Laboratory practice of organic acid analysis based on gas chromatography–mass spectrometry in China

Introduction

Inherited metabolic disorders (IMDs) are a considerable group of genetic disorders with a collective incidence of 1:2000 [1]. On-set IMDs patients usually experience acute acidosis, metabolic decomposition, neurological syndromes, unknown death, and irreversible disability [2]. Due to the non-specific and heterogeneous clinical manifestations in IMDs patients [2], the screening, diagnosis, and patient monitoring management of IMDs extremely rely on laboratory tests. Organic acid (OA) analysis based on gas chromatography–mass spectrometry (GC-MS) is such a crucial first-tier laboratory test for the broad scopes of IMDs, which detects multiple accumulative metabolic products of IMDs in urine and other body fluids [3].

This assay was introduced by some newborn screening laboratories, biogenetic laboratories, and pediatric laboratories in China in recent 20 years. Fifteen years ago, OA analysis was hardly seen in China, and samples were sent out to test [4]. Recently, China has released its clinical guidelines for the diagnosis and treatment of IMDs, in which many disorders are involved with the clinical use of OA analysis detected by GC/MS.

OA analysis is a highly specialized, regulated, non-waived laboratory-developed test (LDT) meeting Clinical Laboratory Improvements Amendments (CLIA) standard. According to CLIA requirements, OA analysis using GC-MS should be regulated in the aspects of external quality assessment (EQA)/proficiency test (PT), facility administration, quality system for the entire analytical phase, and qualified personnel performing GC-MS analysis. In 2018, the American College of Medical Genetics and Genomics (ACMG) has issued a technical standard for this assay [5], while China has not defined its guidelines. The laboratory practice of this assay in China remains unclear. In Authors’ center, EQA results in 2019–2020 reported that the performance of OA analysis in 54 participating laboratories was unsatisfying: although grouped by measurement systems and methods, robust coefficient of variations (RCV) of most OAs were high to 40%, even up to 90%.

It is reported the practice of OA varied in different laboratories [3]. Due to the significance of the OA assay for the screening and diagnosis of IMDs, it is necessary to get a knowledge of laboratory practice in this test in China. To comprehensively understanding the current laboratory practice of OA analysis in China, we issued a survey on the official website of the National Center For Clinical Laboratories of China (cNCCL) and invited all EQA participating laboratories to join in.

Materials and methods

The questionnaire was preliminary designed based on related literature, guidelines, and standards [3], [4], [5], [6], [7], [8]. Then the questionnaire was cross-reviewed by three external reviewers independently, two of whom were practiced technicians in OA analysis and one was an experienced pediatrician. After triple revisions, the survey was issued in October 2020. The notice of survey was issued on our website (available: https://www.nccl.org.cn/mainEn) and sent to 54 OA assay EQA participating labs, and participants could fill out the questionnaire voluntarily, then emailed their completed questionnaires to our laboratory.

A full copy of our survey is presented in the Supplementary Material. The questionnaire covered the aspects of clinical utility, the entire analytical process, and quality assurance. The type of question involves single-choice, multiple-choice, and blanks for different answers. Incomplete questionnaires would be excluded for analysis. Crucial questions and answers about the entire analytical process and quality assurance were extracted for analysis. A Chi-squared test (χ ²) was performed to see whether the difference between public hospitals and private institutes has significance. IBM SPSS Statistics 25.0 and Microsoft Excel 2016 are used for data analysis.

Results

Discussion

Although most laboratory practice in OA analysis was consistent in Chinese laboratories, our survey has identified laboratories were going their own ways in the establishment and verification of RIs, TAT for reporting results, and the quantification of results. Though this assay has introduced for over 20 years, it was in the recent five years that it has been commonly seen in China. It seems not very optimistic for the TAT for the reports releasing. When comes to acute patients, reports released three days later may be disappointing. The practice in some laboratories that RIs in literature were directly used instead of self-established and lacking sufficient validation and verification is of great concern, too. RIs are of great significance for clinicians [9]. A technical standard for OA assay issued by ACMG has recommended that RIs should be established and periodically validated [5]. And the establishment of RIs of OA should be stratified according to ages and sex [10]. Sometimes, it is difficult to validate the RIs, because data used to verify may involve long-term follow-up, which is not always assessable for most laboratories. Cross-verification using results of gene analysis or enzyme assay, or other laboratories may be an effective way to verify.

Besides, it is controversial whether there’s necessary to get the absolute quantitative results for organic acids analysis. 24.2% of respondents showed unclear attitudes towards this issue, 39.4% of respondents insisted that absolutely quantitative results were beneficial for clinicians and better quality management. But 36.4% of respondents agreed it is unnecessary to realize absolute quantification results, some of them thought that it is hard to realize absolute quantification because some standard materials were unavailable, semi-quantitative results could already show the abnormal level, and the level in patients would easily exceed the upper limit of quantification. However, qualitative or semi-quantitative results may be importable between laboratories, making additional costs. And due to the inconsistency of manual addition of quantitative internal standards, semi-quantitative results make it difficult to achieve the consistency within laboratory or interlaboratory, causing poor accuracy of results. Additionally, it can be hard for clinicians to monitor the therapy effect based on semi-quantification results. Though absolute quantitative results are recommended, it is challenging to achieve absolute quantification for some OAs because of the unavailable of isotope-labeled ISs and pure standard materials.

It is worth noting that the interpretation of OA analysis results could be extremely difficult under some interferences [11]. The previous study had comprehensively concluded nutritional, exogenous, iatrogenic, and artificial sources of OA [11]. Effects on results of OA analysis caused by medicines, e.g., valproate, bacterial contamination were also clear [11, 12]. Thus, it was suggested that more information on diet, medicines, pathological status, and the conditions of sample storage and transportation should be provided for clinical laboratory technicians to make more accurate interpretative comments and advice for clinicians.

The survey has its disadvantages. The questionnaire didn’t cover experimental details of sample preparation and GC-MS parameters, in which manual processes were often poorly controlled and not standardized, so methodological differences cannot be identified.

To evaluate and monitor the analytical performance of laboratories implementing this technique-demand test, EQA schemes are warranted [13, 14]. However, simple-designed EQA programs for OA analysis cannot ensure the comparability of results among different laboratories. The harmonization and standardization of OA analysis will make results more accurate and comparable in different clinical settings [15], making contributions for the management of IMDs patients, and the share of RIs among different laboratories. However, the establishment of traceability for OA analysis is challenging due to the lack of calibrators, certified reference materials, isotope internal standards, and reference measurement procedures for most OAs. Clinical laboratories and manufacturers should understand the limitation of OA assay and make efforts for the harmonization and standardization of it.

Conclusions

Considerations in the establishment validation and verification of reference ranges, turnaround time of reporting results, and the quantification of results are especially prominent. Appropriate supervision and efforts for the harmonization and standardization of organic acids analysis are needed.