 EU ‡ ‡ ‡ ‡ ‡     025 AS ‡ „ „ „ „ ‡ ‡ z 026 EU „   „      027 AS „ „ „ „ ‡ ‡ ‡ ‡ 028 AS „   „     029 AS „    „    030 EU „ „ „ „ „ „ „ 031 EU ‡      032 AS z „ z z z 033 EU „     034 EU ‡ ‡    035 EU ‡     036 AS „ „ „ „ „ 037 AS z     038 AS z    039 EU „ „ „ 040 US „ „  041 AS „  Total Serology only 4 2 4 2 2 4 4 4 4 4 4 5 5 Total Microbiology only 0 0 0 0 1 2 2 3 5 4 3 3 3 Total Combination 6 9 1 1 16 18 1 1 20 18 15 16 15 Total Participants 10 11 14 13 19 24 23 25 29 26 22 24 23 or if a specimen proves positive for an unintended agent, the Characterization and Quality Control of PEP Specimens batch would be rejected. 3. When specimens are confirmed, network member laborato- Table 3 summarizes the steps to produce and validate PEP spec- ries send specimens to the distribution center for distribution imens before incorporating them into the program for distribu- to participating laboratories. tion to the participating laboratories. 340 Vergara et al. Table 2. Optional Programs Inside PEP Programs Shipments/Specimens Serology only 1 annual shipment of 10 sera specimens Microbiology only 1 annual shipment of 10 microbiology specimens Combination 1 annual shipment of 10 sera and 10 microbiology specimens PEP, Performance Evaluation Program. Table 3. Production, Characterization, and QC of PEP Specimens Production and Characterization of PEP Specimens PEP Specimen QC • All specimens generated under strict conditions and rigorously characterized. • Acceptance criteria: Pure and Potent • Infectious agents are obtained from known sources and sequenced to • Immune serum confirm identity. − Seropositive to inoculated pathogen only − Moderate to strong reaction by standard assays • Experimental animals are inoculated, and the serum and relevant tissues are • Infectious specimen collected and aliquoted for use as standardized specimens. − Free of extraneous pathogens − Easily detectable concentration of pathogen • Aliquoted specimens are evaluated by 2 laboratories to confirm quality PEP, Performance Evaluation Program; QC, quality control. Table 4. Steps of PEP Application and Participation by Participating Laboratories June–December, year 1 January–May, year 2 1. Participants submit PEP application form 5. Participants receive specimens from PEP distribution center by international courier 2. Participants receive invoice 6. Participants analyze specimens 3. Participants pay participation fee by bank transfer 7. Participants request and are sent expected results (Fig. 2) 4. Participants receive PEP Participation Certificate 8. Participants compare their results with expected results to determine accuracy and/or sensitivity of their assay performance 9. Participants are requested to provide feedback regarding any concerns or discrepancies in their results. These responses are voluntary and are presented anonymously in PEP reports. PEP, Performance Evaluation Program. PEP SELF-ASSESSMENT PEP library is open, and, although it is difficult to have a complete inventory of potential new findings, PEP also tries to include less PEP was designed as a self-assessment program for the diagnos- common agents isolated by the producer laboratories. tic laboratories. Participating laboratories can use the accuracy Second, PEP standard samples are increasingly expensive to of their results to check their own systems and to fulfil the produce, particularly serum samples because they imply the requirements of certifying agencies such as ISO. Table 4 summa- infection of new animals. PEP producer laboratories are investing rizes the steps of PEP application and participation by participat- their own resources and their own quality assurance and quality ing laboratories. After completing their analyses, participating control and are key participants in the quality of biomedical laboratories request the expected results for their samples. An research. example is shown in Figure 1. Although participating labora- tories are not obliged to send back a report to the LAQN, any feedback is appreciated because it helps to detect any problems and gives the opportunity to exchange information with the GENETIC REFERENCE MONITORING PROGRAM producer laboratories. In parallel to the development of PEP, in 2008, the network began to develop a genetic monitoring program. The principal aim FUTURE CHALLENGES FOR PEP was to address the issues of genetic quality assurance arising Quality assurance and quality control are essential in diagnostic from the dramatic increase in rodent strains and stocks and laboratories. PEP provides the means for member laboratories the use of genetically modified animals. Genetic quality assur- to self-assess the quality of their assays. Our current aim is to ance was recognized as crucial for ensuring consistency and increase the number of participating laboratories and to facili- reproducibility in experimental results both within and across tate improvement in laboratory animal diagnostics. research institutions. Of note, researchers in many regions can- PEP and diagnostic laboratories are facing 2 major challenges. not readily purchase stocks or strains of laboratory mice or rats First, the pathogens affecting laboratory animals are changing, of known genetic background from quality commercial vendors. as exemplified by the current rarity of some pathogens that The logistics and costs of international transport are often too were common 20 years ago, for example, Sendai virus, and the daunting, given the challenges of long distances, connecting emergence of potentially new pathogens by the increased use of flights, weather and temperature extremes, insufficient oxygen immunodeficient mice and xenotransplants. For this reason, the in cargo holds, flight and customs delays, and the attendant ILAR Journal, 2021, Vol. 62, No. 3 341 Figure 1: Example of expected results for the 2019 PEP Combination Program. risks of physical trauma or pathogen contamination en route, GENRef was developed in 2 phases: first, a focus on educa- if the animals survive the journey. Thus, many facilities breed tion and training to increase awareness of the importance of their own animals but lack the means to assess and correct for genetic quality monitoring; and second, the establishment of a genetic drift and mutation or to refresh the lines with strains of self-assessment genetic monitoring program. confirmed genetic background. GENRef program eligibility and participation has 3 key fea- tures in common with PEP: IMPLEMENTATION OF THE GENREF PROGRAM 1. Open to any research laboratory worldwide with no specific Education and training on the importance of genetic quality eligibility requirements; monitoring started in 2010, with presentations at various 2. Monitoring is self-assessed; and laboratory animal science conferences such as American 3. Each participant pays in advance to cover the costs of speci- Association for Laboratory Animal Science, Asian Federation men production, shipping, and administration. of Laboratory Animal Science Associations, and Federation of 342 Vergara et al. European Laboratory Animal Science Associations in conjunc- genetic backgrounds are increasing.8,9 ICLAS will continue to tion with 2 papers published in 2013: 1 in Mammalian Genome6 provide information on its website on the equipment, reagents, and the other as a congress paper at the 12th FELASA-SECAL and protocols needed to use the GENRef program as well as via Congress.7 regional, national, and international meetings and via educa- In 2016, the GENRef program was launched and has since tional programs and publications. provided 118 DNA specimens to 13 GENRef participants, mainly The number of DNA strains offered will be expanded to in the Americas and Asia, in accordance with the following aims ensure that most used strains around the world are available. and procedures. For example, due to the increase of CRISPR techniques, there has been an increase in C57BL/6 background mice and less use of other strains. Although GENRef already includes 2 C57BL/6 Aims and Procedures sub-strains, additional C57BL/6 sub-strains will strengthen the Reference DNA from commonly used rodent strains and stocks program. is made available to enable research institutions worldwide (pro- gram participants) to 1. assess if their colonies are genetically sound and representa- CONCLUSION tive of the expected genetic background; and 2. assess animals of mixed or uncertain backgrounds, for exam- Both PEP and GENRef are established programs with functional ple, genetically engineered animals developed from multiple capacity to expand in response to increasing concerns for strains. research quality, rigor, and reproducibility and in response to increasing demands for Quality Assurance resources from diagnostic laboratories and research programs. Species Further details about the ICLAS LAQ Network programs and The initial focus is on the genetic monitoring of rodents publications as well as application forms to participate in the (mice and rats), both inbred and outbred (or closed colony PEP and GENRef programs can be found at the ICLAS website at animals), although to date, the program has only provided inbred www.iclas.org. mouse DNA. Potential conflicts of interest. All authors: No reported con- DNA Strains flicts. The program currently provides reference DNA from 12 common inbred strains/sub-strains of laboratory mice: References • J strains: C57BL/6 J (reg. #664; BALB/cJ (reg. #651); NOD/LtJ (reg. 1. Coates ME; International Council for Laboratory Animal Sci- 1976); A/J (reg.#646) ence. ICLAS guidelines on the selection and formulation for diets for • Tac strains: C57BL/6NTac; BALB/cAnNTac; C3H/HeNTac; animals in biomedical research. London: International Council 129S6/SvEvTac for Laboratory Animal Science; 1987. • Jcl strains: C3H/HeJJcl; DBA/2NJcl; FVB/NJcl 2. The Committee on Standardized Nomenclature for Mice. Guidelines for nomenclature of genetically determined bio- chemical variants in the house mouse, Mus Musculus. Biochem GENREF SPECIMEN PRODUCTION Genet. 1973; 9(4):369–74. 3. Festing M, Kondo K, Loosli, R, Poiley SM, Spiegel A. Inter- 1. DNA providers: DNA for the first batch of samples was pro- national Standardized Nomenclature for Outbred Stocks of vided by 3 internationally recognized breeders (donor breed- Laboratory Animals. ICLA Bulletin. 1972; (30):4–17. ers): The Jackson Laboratory, Taconic Biosciences, and Central 4. Boot R, Reubsaet FA. Identification of bacterial strains by lab- Institute for Experimental Animals. All costs for this first oratories participating in the Deutsches Krebsforschungszen- batch of 100 units per strain were covered by the breeders trum programme quality assurance. Lab Anim. 2007; 41: as a donation to the program. 481–91. 2. DNA production: Donor breeders isolated DNA from 4 mice 5. Goto K, Hayashimoto N, Ishida T, et al. First trial in the devel- of each strain from the following tissues: tail, lungs, heart, opmental phase of the “performance evaluation program” and kidneys. DNA was extracted and placed in tubes (units) based on the ICLAS animal quality network program: self- at concentrations of 25 ng/μL and a total of 10 μL or 250 ng/10 assessment of microbiological monitoring methods using test μL. samples supplied by ICLAS. Exp Anim. 2009; 58(1):47–52. 3. DNA confirmation: Donor breeders provide DNA samples 6. Fahey JR, Katoh H, Malcolm R, et al. The case for genetic to another donor breeder or to an ICLAS Network member monitoring of mice and rats used in biomedical research. laboratory for confirmation. Mamm Genome. 2013; 24(3–4):89–94. 7. Perez A, Fahey JR. Aspects of genetic monitoring in rodents. Paper presented at 12th FELASA-SECAL Congress, Barcelona, June, FUTURE CHALLENGES FOR GENREF 2013. The GENRef program seeks to raise recognition of the need to 8. Threadgill D, Dlugosz A, Hansen L, et al. Targeted disruption of assess and monitor genetic backgrounds of research mice and mouse EGF receptor: effect of genetic background on mutant to increase awareness and participation in the GENRef program. phenotype. Science. 1995; 269:230–4. Examples of sub-strain variations and research complications 9. Mekada K, Yoshiki A. Substrains matter in phenotyping of related to complex, under-characterized, or otherwise uncertain C57BL/6 mice. Exp Anim. 2021; 70(2):145–60.