sample case study rica

RICA Test – “What Every RICA Exam Taker Ought To Know Before Walking Into The Testing Center”

Understanding the creation and development of the RICA test will support your test preparation and even aide in getting answers correct on the real exam. Make sure your RICA test prep book takes the same paradigm as the information below.

Getting your K-8 Multiple Subjects credential in California means you must get a passing score on the Reading Instruction Competency Assessment (RICA).

The California legal code for education is straight to the point: California Commission on Teacher Credentialing (CTC) must “develop, adopt, and administer a reading instruction competence assessment…to measure an individual’s knowledge, skill, and ability relative to effective reading instruction,” according to Section 44283, and is committed to improving student’s reading skills. In order to achieve this legislated mandate, the Reading Instruction Competency Assessment (RICA) has been developed.

The purpose of the RICA exam is to make sure that candidates have the necessary training to deliver effective reading instruction to students. The goal is that students will have the ability to appreciate and interpret all kinds of texts.

You’re advised to begin your test prep as early as possible to avoid risking delaying your credential and having to pay expensive RICA testing fees a second or third time. Be sure you use a high quality RICA study guide from the very beginning. Don’t waste critical review time with free RICA practice test questions and study materials.

California has contracted the Evaluation Systems at Pearson to assist in the development and administration of the exam.

Links To Free RICA Test Prep Tools And Tactics:

• RICA Test Prep
• RICA Pass Now – Secrets

How To Register For The RICA Test

• Candidates can register for the written exam on the RICA test website. For Video Performance Assessments registration must be done by mail.
• In order to register for the RICA exam, applicants must provide their Social Security number and date of birth. This information will allow test-takers to view their scores on line.
• The cost of both the written examination and video assessment is $171. Additional charges will be incurred for extra copies of the test results, or a change in registration.
• Payments can be made using Visa or MasterCard, or a debit or check card that has a VISA or MasterCard logo and doesn’t require a pin number. Payments made by mail must be paid by check or money order.
• RICA test registration should be done as early as possible as appointments are scheduled on a first come, first serve basis, and seating is limited.
• The location Computer based test centers can be found on the RICA website and are available throughout California.

What You’ll See At The RICA Exam Center

Candidates should plan to arrive 30 minutes before their scheduled test time. Arriving more than 15 minutes after the start time could result in not being admitted to the test, and no refund will be offered.

At the test center, part of the verification process involves applicants being photographed, palm scanned and providing an electronic signature. Failure to comply with the verification process will result in not being allowed to take the test and not being reimbursed.

Understanding the contents of the test will bring you one step closer to passing!

There are two RICA test options available, the Written Examination or the Video Performance Assessment. The written test is available year round in a computer based format.

The Video Performance Assessment evaluates an applicant’s knowledge and skills based on classroom performance. A candidate must create a video of themselves as they teach students to read which will then be assessed.

The Reading Instruction Competency Assessment written examination allows applicants 3 hours and 45 minutes to complete the exam. It is divided into five domains and has a total of 70 multiple choice questions.

• Planning, Organizing and Managing Reading Instruction contains 10 multiple choice questions and is weighted at approximately 10 percent of the total score.
• Word Analysis contains 24 multiple choice questions, 1 word response, and represents 33 percent of the test.
• Fluency contains 8 multiple choice questions, 1 word response, and is 13 percent of the final score.
• Vocabulary, Academic Language, and Background knowledge contain 15 multiple choice questions, 1 word response and are weighted at 20 percent of the final total.
• Comprehension has 13 multiple choice questions, 1 word response, and represents 23 percent of the test.
• The RICA exam also includes one case study that assesses all domains and requires 300 to 600 words and is worth 20 percent of the exam.
• The multiple choice questions are computer scored and are based on the number of correct answers. There is no penalty for incorrect answers, so when uncertain, it is better to guess.

Reading And Interpreting Your RICA Score Results

RICA exam results will be available on the published score report date and will be available on line for 45 days thereafter. The raw score from the test is converted to a scaled score that ranges from 100 to 300. In order to pass an applicant must achieve 220 or higher.

The RICA test score report will indicate your passing status. If you failed the exam it will show the score. Other performance indicators include indices for each RICA domain. If you passed the exam, you will receive a Passing Status Verification Slip, which you may need to provide to your college or University before receiving a recommendation to take the Multiple Subject Teaching Credential or the Education Specialist Instruction Credential.

The best way to prepare is to get RICA practice tests and preparation materials by real credentialed California educators who passed.

Teaching young minds is an important job! Make sure you have the necessary skills to succeed!

See: 7 Secrets Learned From RICA Test Takers Who Failed .

Author: Mark Dahlson

• Click here: RICA Secrets >>>

RICA Subtest 3 Study Guide

Subtest 3 of the RICA, or Reading Instruction Competence Assessment, is a test required for individuals seeking to become teachers in California.

This subtest covers content and skills related to the assessment and evaluation of reading development, including the ability to use a variety of assessment tools and techniques to monitor and track student progress.

In other words, Subtest 3 covers important topics like principles of standards-based reading instruction, organizing instruction that meets the needs of all learners in the classroom, alternative assessments for students with an Individualized Education Program (IEP) or Section 504 Plan, much more.

In order to pass Subtest 3, individuals must demonstrate a thorough understanding of the principles of reading assessment and evaluation and be able to apply this knowledge to the reading instruction process.

This includes being able to select and use appropriate assessment tools and techniques, interpret assessment results, and use assessment data to inform instruction and make decisions about student progress.

It is important for teachers in training who are preparing for a career in education to understand the content and skills covered in Subtest 3 of the RICA, as the ability to effectively assess and evaluate student reading development is a critical component of effective reading instruction.

By understanding the principles of reading assessment and learning how to use assessment data to inform instruction, you will be well-prepared to support the reading development of the students in your future classrooms.

In summary, Subtest 3 not only includes a lot of terminology that you’ll need to know, but it is also the subtest where you will write your case study written response.

Challenges of Subtest 3

Through our extensive experience preparing students for the RICA, I can confidently say that the challenges most students experience when taking Subtest 3 are as follows:

Difficulty selecting and using appropriate reading assessment : One common challenge for students taking Subtest 3 is the ability to select and use appropriate assessment tools and techniques to monitor and track student progress. This includes understanding the different types of assessments available and how to use them effectively in the reading instruction process based on individual student’s needs.

Difficulty interpreting assessment results : Another challenge that many students face when taking Subtest 3 is the ability to interpret assessment results and use the data to inform instruction and make decisions about student progress. This requires a thorough understanding of the principles of reading assessment and evaluation and the ability to apply this knowledge to real-world situations.

Time management : The RICA is a timed test, and some students may struggle with managing their time effectively in order to complete all of the questions within the allotted time frame.

Test anxiety : Finally, some students may experience test anxiety, which can impact their performance on the RICA. It is important for students to find strategies to manage their test anxiety and stay focused during the exam.

Overall, these are the main challenges that most students experience when taking Subtest 3 of the RICA. It is important for students to be well-prepared and familiar with the key concepts and principles covered on the test in order to succeed.

To help aspiring teachers fulfill their dream of becoming a credentialed teacher, we have written a study guide that fully prepares teachers in training the techniques to passing the multiple choice and written response sections of the exam.

Here, you can see a preview of the study guide.

Keep in mind that our RICA Test Prep program includes not only the study guide, but self-paced video lessons, interactive quizzes, full-length practice tests, digital flashcards, and even a Subtest audio masterclass for when you’re on the go.

You’re going to love this prep, because it’s going to be your key to pass your RICA!

Pass the RICA Test

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Roula Khalaf, Editor of the FT, selects her favourite stories in this weekly newsletter.

These five sets of case studies highlight how law firms in Asia-Pacific are innovating as businesses.

They feature examples of law firms changing how they manage their own people, and how they are reinventing services and delivery models.

All the case studies were researched, compiled and ranked by RSGI. “Winner” indicates that the organisation won an FT Innovative Lawyers Asia-Pacific award for 2024

Read the other FT Innovative Lawyers Asia-Pacific ‘Best practice case studies’, which showcase the standout innovations made for and by people working in the legal sector:

Practice of law In-house

People and skills

WINNER: Gilbert + Tobin Originality: 8; Leadership: 9; Impact: 8; Total: 25 Last year, the firm ran an “AI Bounty” competition, offering staff a total of A$20,000 in prizes for their best ideas on deploying artificial intelligence at work. The contest attracted 106 submissions, with the money split among five main award winners and 50 smaller prizes. The firm will develop the best ideas identified, including tools to review a privacy policy and enhance due diligence.

Inkling Legal Design O: 9; L: 8; I: 7; Total: 24 The firm developed an online course that encourages writing in plain, straightforward English and provides a benchmark against which other lawyers can compare how well they write about ambiguous areas of law. The application, which launched last year, is designed to improve efficiency and accuracy when writing and has been used by eight clients so far.

Highly commended

Lander & Rogers O: 8; L: 8; I: 7; Total: 23 In its continuing partnership with Melbourne’s Monash University, the Australian firm invited five law students — dubbed “AI investigative agents” — to interview practice group heads and work with the firm’s innovation team to examine scenarios in which the technology might be applied. The training initiative identified more than 40 examples for possible use and the students’ insights on the topic have been published by the firm.

MinterEllison O: 7; L: 8; I: 7; Total: 22 The firm created its own internal cryptocurrency to reward staff for taking part in its online training sessions. The so-called Mintcoins can be exchanged internally for charity donations or gift cards and have helped encourage the completion of more than 1,900 training modules by 850 people.

Rajah & Tann Singapore O: 7; L: 7; I: 8; Total: 22 The firm identified several common skills and qualities required by its lawyers working across the various business sectors, jurisdictions and languages in the region. These include project management and communication skills aside from specific legal knowledge. Lawyers can receive 50-plus hours of training and at least half of the programme comprises practical activities.

Ashurst O: 6; L: 7; I: 8; Total: 21 Lawyers at the firm can now use an online tool to highlight their availability and expertise, to help with allocation of work. In the first eight months of use, it received more than 720 notifications of availability from lawyers across the firm’s Asia-Pacific offices.

Khaitan & Co O: 7; L: 7; I: 7; Total: 21 The firm worked with a consultancy to set up a process that assesses lawyers in areas such as productivity and business development skills, to decide if they are ready for partnership promotion.

Nagashima Ohno & Tsunematsu O: 8; L: 6; I: 6; Total: 20 The firm created a programme where associates work with partners to develop new client relationships in emerging practice areas. These areas include the latest tech developments, sports, and agriculture and fishing industries.

King & Wood Mallesons O: 6; L: 6; I: 7; Total: 19 The firm added new modules in AI, process improvement and change management to its “legal transformation belts” programme, which grades and certifies digital skills and is also available to clients.

Knowledge and data

WINNER: MinterEllison Originality: 7; Leadership: 9; Impact: 8; Total: 24 In December 2023, the firm’s environment and planning team led a pilot of a generative AI tool that can draft legal documents that are roughly 80 per cent assembled in under a minute. Replicating a junior lawyer’s work, the model draws information from a repository of the firm’s historic advice, and other sources, to provide a draft that senior lawyers can check and amend. The pilot involved 50 lawyers and the firm now plans to roll out the tool across the practices. Commended individual: Simon Ball

King & Wood Mallesons O: 7; L: 8; I: 8; Total: 23 The firm’s commercial real estate practice developed a system based on records of past property transactions to identify market trends across Asia. Using data visualisation software, the tool helps identify patterns across the firm’s global property work, such as popular drafting clauses and market standards. According to the firm, the tool has reduced typical time taken for some research tasks from up to four hours to just minutes.

JunHe O: 6; L: 8; I: 8; Total: 22 To encourage use of its knowledge-management system, the Chinese firm added training materials along with a feature that automatically logs when training sessions are taken. The firm also rewards its 700-plus lawyers for adding good quality data to the platform by tying this to their bonuses. This has led to an increase in activity on the platform, with 25,000 clicks recorded per month in 2023.

Khaitan & Co O: 8; L: 7; I: 7; Total: 22 The firm sifted 17mn items to identify 841,000 relevant documents for future case work and integrate them into a searchable system. AI tools, under development, will be able to create summaries of the documents and perform predictive analysis on contracts.

Anand and Anand O: 7; L: 7; I: 7; Total: 21 The Indian firm created a “matter knowledge” bank where users can search for a case and generate a summary of relevant details. It spent three years digitising decades’ worth of physical documents and started using the repository in June 2023. The firm hopes to improve processes such as document drafting and trial preparation and encourage internal collaboration.

Clifford Chance O: 6; L: 7; I: 7; Total: 20 The firm developed a tool to help with billing when a client’s requirements change during a transaction, and launched it in the Asia-Pacific region. The spreadsheet-based system gives an improved overview of work done on particular case work with live projections, allowing significantly faster billing decisions.

Sprintlaw O: 7; L: 8; I: 5; Total: 20 In 2023, the Australian firm launched a knowledge-sharing platform for staff servicing smaller businesses. The internal resource has halved the time taken for its lawyers to create some documents, such as standard shareholder agreements.

Hogan Lovells O: 6; L: 7; I: 6; Total: 19 The lawyers conducted a document review for an anti-bribery investigation using AI software that required training in Vietnamese. The subsequent search has identified 150,000 documents for scrutiny by lawyers.

Digital tools

WINNER: Clifford Chance Originality: 8; Leadership: 9; Impact: 8; Total: 25 The firm partnered with tech company Microsoft to create a generative artificial intelligence bot, launched in late October, that tracks and summarises press releases published by Hong Kong regulators.

Lawyers in the region took the lead on ensuring the tool had appropriate understanding of legal jargon and included a summarisation and context extraction function that will save the firm an estimated 480 hours of associate and trainee time per year. Output can be turned into interactive graphs and trends, which the lawyers use to advise clients.

Yulchon O: 8; L: 8; I: 7; Total: 23 The South Korean firm created a service to help clients better comply with the country’s Serious Accidents Punishment Act (SAPA), introduced in 2022, which puts greater responsibility on businesses to ensure safety in their operations and facilities.

The service involves a free self-diagnosis tool that shows clients where they are most at risk of violating SAPA. The firm also uses automation to track news reports of SAPA-related incidents and has created a training programme with videos offering insights into the law. The programme uses an AI-powered search engine to help clients find specific information.

Clayton Utz O: 7; L: 8; I: 7; Total: 22 The Sydney-based firm launched an AI-generated compliance portal, Obligations Navigator, in December 2023. In a client assessment, the portal analysed 100 examples of case law and produced a hyperlinked list of 42,000 obligations, described in plain English and checked by human lawyers. Summarising compliance requirements can be labour-intensive, so the portal saves time and resources and gives the client a clear and comprehensive compliance process — helping them understand which obligations they must comply with, and how.

Lander & Rogers O: 7; L: 7; I: 7; Total: 21 The Australian firm developed an AI tool to extract relevant information from files submitted alongside compensation claims. The innovation and compensation law teams partnered with the firm’s legal tech incubator, Halisok, to digitise the manually-intensive sorting process in mass litigation and class action suits.

Nagashima Ohno & Tsunematsu O: 5; L: 8; I: 8; Total: 21 Led by managing partner Soichiro Fujiwara, the firm’s technology start-up MNTSQ designed an AI-powered search engine that allows lawyers to search an internal contract database for relevant Japanese clauses and provisions, after the firm found public search engines such as Google were insufficient.

Rajah & Tann Singapore O: 7; L: 7; I: 6; Total: 20 In June 2023, Singapore’s Urban Redevelopment Authority implemented new requirements for anti-money laundering checks in residential property purchases. The Singaporean law firm launched an AI tool that automates these due diligence checks of potential buyers, which is now used by 30 property developers in the country.

MinterEllison O: 5; L: 7; I: 7; Total: 19 The firm developed a contract review tool to help mining group Anglo American manage its supply chain and contracts. The firm approached the miner — a long-term client — after it struggled with the management of contracts. Lawyers used the company’s data to build the tool from scratch in 2023. Commended individual: Benjamin Fox

Digital strategy

WINNER: A&O Shearman Originality: 8; Leadership: 9; Impact: 8; Total: 25 In partnership with legal tech company Harvey, A&O Shearman (formerly Allen & Overy) was among the first law firms to make wide use of a generative AI tool in early 2023.

Capitalising on the publicity this created, it then launched an AI client working group in Asia Pacific where 81 participants from 19 companies paid the firm for advice on generative AI’s potential legal implications and practical lessons about adoption of the technology in a big organisation.

Highly Commended

Ashurst O: 7; L: 8; I: 8; Total: 23 The firm has run pilots and trials of generative AI that involved more than 400 staff in 23 offices including a competition to identify future applications and blind trials testing applications against humans. The strategy has been implemented worldwide, with a prominent role played by the team from Australia. The firm says nearly 90 per cent of staff felt its technology focus was preparing them for the coming years.

Mayer Brown O: 7; L: 8; I: 8; Total: 23 The Hong Kong office led the rollout in 2023 of AI tool Harvey for use in research, drafting, and data analysis. It is also used to cut the time spent summarising local case law, to improve due diligence, and Chinese-to-English translations.

PwC Asia Pacific O: 7; L: 8; I: 7; Total: 22 The firm’s Asia-Pacific business merged its legal and NewLaw legal services divisions to help clients implement related technology.

Internally, the firm is using AI tool Harvey and its own virtual assistant ChatPwC, as well as experimenting with other relevant tools. The firm recorded more than 18,000 queries being submitted to Harvey in the first six months of using it in the region and estimates that the application saved the firm 9,000 hours of time in that period.

Rajah & Tann Singapore O: 6; L: 8; I: 7; Total: 21 The firm is working with its technology arm Rajah & Tann Technologies to bring in external software that will encourage lawyers to embrace digitisation fully and prepare for the future adoption of AI systems. Examples include applications that are designed to cut the time spent on research and to locate the relevant contract clauses from a centralised database.

JunHe O: 6; L: 7; I: 7; Total: 20 The Chinese law firm’s tech team created a tool that automates the identification and redaction of sensitive material from documents. This tool helps lawyers protect sensitive data and better comply with data protection laws in China when using generative AI.

Trilegal O: 6; L: 7; I: 7; Total: 20 Trilegal’s digital innovation group is leading technological advances at the Indian law firm, creating a knowledge management system and AI-based dashboards to monitor work progress and preparing existing systems to incorporate generative AI.

New solutions

WINNER: Inkling Legal Design Originality: 8; Leadership: 9; Impact: 8; Total: 25 The firm advised the Australian Nuclear Science and Technology Organisation (ANSTO) on redesigning its commercial project agreements. The lawyers simplified the contracts used by the public research body for project partners, which predominantly include scientists with a non-legal background. Clauses were simplified and legal jargon removed, while retaining legal compliance and addressing complex scientific issues. Redesigning these contracts has led to greater collaboration between the legal department at ANSTO and other parties, internally and externally. Using digital tools and visuals made the contracts easier to navigate.

A&O Shearman O: 7; L: 8; I: 9; Total: 24 In response to China’s property market crash, the firm developed an interactive portal to help co-ordinating committees representing bank lenders to navigate complex, large-scale corporate restructurings. The portal provides clients with access to resources relating to relevant restructurings, a Q&A tool and document review capabilities. Rapid access to comprehensive information and support saves clients time and money.

Lawpath O: 6; L: 8; I: 8; Total: 22 The Australian firm is targeting smaller companies by providing low-cost access to software and document libraries that use AI to fill out contracts and agreements. A human lawyer can be involved to check documents or deal with more complex work if required.

Atsumi & Sakai O: 6; L: 8; I: 7; Total: 21 The Tokyo-based law firm’s Policy Research Institute advised the Japanese government on emerging technology topics, such as developing AI regulation and the construction of a semiconductor factory.

Pinsent Masons O: 7; L: 7; I: 7; Total: 21 The firm has broadened the legal services it offers at each stage of big renewable energy projects to offer clients a more integrated service — ranging from environmental, social and governance assessments to licensing and property transactions. Commended individual: Mark Hu

KPMG Law O: 5; L: 8; I: 7; Total: 20 The firm created a platform to assess how well equipped a company’s legal team is. The system, launched in 2022, uses a digital tool and questionnaires to gather data about the company and automates a report to help in-house teams assess their resourcing.

Keypoint Law O: 5; L: 7; I: 7; Total: 19 The Australian firm is celebrating a decade of operating without billable hour targets or budgets. The policy aims to offer a more flexible working arrangement to lawyers. Partners are typically paid 70 per cent of the services they charge to clients but, if they do not earn, there is no guaranteed pay. The firm has grown to 75 partners since it was launched in 2014.

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International Edition

• Open access
• Published: 14 May 2024

Developing a survey to measure nursing students’ knowledge, attitudes and beliefs, influences, and willingness to be involved in Medical Assistance in Dying (MAiD): a mixed method modified e-Delphi study

• Jocelyn Schroeder 1 ,
• Barbara Pesut 1 , 2 ,
• Lise Olsen 2 ,
• Nelly D. Oelke 2 &
• Helen Sharp 2  

BMC Nursing volume  23 , Article number:  326 ( 2024 ) Cite this article

31 Accesses

Metrics details

Medical Assistance in Dying (MAiD) was legalized in Canada in 2016. Canada’s legislation is the first to permit Nurse Practitioners (NP) to serve as independent MAiD assessors and providers. Registered Nurses’ (RN) also have important roles in MAiD that include MAiD care coordination; client and family teaching and support, MAiD procedural quality; healthcare provider and public education; and bereavement care for family. Nurses have a right under the law to conscientious objection to participating in MAiD. Therefore, it is essential to prepare nurses in their entry-level education for the practice implications and moral complexities inherent in this practice. Knowing what nursing students think about MAiD is a critical first step. Therefore, the purpose of this study was to develop a survey to measure nursing students’ knowledge, attitudes and beliefs, influences, and willingness to be involved in MAiD in the Canadian context.

The design was a mixed-method, modified e-Delphi method that entailed item generation from the literature, item refinement through a 2 round survey of an expert faculty panel, and item validation through a cognitive focus group interview with nursing students. The settings were a University located in an urban area and a College located in a rural area in Western Canada.

During phase 1, a 56-item survey was developed from existing literature that included demographic items and items designed to measure experience with death and dying (including MAiD), education and preparation, attitudes and beliefs, influences on those beliefs, and anticipated future involvement. During phase 2, an expert faculty panel reviewed, modified, and prioritized the items yielding 51 items. During phase 3, a sample of nursing students further evaluated and modified the language in the survey to aid readability and comprehension. The final survey consists of 45 items including 4 case studies.

Systematic evaluation of knowledge-to-date coupled with stakeholder perspectives supports robust survey design. This study yielded a survey to assess nursing students’ attitudes toward MAiD in a Canadian context.

The survey is appropriate for use in education and research to measure knowledge and attitudes about MAiD among nurse trainees and can be a helpful step in preparing nursing students for entry-level practice.

Peer Review reports

Medical Assistance in Dying (MAiD) is permitted under an amendment to Canada’s Criminal Code which was passed in 2016 [ 1 ]. MAiD is defined in the legislation as both self-administered and clinician-administered medication for the purpose of causing death. In the 2016 Bill C-14 legislation one of the eligibility criteria was that an applicant for MAiD must have a reasonably foreseeable natural death although this term was not defined. It was left to the clinical judgement of MAiD assessors and providers to determine the time frame that constitutes reasonably foreseeable [ 2 ]. However, in 2021 under Bill C-7, the eligibility criteria for MAiD were changed to allow individuals with irreversible medical conditions, declining health, and suffering, but whose natural death was not reasonably foreseeable, to receive MAiD [ 3 ]. This population of MAiD applicants are referred to as Track 2 MAiD (those whose natural death is foreseeable are referred to as Track 1). Track 2 applicants are subject to additional safeguards under the 2021 C-7 legislation.

Three additional proposed changes to the legislation have been extensively studied by Canadian Expert Panels (Council of Canadian Academics [CCA]) [ 4 , 5 , 6 ] First, under the legislation that defines Track 2, individuals with mental disease as their sole underlying medical condition may apply for MAiD, but implementation of this practice is embargoed until March 2027 [ 4 ]. Second, there is consideration of allowing MAiD to be implemented through advanced consent. This would make it possible for persons living with dementia to receive MAID after they have lost the capacity to consent to the procedure [ 5 ]. Third, there is consideration of extending MAiD to mature minors. A mature minor is defined as “a person under the age of majority…and who has the capacity to understand and appreciate the nature and consequences of a decision” ([ 6 ] p. 5). In summary, since the legalization of MAiD in 2016 the eligibility criteria and safeguards have evolved significantly with consequent implications for nurses and nursing care. Further, the number of Canadians who access MAiD shows steady increases since 2016 [ 7 ] and it is expected that these increases will continue in the foreseeable future.

Nurses have been integral to MAiD care in the Canadian context. While other countries such as Belgium and the Netherlands also permit euthanasia, Canada is the first country to allow Nurse Practitioners (Registered Nurses with additional preparation typically achieved at the graduate level) to act independently as assessors and providers of MAiD [ 1 ]. Although the role of Registered Nurses (RNs) in MAiD is not defined in federal legislation, it has been addressed at the provincial/territorial-level with variability in scope of practice by region [ 8 , 9 ]. For example, there are differences with respect to the obligation of the nurse to provide information to patients about MAiD, and to the degree that nurses are expected to ensure that patient eligibility criteria and safeguards are met prior to their participation [ 10 ]. Studies conducted in the Canadian context indicate that RNs perform essential roles in MAiD care coordination; client and family teaching and support; MAiD procedural quality; healthcare provider and public education; and bereavement care for family [ 9 , 11 ]. Nurse practitioners and RNs are integral to a robust MAiD care system in Canada and hence need to be well-prepared for their role [ 12 ].

Previous studies have found that end of life care, and MAiD specifically, raise complex moral and ethical issues for nurses [ 13 , 14 , 15 , 16 ]. The knowledge, attitudes, and beliefs of nurses are important across practice settings because nurses have consistent, ongoing, and direct contact with patients who experience chronic or life-limiting health conditions. Canadian studies exploring nurses’ moral and ethical decision-making in relation to MAiD reveal that although some nurses are clear in their support for, or opposition to, MAiD, others are unclear on what they believe to be good and right [ 14 ]. Empirical findings suggest that nurses go through a period of moral sense-making that is often informed by their family, peers, and initial experiences with MAID [ 17 , 18 ]. Canadian legislation and policy specifies that nurses are not required to participate in MAiD and may recuse themselves as conscientious objectors with appropriate steps to ensure ongoing and safe care of patients [ 1 , 19 ]. However, with so many nurses having to reflect on and make sense of their moral position, it is essential that they are given adequate time and preparation to make an informed and thoughtful decision before they participate in a MAID death [ 20 , 21 ].

It is well established that nursing students receive inconsistent exposure to end of life care issues [ 22 ] and little or no training related to MAiD [ 23 ]. Without such education and reflection time in pre-entry nursing preparation, nurses are at significant risk for moral harm. An important first step in providing this preparation is to be able to assess the knowledge, values, and beliefs of nursing students regarding MAID and end of life care. As demand for MAiD increases along with the complexities of MAiD, it is critical to understand the knowledge, attitudes, and likelihood of engagement with MAiD among nursing students as a baseline upon which to build curriculum and as a means to track these variables over time.

Aim, design, and setting

The aim of this study was to develop a survey to measure nursing students’ knowledge, attitudes and beliefs, influences, and willingness to be involved in MAiD in the Canadian context. We sought to explore both their willingness to be involved in the registered nursing role and in the nurse practitioner role should they chose to prepare themselves to that level of education. The design was a mixed-method, modified e-Delphi method that entailed item generation, item refinement through an expert faculty panel [ 24 , 25 , 26 ], and initial item validation through a cognitive focus group interview with nursing students [ 27 ]. The settings were a University located in an urban area and a College located in a rural area in Western Canada.

Participants

A panel of 10 faculty from the two nursing education programs were recruited for Phase 2 of the e-Delphi. To be included, faculty were required to have a minimum of three years of experience in nurse education, be employed as nursing faculty, and self-identify as having experience with MAiD. A convenience sample of 5 fourth-year nursing students were recruited to participate in Phase 3. Students had to be in good standing in the nursing program and be willing to share their experiences of the survey in an online group interview format.

The modified e-Delphi was conducted in 3 phases: Phase 1 entailed item generation through literature and existing survey review. Phase 2 entailed item refinement through a faculty expert panel review with focus on content validity, prioritization, and revision of item wording [ 25 ]. Phase 3 entailed an assessment of face validity through focus group-based cognitive interview with nursing students.

Phase I. Item generation through literature review

The goal of phase 1 was to develop a bank of survey items that would represent the variables of interest and which could be provided to expert faculty in Phase 2. Initial survey items were generated through a literature review of similar surveys designed to assess knowledge and attitudes toward MAiD/euthanasia in healthcare providers; Canadian empirical studies on nurses’ roles and/or experiences with MAiD; and legislative and expert panel documents that outlined proposed changes to the legislative eligibility criteria and safeguards. The literature review was conducted in three online databases: CINAHL, PsycINFO, and Medline. Key words for the search included nurses , nursing students , medical students , NPs, MAiD , euthanasia , assisted death , and end-of-life care . Only articles written in English were reviewed. The legalization and legislation of MAiD is new in many countries; therefore, studies that were greater than twenty years old were excluded, no further exclusion criteria set for country.

Items from surveys designed to measure similar variables in other health care providers and geographic contexts were placed in a table and similar items were collated and revised into a single item. Then key variables were identified from the empirical literature on nurses and MAiD in Canada and checked against the items derived from the surveys to ensure that each of the key variables were represented. For example, conscientious objection has figured prominently in the Canadian literature, but there were few items that assessed knowledge of conscientious objection in other surveys and so items were added [ 15 , 21 , 28 , 29 ]. Finally, four case studies were added to the survey to address the anticipated changes to the Canadian legislation. The case studies were based upon the inclusion of mature minors, advanced consent, and mental disorder as the sole underlying medical condition. The intention was to assess nurses’ beliefs and comfort with these potential legislative changes.

Phase 2. Item refinement through expert panel review

The goal of phase 2 was to refine and prioritize the proposed survey items identified in phase 1 using a modified e-Delphi approach to achieve consensus among an expert panel [ 26 ]. Items from phase 1 were presented to an expert faculty panel using a Qualtrics (Provo, UT) online survey. Panel members were asked to review each item to determine if it should be: included, excluded or adapted for the survey. When adapted was selected faculty experts were asked to provide rationale and suggestions for adaptation through the use of an open text box. Items that reached a level of 75% consensus for either inclusion or adaptation were retained [ 25 , 26 ]. New items were categorized and added, and a revised survey was presented to the panel of experts in round 2. Panel members were again asked to review items, including new items, to determine if it should be: included, excluded, or adapted for the survey. Round 2 of the modified e-Delphi approach also included an item prioritization activity, where participants were then asked to rate the importance of each item, based on a 5-point Likert scale (low to high importance), which De Vaus [ 30 ] states is helpful for increasing the reliability of responses. Items that reached a 75% consensus on inclusion were then considered in relation to the importance it was given by the expert panel. Quantitative data were managed using SPSS (IBM Corp).

Phase 3. Face validity through cognitive interviews with nursing students

The goal of phase 3 was to obtain initial face validity of the proposed survey using a sample of nursing student informants. More specifically, student participants were asked to discuss how items were interpreted, to identify confusing wording or other problematic construction of items, and to provide feedback about the survey as a whole including readability and organization [ 31 , 32 , 33 ]. The focus group was held online and audio recorded. A semi-structured interview guide was developed for this study that focused on clarity, meaning, order and wording of questions; emotions evoked by the questions; and overall survey cohesion and length was used to obtain data (see Supplementary Material 2  for the interview guide). A prompt to “think aloud” was used to limit interviewer-imposed bias and encourage participants to describe their thoughts and response to a given item as they reviewed survey items [ 27 ]. Where needed, verbal probes such as “could you expand on that” were used to encourage participants to expand on their responses [ 27 ]. Student participants’ feedback was collated verbatim and presented to the research team where potential survey modifications were negotiated and finalized among team members. Conventional content analysis [ 34 ] of focus group data was conducted to identify key themes that emerged through discussion with students. Themes were derived from the data by grouping common responses and then using those common responses to modify survey items.

Ten nursing faculty participated in the expert panel. Eight of the 10 faculty self-identified as female. No faculty panel members reported conscientious objector status and ninety percent reported general agreement with MAiD with one respondent who indicated their view as “unsure.” Six of the 10 faculty experts had 16 years of experience or more working as a nurse educator.

Five nursing students participated in the cognitive interview focus group. The duration of the focus group was 2.5 h. All participants identified that they were born in Canada, self-identified as female (one preferred not to say) and reported having received some instruction about MAiD as part of their nursing curriculum. See Tables  1 and 2 for the demographic descriptors of the study sample. Study results will be reported in accordance with the study phases. See Fig.  1 for an overview of the results from each phase.

Fig. 1  Overview of survey development findings

Phase 1: survey item generation

Review of the literature identified that no existing survey was available for use with nursing students in the Canadian context. However, an analysis of themes across qualitative and quantitative studies of physicians, medical students, nurses, and nursing students provided sufficient data to develop a preliminary set of items suitable for adaptation to a population of nursing students.

Four major themes and factors that influence knowledge, attitudes, and beliefs about MAiD were evident from the literature: (i) endogenous or individual factors such as age, gender, personally held values, religion, religiosity, and/or spirituality [ 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 ], (ii) experience with death and dying in personal and/or professional life [ 35 , 40 , 41 , 43 , 44 , 45 ], (iii) training including curricular instruction about clinical role, scope of practice, or the law [ 23 , 36 , 39 ], and (iv) exogenous or social factors such as the influence of key leaders, colleagues, friends and/or family, professional and licensure organizations, support within professional settings, and/or engagement in MAiD in an interdisciplinary team context [ 9 , 35 , 46 ].

Studies of nursing students also suggest overlap across these categories. For example, value for patient autonomy [ 23 ] and the moral complexity of decision-making [ 37 ] are important factors that contribute to attitudes about MAiD and may stem from a blend of personally held values coupled with curricular content, professional training and norms, and clinical exposure. For example, students report that participation in end of life care allows for personal growth, shifts in perception, and opportunities to build therapeutic relationships with their clients [ 44 , 47 , 48 ].

Preliminary items generated from the literature resulted in 56 questions from 11 published sources (See Table  3 ). These items were constructed across four main categories: (i) socio-demographic questions; (ii) end of life care questions; (iii) knowledge about MAiD; or (iv) comfort and willingness to participate in MAiD. Knowledge questions were refined to reflect current MAiD legislation, policies, and regulatory frameworks. Falconer [ 39 ] and Freeman [ 45 ] studies were foundational sources for item selection. Additionally, four case studies were written to reflect the most recent anticipated changes to MAiD legislation and all used the same open-ended core questions to address respondents’ perspectives about the patient’s right to make the decision, comfort in assisting a physician or NP to administer MAiD in that scenario, and hypothesized comfort about serving as a primary provider if qualified as an NP in future. Response options for the survey were also constructed during this stage and included: open text, categorical, yes/no , and Likert scales.

Phase 2: faculty expert panel review

Of the 56 items presented to the faculty panel, 54 questions reached 75% consensus. However, based upon the qualitative responses 9 items were removed largely because they were felt to be repetitive. Items that generated the most controversy were related to measuring religion and spirituality in the Canadian context, defining end of life care when there is no agreed upon time frames (e.g., last days, months, or years), and predicting willingness to be involved in a future events – thus predicting their future selves. Phase 2, round 1 resulted in an initial set of 47 items which were then presented back to the faculty panel in round 2.

Of the 47 initial questions presented to the panel in round 2, 45 reached a level of consensus of 75% or greater, and 34 of these questions reached a level of 100% consensus [ 27 ] of which all participants chose to include without any adaptations) For each question, level of importance was determined based on a 5-point Likert scale (1 = very unimportant, 2 = somewhat unimportant, 3 = neutral, 4 = somewhat important, and 5 = very important). Figure  2 provides an overview of the level of importance assigned to each item.

Ranking level of importance for survey items

After round 2, a careful analysis of participant comments and level of importance was completed by the research team. While the main method of survey item development came from participants’ response to the first round of Delphi consensus ratings, level of importance was used to assist in the decision of whether to keep or modify questions that created controversy, or that rated lower in the include/exclude/adapt portion of the Delphi. Survey items that rated low in level of importance included questions about future roles, sex and gender, and religion/spirituality. After deliberation by the research committee, these questions were retained in the survey based upon the importance of these variables in the scientific literature.

Of the 47 questions remaining from Phase 2, round 2, four were revised. In addition, the two questions that did not meet the 75% cut off level for consensus were reviewed by the research team. The first question reviewed was What is your comfort level with providing a MAiD death in the future if you were a qualified NP ? Based on a review of participant comments, it was decided to retain this question for the cognitive interviews with students in the final phase of testing. The second question asked about impacts on respondents’ views of MAiD and was changed from one item with 4 subcategories into 4 separate items, resulting in a final total of 51 items for phase 3. The revised survey was then brought forward to the cognitive interviews with student participants in Phase 3. (see Supplementary Material 1 for a complete description of item modification during round 2).

Phase 3. Outcomes of cognitive interview focus group

Of the 51 items reviewed by student participants, 29 were identified as clear with little or no discussion. Participant comments for the remaining 22 questions were noted and verified against the audio recording. Following content analysis of the comments, four key themes emerged through the student discussion: unclear or ambiguous wording; difficult to answer questions; need for additional response options; and emotional response evoked by questions. An example of unclear or ambiguous wording was a request for clarity in the use of the word “sufficient” in the context of assessing an item that read “My nursing education has provided sufficient content about the nursing role in MAiD.” “Sufficient” was viewed as subjective and “laden with…complexity that distracted me from the question.” The group recommended rewording the item to read “My nursing education has provided enough content for me to care for a patient considering or requesting MAiD.”

An example of having difficulty answering questions related to limited knowledge related to terms used in the legislation such as such as safeguards , mature minor , eligibility criteria , and conscientious objection. Students were unclear about what these words meant relative to the legislation and indicated that this lack of clarity would hamper appropriate responses to the survey. To ensure that respondents are able to answer relevant questions, student participants recommended that the final survey include explanation of key terms such as mature minor and conscientious objection and an overview of current legislation.

Response options were also a point of discussion. Participants noted a lack of distinction between response options of unsure and unable to say . Additionally, scaling of attitudes was noted as important since perspectives about MAiD are dynamic and not dichotomous “agree or disagree” responses. Although the faculty expert panel recommended the integration of the demographic variables of religious and/or spiritual remain as a single item, the student group stated a preference to have religion and spirituality appear as separate items. The student focus group also took issue with separate items for the variables of sex and gender, specifically that non-binary respondents might feel othered or “outed” particularly when asked to identify their sex. These variables had been created based upon best practices in health research but students did not feel they were appropriate in this context [ 49 ]. Finally, students agreed with the faculty expert panel in terms of the complexity of projecting their future involvement as a Nurse Practitioner. One participant stated: “I certainly had to like, whoa, whoa, whoa. Now let me finish this degree first, please.” Another stated, “I'm still imagining myself, my future career as an RN.”

Finally, student participants acknowledged the array of emotions that some of the items produced for them. For example, one student described positive feelings when interacting with the survey. “Brought me a little bit of feeling of joy. Like it reminded me that this is the last piece of independence that people grab on to.” Another participant, described the freedom that the idea of an advance request gave her. “The advance request gives the most comfort for me, just with early onset Alzheimer’s and knowing what it can do.” But other participants described less positive feelings. For example, the mature minor case study yielded a comment: “This whole scenario just made my heart hurt with the idea of a child requesting that.”

Based on the data gathered from the cognitive interview focus group of nursing students, revisions were made to 11 closed-ended questions (see Table  4 ) and 3 items were excluded. In the four case studies, the open-ended question related to a respondents’ hypothesized actions in a future role as NP were removed. The final survey consists of 45 items including 4 case studies (see Supplementary Material 3 ).

The aim of this study was to develop and validate a survey that can be used to track the growth of knowledge about MAiD among nursing students over time, inform training programs about curricular needs, and evaluate attitudes and willingness to participate in MAiD at time-points during training or across nursing programs over time.

The faculty expert panel and student participants in the cognitive interview focus group identified a need to establish core knowledge of the terminology and legislative rules related to MAiD. For example, within the cognitive interview group of student participants, several acknowledged lack of clear understanding of specific terms such as “conscientious objector” and “safeguards.” Participants acknowledged discomfort with the uncertainty of not knowing and their inclination to look up these terms to assist with answering the questions. This survey can be administered to nursing or pre-nursing students at any phase of their training within a program or across training programs. However, in doing so it is important to acknowledge that their baseline knowledge of MAiD will vary. A response option of “not sure” is important and provides a means for respondents to convey uncertainty. If this survey is used to inform curricular needs, respondents should be given explicit instructions not to conduct online searches to inform their responses, but rather to provide an honest appraisal of their current knowledge and these instructions are included in the survey (see Supplementary Material 3 ).

Some provincial regulatory bodies have established core competencies for entry-level nurses that include MAiD. For example, the BC College of Nurses and Midwives (BCCNM) requires “knowledge about ethical, legal, and regulatory implications of medical assistance in dying (MAiD) when providing nursing care.” (10 p. 6) However, across Canada curricular content and coverage related to end of life care and MAiD is variable [ 23 ]. Given the dynamic nature of the legislation that includes portions of the law that are embargoed until 2024, it is important to ensure that respondents are guided by current and accurate information. As the law changes, nursing curricula, and public attitudes continue to evolve, inclusion of core knowledge and content is essential and relevant for investigators to be able to interpret the portions of the survey focused on attitudes and beliefs about MAiD. Content knowledge portions of the survey may need to be modified over time as legislation and training change and to meet the specific purposes of the investigator.

Given the sensitive nature of the topic, it is strongly recommended that surveys be conducted anonymously and that students be provided with an opportunity to discuss their responses to the survey. A majority of feedback from both the expert panel of faculty and from student participants related to the wording and inclusion of demographic variables, in particular religion, religiosity, gender identity, and sex assigned at birth. These and other demographic variables have the potential to be highly identifying in small samples. In any instance in which the survey could be expected to yield demographic group sizes less than 5, users should eliminate the demographic variables from the survey. For example, the profession of nursing is highly dominated by females with over 90% of nurses who identify as female [ 50 ]. Thus, a survey within a single class of students or even across classes in a single institution is likely to yield a small number of male respondents and/or respondents who report a difference between sex assigned at birth and gender identity. When variables that serve to identify respondents are included, respondents are less likely to complete or submit the survey, to obscure their responses so as not to be identifiable, or to be influenced by social desirability bias in their responses rather than to convey their attitudes accurately [ 51 ]. Further, small samples do not allow for conclusive analyses or interpretation of apparent group differences. Although these variables are often included in surveys, such demographics should be included only when anonymity can be sustained. In small and/or known samples, highly identifying variables should be omitted.

There are several limitations associated with the development of this survey. The expert panel was comprised of faculty who teach nursing students and are knowledgeable about MAiD and curricular content, however none identified as a conscientious objector to MAiD. Ideally, our expert panel would have included one or more conscientious objectors to MAiD to provide a broader perspective. Review by practitioners who participate in MAiD, those who are neutral or undecided, and practitioners who are conscientious objectors would ensure broad applicability of the survey. This study included one student cognitive interview focus group with 5 self-selected participants. All student participants had held discussions about end of life care with at least one patient, 4 of 5 participants had worked with a patient who requested MAiD, and one had been present for a MAiD death. It is not clear that these participants are representative of nursing students demographically or by experience with end of life care. It is possible that the students who elected to participate hold perspectives and reflections on patient care and MAiD that differ from students with little or no exposure to end of life care and/or MAiD. However, previous studies find that most nursing students have been involved with end of life care including meaningful discussions about patients’ preferences and care needs during their education [ 40 , 44 , 47 , 48 , 52 ]. Data collection with additional student focus groups with students early in their training and drawn from other training contexts would contribute to further validation of survey items.

Future studies should incorporate pilot testing with small sample of nursing students followed by a larger cross-program sample to allow evaluation of the psychometric properties of specific items and further refinement of the survey tool. Consistent with literature about the importance of leadership in the context of MAiD [ 12 , 53 , 54 ], a study of faculty knowledge, beliefs, and attitudes toward MAiD would provide context for understanding student perspectives within and across programs. Additional research is also needed to understand the timing and content coverage of MAiD across Canadian nurse training programs’ curricula.

The implementation of MAiD is complex and requires understanding of the perspectives of multiple stakeholders. Within the field of nursing this includes clinical providers, educators, and students who will deliver clinical care. A survey to assess nursing students’ attitudes toward and willingness to participate in MAiD in the Canadian context is timely, due to the legislation enacted in 2016 and subsequent modifications to the law in 2021 with portions of the law to be enacted in 2027. Further development of this survey could be undertaken to allow for use in settings with practicing nurses or to allow longitudinal follow up with students as they enter practice. As the Canadian landscape changes, ongoing assessment of the perspectives and needs of health professionals and students in the health professions is needed to inform policy makers, leaders in practice, curricular needs, and to monitor changes in attitudes and practice patterns over time.

Availability of data and materials

The datasets used and/or analysed during the current study are not publicly available due to small sample sizes, but are available from the corresponding author on reasonable request.

Abbreviations

British Columbia College of Nurses and Midwives

Medical assistance in dying

Nurse practitioner

Registered nurse

University of British Columbia Okanagan

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Acknowledgements

We would like to acknowledge the faculty and students who generously contributed their time to this work.

JS received a student traineeship through the Principal Research Chairs program at the University of British Columbia Okanagan.

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JS made substantial contributions to the conception of the work; data acquisition, analysis, and interpretation; and drafting and substantively revising the work. JS has approved the submitted version and agreed to be personally accountable for the author's own contributions and to ensure that questions related to the accuracy or integrity of any part of the work, even ones in which the author was not personally involved, are appropriately investigated, resolved, and the resolution documented in the literature. BP made substantial contributions to the conception of the work; data acquisition, analysis, and interpretation; and drafting and substantively revising the work. BP has approved the submitted version and agreed to be personally accountable for the author's own contributions and to ensure that questions related to the accuracy or integrity of any part of the work, even ones in which the author was not personally involved, are appropriately investigated, resolved, and the resolution documented in the literature. LO made substantial contributions to the conception of the work; data acquisition, analysis, and interpretation; and substantively revising the work. LO has approved the submitted version and agreed to be personally accountable for the author's own contributions and to ensure that questions related to the accuracy or integrity of any part of the work, even ones in which the author was not personally involved, are appropriately investigated, resolved, and the resolution documented in the literature. NDO made substantial contributions to the conception of the work; data acquisition, analysis, and interpretation; and substantively revising the work. NDO has approved the submitted version and agreed to be personally accountable for the author's own contributions and to ensure that questions related to the accuracy or integrity of any part of the work, even ones in which the author was not personally involved, are appropriately investigated, resolved, and the resolution documented in the literature. HS made substantial contributions to drafting and substantively revising the work. HS has approved the submitted version and agreed to be personally accountable for the author's own contributions and to ensure that questions related to the accuracy or integrity of any part of the work, even ones in which the author was not personally involved, are appropriately investigated, resolved, and the resolution documented in the literature.

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Schroeder, J., Pesut, B., Olsen, L. et al. Developing a survey to measure nursing students’ knowledge, attitudes and beliefs, influences, and willingness to be involved in Medical Assistance in Dying (MAiD): a mixed method modified e-Delphi study. BMC Nurs 23 , 326 (2024). https://doi.org/10.1186/s12912-024-01984-z

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Lipidomic studies revealing serological markers associated with the occurrence of retinopathy in type 2 diabetes

• Mingqian He 1   na1 ,
• Guixue Hou 2   na1 ,
• Mengmeng Liu 1   na1 ,
• Zhaoyi Peng 1 ,
• Hui Guo 1 ,
• Yue Wang 1 ,
• Jing Sui 3 ,
• Hui Liu 4 ,
• Xiaoming Yin 5 ,
• Meng Zhang 1 ,
• Ziyi Chen 1 ,
• Patrick C.N. Rensen 1 , 6 ,
• Liang Lin 2 , 8 ,
• Yanan Wang 1 , 7 &
• Bingyin Shi 1  

Journal of Translational Medicine volume  22 , Article number:  448 ( 2024 ) Cite this article

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The duration of type 2 diabetes mellitus (T2DM) and blood glucose levels have a significant impact on the development of T2DM complications. However, currently known risk factors are not good predictors of the onset or progression of diabetic retinopathy (DR). Therefore, we aimed to investigate the differences in the serum lipid composition in patients with T2DM, without and with DR, and search for potential serological indicators associated with the development of DR.

A total of 622 patients with T2DM hospitalized in the Department of Endocrinology of the First Affiliated Hospital of Xi’an JiaoTong University were selected as the discovery set. One-to-one case–control matching was performed according to the traditional risk factors for DR (i.e., age, duration of diabetes, HbA1c level, and hypertension). All cases with comorbid chronic kidney disease were excluded to eliminate confounding factors. A total of 42 pairs were successfully matched. T2DM patients with DR (DR group) were the case group, and T2DM patients without DR (NDR group) served as control subjects. Ultra-performance liquid chromatography–mass spectrometry (LC–MS/MS) was used for untargeted lipidomics analysis on serum, and a partial least squares discriminant analysis (PLS-DA) model was established to screen differential lipid molecules based on variable importance in the projection (VIP) > 1. An additional 531 T2DM patients were selected as the validation set. Next, 1:1 propensity score matching (PSM) was performed for the traditional risk factors for DR, and a combined 95 pairings in the NDR and DR groups were successfully matched. The screened differential lipid molecules were validated by multiple reaction monitoring (MRM) quantification based on mass spectrometry.

The discovery set showed no differences in traditional risk factors associated with the development of DR (i.e., age, disease duration, HbA1c, blood pressure, and glomerular filtration rate). In the DR group compared with the NDR group, the levels of three ceramides (Cer) and seven sphingomyelins (SM) were significantly lower, and one phosphatidylcholine (PC), two lysophosphatidylcholines (LPC), and two SMs were significantly higher. Furthermore, evaluation of these 15 differential lipid molecules in the validation sample set showed that three Cer and SM(d18:1/24:1) molecules were substantially lower in the DR group. After excluding other confounding factors (e.g., sex, BMI, lipid-lowering drug therapy, and lipid levels), multifactorial logistic regression analysis revealed that a lower abundance of two ceramides, i.e., Cer(d18:0/22:0) and Cer(d18:0/24:0), was an independent risk factor for the occurrence of DR in T2DM patients.

Disturbances in lipid metabolism are closely associated with the occurrence of DR in patients with T2DM, especially in ceramides. Our study revealed for the first time that Cer(d18:0/22:0) and Cer(d18:0/24:0) might be potential serological markers for the diagnosis of DR occurrence in T2DM patients, providing new ideas for the early diagnosis of DR.

Introduction

Type 2 diabetes mellitus (T2DM) is a common chronic disease in many countries, and its prevalence is growing as people’s lifestyles are changing [ 1 ]. Diabetes causes various complications, classified as either macrovascular complications (such as cardiovascular disease and stroke) or microvascular complications (such as kidney disease) [ 2 ]. Diabetic retinopathy (DR), a specific microvascular complication of diabetes, is the most common cause of vision loss in people of working age [ 3 , 4 ]. Poor glycemic control, hypertension, and diabetes duration are major risk factors for DR [ 5 ]. Although intensive risk factor control reduces the risk of DR progression and vision loss, many diabetic patients continue to develop DR with strict glycemic and blood pressure control [ 6 ]. Despite increasing research supporting the efficacy of routine DR screening to prevent DR and early treatment to reduce the risk of vision loss, there are no specific biomarkers for diagnosing the onset and early progression of DR. Additionally, new and more effective strategies are awaited to prevent and treat the progression of DR.

Accumulating evidence suggests that disruption in lipid metabolism is an early event in the pathogenesis of diabetes complications. Previous studies found that levels of multiple lipid species, including glycerophospholipids, sphingolipids and glycerolipids, are critical risk factors for T2DM and its complications [ 7 , 8 ]. Lysophosphatidylcholine (LPC) is a main glycerophospholipid known for its essential role in lipid and glucose metabolism, and LPC has been intensively studied in the development of metabolic diseases including T2DM [ 9 ]. Sphingolipids, including ceramides (Cer), sphingomyelins (SM) and gangliosides, have a variety of intra- and extracellular effects on glucose homeostasis and metabolic disease [ 10 ] Numerous studies suggest Cer, a crucial lipid intermediate in sphingolipid metabolism, is a major contributing factor for insulin resistance, and inhibition or depletion of enzymes driving de novo ceramide synthesis can prevent the development of diabetes in mice [ 7 , 11 , 12 ]. In contrast, a decrease in very long chain Cer is correlated with the development of macroalbuminuria in diabetes [ 13 ]. Accelerated sphingolipid catabolism’ leading to an increase in glucosylceramide or glycosphingolipids might contribute to the neuronal pathologies of DR [ 14 ]. In addition, SM produced by the transfer of a phosphocholine moiety from phosphatidylcholine to the ceramide backbone has been linked to insulin resistance [ 15 , 16 ] and is also an independent marker of cardiovascular disease [ 17 ]. Thus, dysregulated lipid metabolism is a major contributor to the pathogenesis of T2DM and its complications, and specific lipid species that are responsible for the occurrence of DR are rather obscure.

Lipidomics offers solid platforms for identifying novel lipid mediates in biochemical processes of lipid metabolism, thus providing new opportunities for disease prediction and detection [ 18 , 19 ]. Lipidome analysis is performed by liquid chromatography and electrospray ionization-tandem mass spectrometry (LC–MS/MS) for molecular lipid identification and quantification and multiple reaction monitoring (MRM) for targeted quantification of those lipid species. Lipid-based biomarkers offer unique options for precision medicine by providing sensitive diagnostic tools for disease prediction and monitoring [ 20 ]. Using a quantitative metabolomics approach, Emil et al. compared the aqueous humor and serum concentrations of metabolites in senior adults with an without diabetes who underwent cataract surgery [ 21 ]. However, the field of lipidomics studies of DR is still in its early stages, with few studies published and little replication of results [ 22 ].

In this study, we aimed to find reliable serum lipid-based biomarkers for the presence of DR in patients with T2DM by using two cohorts. To this end, serum samples of the discovery cohort was subjected to untargeted lipidomics analysis to search for differentially abundant lipids between individuals without and with DR. In the validation cohort, the observed differential lipid molecules were validated using mass spectrometry MRM targeting techniques. We hypothesized that DR has a distinctive serum lipid signature and that particular lipid species can act as biomarkers for T2DM patients with DR.

Research design and methods

Participants.

A total of 622 participants with T2DM hospitalized in the Endocrinology Department of the First Affiliated Hospital of Xi’an JiaoTong University were screened as the discovery set. Participants with chronic kidney disease [estimated glomerular filtration rate (eGFR) < 90 (mL/min/1.73 m 2 )] were excluded from the selection. We conducted pair matching according to the traditional risk factors for DR (including age, duration of diabetes, HbA1c level, and hypertension). For the discovery cohort, we selected 42 T2DM patients with DR (DR group). The control participants were 42 T2DM patients without DR (NDR group), and they were matched to patients in the DR group by age (in 5-year bands), diabetes duration (in 5-year bands), HbA1c levels (in 0.5% bands), and hypertension status.

Lipid markers of DR identified from the discovery cohort were quantified in a separate sample cohort (validation cohort). We first screened 531 T2DM patients. Individuals with chronic kidney disease [eGFR < 90 (mL/min/1.73 m 2 )] were excluded from the selection. Then, we conducted 1:1 propensity score matching (PSM) (matching tolerance = 0.02) by age, diabetes duration, HbA1c level, hypertension status, sex, BMI, systolic blood pressure (SBP), diastolic blood pressure (DBP), and eGFR. For the validation cohort, 95 T2DM patients with DR (DR group) and 95 T2DM patients without DR (NDR group) were included.

Sample collection

Fasting blood samples and clinical data were collected from the individuals. All blood samples were collected at the First Affiliated Hospital of Xi’an JiaoTong University physical examination center. Blood samples were centrifuged for 20 min at 1500 rpm and 4 °C. Then, serum was collected and stored at -80 °C until analysis. HbA1c was measured using an automatic HbA1c analyzer (TOSOH BIOSCIENCE, INC.; HLC-723G8). Total cholesterol (CHOL), triglyceride (TG), high density lipoprotein-cholesterol (HDL-c), low density lipoprotein-cholesterol (LDL-c), uric acid (UA), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), gamma-glutamyl transpeptidase (GGT), total bilirubin (TBIL), direct bilirubin (DBIL), total protein (TP), albumin (ALB), glucose (GLU), blood urea nitrogen (BUN), creatinine (CRE) were measured using standard reagents on an automatic biochemistry analyzer (HITACHI, Inc.; LAbOSPECT, 008AS). Blood pressure was measured in triplicate using an Omron HBP-9020 digital automatic blood pressure machine (Kyoto, Japan).

Lipid extraction

The serum samples were thawed slowly at 4 °C, 100 µL of the sample was placed in a 96-well plate, 300 µL of isopropanol (prechilled at -20 °C) spiked with internal standards (SPLASH® LIPIDOMIX® Mass Spec Standard, Avanti, USA) was added, and the samples were vortexed and mixed for 1 min and then centrifuged at 4 °C for 20 min at 4000 rcf after resting overnight at -20 °C as previously reported [ 23 ]. The supernatant was injected for LC–MS/MS analysis, and 10 µL of each supernatant was mixed into quality control (QC) samples to assess the reproducibility and stability of the LC–MS analysis process.

LC–MS/MS analysis

Lipids were separated and detected by an UPLC (CSH C18 column, 1.7 μm 2.1*100 mm, Waters, USA) equipped with a Q Exactive Plus high-resolution mass spectrometer (Thermo Fisher Scientific, USA) as previously reported [ 24 ]. The following gradient was used for elution: 0–2 min, 40-43% mobile phase B (10 mM ammonia formate, 0.1% formic acid, 90% isopropyl alcohol, and 10% acetonitrile); 2–2.1 min, 43-50% liquid B; 2.1–7 min, 50-54% solution B; 7–7.1 min, 54-70% liquid B; 7.1–13 min, 70-99% liquid B with a flow rate of 0.35 mL/min. Mobile phase A was an aqueous solution containing 10 mM ammonia formate, 0.1% formic acid and 60% acetonitrile in water.

All samples were analyzed in data-dependent acquisition (DDA) mode with the following positive/negative ionization settings: spray voltage, 3.8/–3.2 kV; aux gas heater temperature, 350 °C; and capillary temperature, 320 °C. The full scan mass range was 200–2000 m/z with 70,000 mass resolution at m/z 200 and AGC set to 3e6 with a maximum ion injection time of 100 ms. The top three precursors were selected for subsequent MS fragmentation with a maximum ion injection time of 50 ms and resolution of 17,500 at m/z 200, and the AGC was 1e5. The stepped normalized collision energy was set to 15, 30, and 45 eV.

Data preprocessing and quality control

The raw data obtained from the LC–MS/MS detection were imported into LipidSearch v.4.1 (Thermo Fisher Scientific, USA) for lipid identification and quantification. The following parameters were used for lipid identification and peak extraction: the type of identification was Product, the mass deviation of the parent and daughter ions was 5 ppm, and the response threshold was set to 5.0% of the relative response deviation of the daughter ions; the quantitative parameters were set to calculate the peak areas of all identified lipids, and the peak extraction mass deviation was set to 5 ppm. For ESI + data, [M + H]+, [M + NH4]+, and [M + Na] + were selected as adducts, while for ESI- data, [M-H]-, [M-2 H]-, and [M-HCOO]- were selected as adducts. The peak alignment was performed for all identified lipids, and those not marked as “rejected” were considered for inclusion in the subsequent analysis.

For data preprocessing, raw data exported from LipidSearch were further analyzed by meta X [ 25 ]. The data preprocessing included (1) Removing lipid molecules with more than 50% missing information in QC samples and more than 80% missing information in experimental samples (i.e., LipidIon in the table); (2) Filling the missing values using the k-nearest neighbor (KNN) algorithm; (3) Correcting the batch effect using quality control-based robust LOESS signal correction (QC-RLSC); (4) Using probabilistic quotient normalization (PQN) to normalize the data to obtain the relative peak areas; and (5) Removing the lipid molecules with a coefficient of variation (CV) greater than 30% of the relative peak areas from all QC samples.

Data quality was assessed by the reproducibility of QC sample assays. The assessment included chromatogram overlap of QC samples, principal component analysis (PCA), number of extracted peaks, and differences in peak response intensity.

Data processing

A combination of multivariate statistical analysis and univariate analysis was used to screen for lipids of which the abundance differed between groups. The multivariate statistical analysis methods used were principal component analysis (PCA) and partial least squares method-discriminant analysis (PLS-DA). PCA is an unsupervised pattern recognition method, and PLS-DA is a supervised pattern recognition method. The univariate analyses were fold change (FC) and Student’s t test. The FC was obtained by fold change analysis, and the p  value pairs of the t test were corrected for the false discovery rate (FDR) to obtain a q-value. The differential lipid molecule screening conditions were as follows: (1) variable importance in the projection (VIP) ≥ 1 for the first two principal components of the PLS-DA model; (2) fold change ≥ 1.2 or ≤ 0.83; and (3) p  value < 0.05.

Targeted lipid quantification by MRM in validation samples

The identified differential lipids were further quantified by multiple reaction monitoring (MRM). For lipid extraction, the procedure was consistent with the untargeted experiment as described. The MRM transition list is shown in Table S1 . For MRM quantification, all validation samples were analyzed on a QTRAP 5500 mass spectrometer with a CSH C18 column (1.7 μm 2.1*100 mm, Waters, USA) for separation. All lipids were subjected to targeted quantification in ESI + mode with a specific transition setting.

Statistical analysis

The clinical data of samples are presented as the mean ± standard deviation (SD) for normally distributed variables or the median (interquartile range) for abnormal distribution. Comparisons between the case group and the control group were made using a two-tailed t test or Mann-Whitney U test for continuous data and the X 2 test for categorical data. The calculation of the area under the curve (AUC) in receiver operating characteristic (ROC) curve analysis was used to evaluate the discriminatory ability of the markers. Logistic regression models were applied to assess the relationship between lipid molecules and the presence of DR. The odds ratios (ORs) with 95% confidence intervals (CIs) were calculated for the molecules with 1-SD changes. The known risk factors for DR, such as CHOL, TGs, LDL-c, and HDL-c, were added to multivariate logistic regression to calculate the adjusted odds ratios. Ordinal logistic regression models were used to assess the relationships between lipid molecules and DR stages [NDR, nonproliferative DR (NPDR) and proliferative DR (PDR)].

Characteristics of the discovery cohort

Table  1 shows the clinical characteristics of individuals selected for the discovery cohort. There were no significant differences in age and sex between the DR and NDR groups. In fact, these groups were comparable for most metabolic characteristics, such as BMI, diabetes duration, and HbA1c, and there were no significant between-group differences for hypertension status, antihypertensive agent use, hypoglycemic therapy status or NSAID use. The blood pressure and glucose of the participants were treated and controlled. Compared with control subjects with T2DM, T2DM patients with DR had higher levels of LDL-c levels, AST, TBIL, and BUN (Table  1 and Table S2 ).

Untargeted lipidome-derived biomarkers for diabetic retinopathy: results from the discovery cohort

A total of 1721 lipids were detected. The number of lipids with an RSD (CV) less than or equal to 30% in the QC samples was 1421. The ratio of the number of lipids with CV less than or equal to 30% to the number of all detected lipids in QC samples was 81%.

Fifteen candidate lipids were identified from the discovery cohort. Compared with those of the NDR group, the levels of three Cer and seven SM were significantly lower in the DR group. In contrast, two SM, two LPC and one PC were significantly higher in the DR group (Fig.  1 A and B). More specifically, compared with T2DM patients without DR, T2DM patients with DR showed lower levels of Cer(d18:0/24:0), Cer(d18:0/22:0), Cer(d42:3), SM(d22:0/16:0), SM(d18:1/24:1), SM(d42:0), SM(d40:0), SM(d39:0), SM(d38:0), and SM(d36:0), and higher levels of SM(d20:1/16:1), SM(d34:1), LPC(18:2), LPC(16:0) and PC(34:2). The heat map shows the distribution of these lipids between individuals of the NDR and DR groups (Fig.  1 C). The results of ROC analysis and the odds ratios of the lipid markers in the basic logistic regression models are shown in Table  2 . The AUC values for the 15 lipids ranged from 0.72 to 0.94. All lipids retained significant ORs after adjusted for CHOL, TG, LDL-c, and HDL-c (adjusted ORs are shown in Table  2 ). Furthermore, we used ordinal logistic regression, which estimated the odds of being in one higher category of the DR stage (from NDR to PDR) for lipid species, to test the associations between lipid species and DR stage (Table S3 ; n  = 42 in the NDR group, n  = 37 in the NPDR group, n  = 5 in the PDR group), and we analyzed the data while excluding participants with diabetic macular edema (DME) ( n  = 4 in the DR group), as before, all lipids retained significant ORs (Table S4 ).

Lipidome-derived markers identified from the discovery cohort. Lipidomic analysis identified fifteen candidate lipids of which serum levels were different between 42 T2DM patients with DR (DR group) and 42 T2DM patients without DR (NDR group) from the discovery cohort. ( A ) Mean peak intensity of lipids was analyzed after Log2 transformation of the data. ( B ) Fold change in DR/NDR was analyzed after Log2 transformation of the data. ( C ) Heatmap showing the distribution of lipid markers. Each row in the figure represents a different lipid, and each column represents a sample. Different colors indicate different intensities, and Log2 conversion was used for the data

Characteristics of the validation cohort and targeted lipidomics analysis

The 15 differential lipids found from the discovery cohort were validated in another set of samples. The clinical characteristics of individuals selected for the validation cohort are shown in Table  3 . Most metabolic and clinical features were comparable (Table S5 ), and there was no significant difference in LDL-c between the DR and NDR groups.

In the validation cohort, when compared with subjects in the NDR group, T2DM patients with DR showed lower levels of Cer(d18:0/24:0), Cer(d18:0/22:0), Cer(d42:3) and SM(d18:1/24:1) by univariate logistic regression, which was consistent with the results of the discovery cohort. However, the levels of SM(d20:1/16:1), LPC(18:2) and LPC(16:0) were lower in T2DM patients with DR from the validation cohort, opposite to the result obtained in the discovery cohort (Fig.  2 A and B). The AUC values for these lipids were higher than 0.61. The other 8 lipids did not significantly differ between the DR and NDR groups in the validation cohort (Table  4 ). Of note, compared with those in T2DM patients, the peak area (after Log2 transformation) of Cer(d18:0/24:0) (20.48 ± 0.82 vs. 20.12 ± 0.99, p  = 0.006, Fig.  2 C) and Cer(d18:0/22:0) (19.91 ± 0.75 vs. 19.64 ± 0.92, p  = 0.028, Fig.  2 C) remained significantly lower in T2DM patients with DR, and the levels of these two lipids retained significant ORs when adjusted for known risk factors (i.e., CHOL, TG, LDL-c and HDL-c). In the ordinal regression, these two lipids maintained significant ORs (Table S7 , n  = 95 in the NDR group, n  = 87 in the NPDR group; n  = 8 in the PDR group), and were also significant while excluding patients with DME (Table S6 , n  = 2 in the DR group). These findings imply that levels of Cer(d18:0/24:0) and Cer(d18:0/22:0) were independent markers for T2DM patients with DR in both the discovery cohort (Table  2 ) and validation cohort (Table  4 ).

The results of targeted lipidomics analysis in the validation cohort. For the validation cohort, the cases were 95 T2DM patients with DR (DR group), and the control subjects were 95 T2DM patients who had no DR (NDR group). ( A ) Peak area of lipids was analyzed after Log2 transformation of the data. ( B ) Fold change in DR/NDR was analyzed after Log2 transformation of the data. ( C ) The log2 conversion was used for the intensities of Cer(d18:0/24:0) and Cer(d18:0/22:0). All data are presented as the mean ± standard deviation (SD). Each symbol represents an individual participant. * p  < 0.05, ** p  < 0.01, pairwise comparisons of change scores between the groups were evaluated by t test

DR is the most common microvascular complication of diabetes and the main factor contributing to visual impairment in working-age individuals [ 3 ]. T2DM patients often develop DR despite of proper control of systemic risk factors, indicating the involvement of other pathogenic factors for DR development. To find new and more effective strategies for preventing and treating DR, it is necessary for us to identify novel biomarkers for DR screening or detection. Lipidomics will aid in understanding the mechanism of DR at various stages of the disease, early diagnosis, and the identification of new therapeutic targets. In this study, by using two clinical cohorts, we found that the serum lipidomic profiles in T2DM patients with DR showed significant differences from those in T2DM patients without DR. The differential lipid species in the DR group were linked to disturbances in sphingolipid metabolism. Compared with those in the NDR group, the levels of Cer(d18:0/24:0) and Cer(d18:0/22:0) were significantly lower in the DR group after adjusting for covariates, i.e. known risk factors in both the discovery and validation cohorts. These findings suggest that these two lipid species may be potential serological markers for the diagnosis of DR in patients with T2DM.

In this study, we found two ceramide molecules that were significantly lower in T2DM patients with DR, indicating that they may have disturbed ceramide metabolism compared to T2DM patients without DR. Ceramide is sphingolipid [ 11 ] and can be found in VLDL, LDL, and HDL. Consistent with our findings, Fort et al. found a significantly lower abundance of Cer in central retinal tissue obtained postmortem from T2DM patients with DR compared to those without DR [ 26 ]. Similarly, ceramide levels were shown to be lower and glucosylceramide levels higher in the retinas of diabetic rodents [ 27 ]. This indicates that diabetes reduces the retinal ceramide content and may suggest that dysregulated sphingolipid metabolism may cause retinal resistance to insulin action [ 27 ]. These findings imply that ceramide is diverted from the overall pools of retinal sphingolipids toward the glycosylated forms due to hyperglycemia. In contrast, Levitsky et al. found that diabetes-induced increases in mitochondrial ceramide led to impaired mitochondrial function in the retinal pigment epithelial (RPE) cells of the retina [ 28 ], and disruption of the blood-retinal barrier might be caused by diabetes-induced overexpression of acid sphingomyelinase. Additionally, inflammation is a common underlying factor in DR, and inflammation generates Cer from SM in the serum membrane. This induces death receptor ligand formation and leads to apoptosis of RPE and photoreceptor cells [ 29 ]. In addition to diabetes, circulating Cer was shown to strongly correlate with future adverse cardiovascular events. It has recently been discovered that in individuals with atherosclerotic CVD, serum levels of specific Cer species can predict the future risk of cardiovascular death. In the Corogene study, higher concentrations of Cer(d18:1/16:0), Cer(d18:1/18:0), and Cer(d18:1/24:1) and lower concentrations of Cer(d18:1/24:0) were associated with a higher risk of fatal myocardial infarction [ 30 ]. Our study found that Cer(d18:0/24:0) and Cer(d18:0/22:0) were significantly lower in T2DM patients with DR compared to those without DR, which suggests that different numbers of carbons and double bonds in ceramides might play differential roles in DR and CVD. The distinct ceramides and ceramide metabolites involved in metabolic regulation play unanticipated roles [ 31 ]. Watt et al. discovered that circulating ceramides present in LDL particles were sufficient to induce insulin resistance in vitro and in vivo [ 32 ]. However, how these two identified ceramides influence lipid metabolism in T2DM remains unclear and needs further exploration. Thus, disturbed Cer metabolism may contribute to dysfunction in DR, and therapeutic strategies to restore normal Cer metabolism might be an effective approach for treatment of DR.

In the discovery cohort, LPC(18:2) and LPC(16:0) were significantly higher in T2DM patients with DR. However, these two lipids were significantly lower in DR in the validation cohort. The previous findings point to a change in sphingolipid composition between control and T2DM [ 33 ]. LPC is an inflammatory phospholipid and an important atherogenic substance in LDL that contributes to diabetic complications [ 34 ]. Lipoprotein-associated phospholipase A2 (Lp-PLA2) plays a crucial role in diabetes-related retinal vasopermeability, a response mediated by LPC, and inhibiting Lp-PLA2 reduces diabetes-induced retinal vasopermeability [ 35 ]. LPC O-16:0, LPC P-16:0, LPC O-18:0, and LPC 18:1 were all found to be inversely related to incident T2DM [ 36 ]. The differences between the discovery and validation cohorts may be related to the populations studied, medications used, and stages of diabetic retinopathy [ 37 ].

There are some limitations of this study. First, only a Chinese ethnic group was selected, and future validation of our findings in other races or ethnic groups is warranted. Second, instead of chronic risk factors associated with the development of DR, some of the identified lipid markers might only represent temporary metabolic perturbations in this cross-sectional study. Third, the exact mechanism of DR development in patients with T2DM through which ceramide functions has not been explained. Therefore, more extensive preclinical and clinical studies are needed to clarify the mechanisms behind the potential effects of specific lipids.

Overall, the deregulation of sphingolipid metabolism in the diabetic retina appears to be a significant and seldom-studied element of DR pathophysiology. The precise mechanism underlying this disease is still unknown and requires further investigation. We showed the potential value of lipidomics research in understanding the pathophysiology of DR, and the results suggest that lipidomics profiling may be capable of identifying early-stage DR diagnostic indicators in high-risk Chinese populations. In addition, the findings from this study may help in the elucidation of new therapeutic targets for DR prevention and treatment.

Data availability

All relevant data and materials have been included in the article and its supplementary data files. Further inquiries can be directed to the corresponding authors.

Abbreviations

• Type 2 diabetes mellitus

Diabetic retinopathy

Liquid chromatography–mass spectrometry

Partial least squares discriminant analysis

Variable importance in the projection

Propensity score matching

Sphingomyelins

Phosphatidylcholine

Lysophosphatidylcholines

Multiple reaction monitoring

Systolic blood pressure

Diastolic blood pressure

Total cholesterol

Triglyceride

High density lipoprotein-cholesterol

Low density lipoprotein-cholesterol

Aspartate aminotransferase

Alanine aminotransferase

Alkaline phosphatase

Gamma-glutamyl transpeptidase

Total bilirubin

Direct bilirubin

Total protein

Blood urea nitrogen

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Acknowledgements

Y.N.W. is supported by the China “Thousand Talents Plan” (Young Talents), Shaanxi province “Thousand Talents Plan” (Young Talents) and Foundation of Xi’an Jiaotong University (Plan A).

This study was supported by grants from The Natural Science Foundation Program of Shaanxi (2024JC-YBQN-0828) and National Key R&D Program of China (No. 2018YFC1311501).

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Mingqian He, Guixue Hou and Mengmeng Liu contributed equally to this work.

Authors and Affiliations

Department of Endocrinology, the First Affiliated Hospital of Xi’an JiaoTong University, No.277, West Yanta Road, Xi’an, Shaanxi, 710061, P.R. China

Mingqian He, Mengmeng Liu, Zhaoyi Peng, Hui Guo, Yue Wang, Meng Zhang, Ziyi Chen, Patrick C.N. Rensen, Yanan Wang & Bingyin Shi

BGI-SHENZHEN, No. 21 Hongan 3rd Street, Yantian District, Shenzhen, Guangdong, 518083, P.R. China

Guixue Hou & Liang Lin

Department of Endocrinology and International Medical Center, the First Affiliated Hospital of Xi’an JiaoTong University, No.277, West Yanta Road, Xi’an, Shaanxi, 710061, P.R. China

Biobank, The First Affiliated Hospital of Xi’an JiaoTong University, Xi’an, Shaanxi, 710061, China

Chengdu HuiXin Life Technology, Chengdu, Sichuan, 610091, P.R. China

Xiaoming Yin

Department of Medicine, Division of Endocrinology, Leiden University Medical Center, P.O. Box 9600, Leiden, 2300 RA, The Netherlands

Patrick C.N. Rensen

Med-X institute, Center for Immunological and Metabolic Diseases, the First Affiliated Hospital of Xi’an JiaoTong University, Xi’an JiaoTong university, Xi’an, Shaanxi, 710061, P.R. China

Building NO.7, BGI Park, No. 21 Hongan 3rd Street, Yantian District, Shenzhen, Guangdong, 518083, P.R. China

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B.S., Y.W., and L.L. conceived this review and critically revised the manuscript. M.H., G.H., and M.L. drafted the manuscript. Z.P., H.G., Y.W., and J.S. drew the figures and collected the related references. H.L., X.Y., M.Z., Z.C. and P.C.N. supervised and revised the manuscript. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Liang Lin , Yanan Wang or Bingyin Shi .

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He, M., Hou, G., Liu, M. et al. Lipidomic studies revealing serological markers associated with the occurrence of retinopathy in type 2 diabetes. J Transl Med 22 , 448 (2024). https://doi.org/10.1186/s12967-024-05274-9

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Received : 21 August 2023

Accepted : 04 May 2024

Published : 13 May 2024

DOI : https://doi.org/10.1186/s12967-024-05274-9

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