The Harmonization Hurdle—A Case for Pyridoxal-5′-Phosphate

To the Editor: We read with interest the report by Martínez-Rodríguez et al. on the value of investigating analytical interferences in measurements of alanine aminotransferase (ALT) activity on the Abbott Alinity c platform (1). Specifically, their investigational findings highlight an absorbance error that prevents the determination of ALT activity in viscous samples coinciding with monoclonal gammopathies. However, the report did not indicate that this interference was specific to the formulation of ALT (called ALT2) that lacks the cofactor pyridoxal-5′-phosphate (P5P). Our studies illustrate that these errors are not evident with the formulation of ALT supplemented with P5P (Table 1). The IFCC recommends incorporating P5P in the measurement of ALT and aspartate aminotransferase (2). Without P5P, the results of ALT activities can be underestimated as the measured activity relies on the patients’ endogenous vitamin B6 levels, which may lead to inaccurate results in individuals with vitamin B6 deficiency (2, 3). Comparison of ALT measurements by 2 methods on the Abbott Alinity c, ALT2 formulation (without P5P) and A-ALT (supplemented with P5P). Error in ALT2 formulation specifically refers to “Error:1040—Unable to calculate result. No absorbance reads within absorbance range” on the factor-based calibration method lacking P5P on the Abbot Alinity c. A-ALT, activated alanine aminotransferase (P5P supplemented); RI, reference interval; IgM, immunoglobulin M. aSamples 14 and 30 were the post- and preapheresis sample from the same patient. *Samples were run without any dilution for ALT measurements. We have also encountered the absorbance errors with the ALT2 method that lacks P5P, and such interference or errors are indeed often associated with elevated sample viscosity and/or unusually high immunoglobulin concentrations (Table 1). However, the precise threshold of viscosity and/or concentrations of paraproteins required to cause an absorbance error remains unclear and was not established by the authors in their report (1). Defining such a threshold is difficult as these absorbance errors can be present even with normal viscosity levels (e.g., sample 14, Table 1) or at abnormally low viscosity levels. Conversely, many samples with abnormally high viscosity and immunoglobulin M levels also do not exhibit these absorbance errors and do not interfere with determining ALT activity without P5P supplementation. Furthermore, irrespective of viscosity or immunoglobulin levels, such interferences are nonexistent when using the P5P-supplemented methods that are also available from Abbott (Table 1) or Roche. Thus, while the authors provide a rationale for using reducing agents to alleviate absorbance errors, the alternative solution to the problem is the adoption of an appropriate ALT method with P5P supplementation, which is truly traceable to the IFCC procedure (2). Complicating the adoption of methods with P5P is the manufacturer's claim of traceability to the IFCC method, even when P5P is absent in the reagent formulation (4, 5). Specifically, for a given method that does not use P5P (such as the ALT2 formulation), it is important to note that full traceability to the IFCC reference method cannot be achieved through calibration alone, since the IFCC's reference measurement procedure incorporates P5P by design (4). While there are some disadvantages to P5P supplementation such as limited dynamic range or onboard stability (2, 5), true traceability requires P5P in the assay formulation. Recently, the Clinical Chemistry Committee of the College of American Pathologists indicated that more than 60% of laboratories in the United States and internationally used ALT assays that were not supplemented with P5P. Surprisingly, this degree of variation was even more pronounced in academic medical centers, although the adoption of assays with P5P is reportedly increasing over time (3). As suggested recently by Panteghini et al., to fully solve “the (dis)harmonization of ALT and improve the interpretation of values,” several “urgent actions” are needed, including laboratory professionals taking responsibility for the selection of ALT methods incorporating P5P (4). Overall, the report by Martínez-Rodríguez et al. (1) confirms that adherence to international recommendations remains poor, despite evidence that P5P-supplemented reagents enable greater accuracy in determining ALT activity (2, 3). Our data illustrate that the cited interference (1) is not evident when using an ALT method that is supplemented with P5P (Table 1). While acting on these absorbance errors to investigate potential monoclonal gammopathies is appropriate, these interferences—which hinder reporting ALT results—pose a significant inconvenience in high-volume laboratories and for patients with known monoclonal gammopathies. In addition, the inaccuracy and lack of readily available results also impact other calculated parameters that make use of ALT such as the Fibrosis-4 index (6). Nonstandard Abbreviations: ALT, alanine aminotransferase; P5P, pyridoxal-5′-phosphate. Author Contributions: The corresponding author takes full responsibility that all authors on this publication have met the following required criteria of eligibility for authorship: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; (c) final approval of the published article; and (d) agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved. Nobody who qualifies for authorship has been omitted from the list. Davor Brinc (Conceptualization-Equal, Data curation-Equal, Formal analysis-Equal, Investigation-Equal, Methodology-Equal, Resources-Equal, Supervision-Equal, Writing—review & editing-Equal), Terence A Agbor (Conceptualization-Equal, Data curation-Equal, Formal analysis-Equal, Investigation-Equal, Methodology-Equal, Resources-Equal, Supervision-Equal, Writing—original draft-Equal, Writing—review & editing-Equal), Anselmo Fabros (Conceptualization-Equal, Data curation-Equal, Investigation-Equal, Methodology-Equal, Resources-Equal, Validation-Equal, Writing—review & editing-Equal), Pow Lee Cheng (Conceptualization-Equal, Data curation-Equal, Methodology-Equal, Resources-Equal, Validation-Equal, Writing—review & editing-Equal), Vathany Kulasingam (Conceptualization-Equal, Data curation-Equal, Funding acquisition-Equal, Resources-Equal, Supervision-Equal, Writing—review & editing-Equal), and Rajeevan Selvaratnam (Conceptualization-Equal, Data curation-Equal, Formal analysis-Equal, Investigation-Equal, Methodology-Equal, Resources-Equal, Supervision-Equal, Validation-Equal, Visualization-Equal, Writing—original draft-Equal, Writing—review & editing-Equal). Authors’ Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Research Funding: Abbott Diagnostics provided the reagents free of charge (to V. Kulasingam). Disclosures: V. Kulasingam serves as a consultant for Abbott Diagnostics.