Response to: Be aware of legally required metrology and higher-order reference measurement services run in calibration laboratories

In this issue of Clinical Chemistry and Laboratory Medicine (CCLM), van der Helm and colleagues provide a reply [1] to an Editorial that I previously wrote in this journal [2].In that Editorial, I questioned the contemporary relevance of the manual tilt tube method (MTTM) for prothrombin time (PT), particularly in the age of automation, artificial intelligence (AI) and machine learning (ML).Notably, the MTTM for PT is used for assignment of the international sensitivity index (ISI) for reference thromboplastin reagents [1,2], and may also be potentially used to establish a mean normal PT (MNPT) [2].These two indices (ISI, MNPT) are utilised for calculating the international normalised ratio (INR) from the patient's PT; in turn, the INR is used for monitoring vitamin K antagonist (VKA) therapy, including warfarin and coumadin.My concerns included the fact that the MTTM for PT is no longer used by most laboratories performing PT/INR measurements, and thus there is a disconnect between the assignment of ISI (and potentially MNPT) values and what actually happens with PT/INR testing in the real world.I also argued that in the age of AI and ML, that there may be better approaches to this task [2].I also cited data from an external quality assessment (EQA) program utilised in my geographic locality, which showed considerable remaining major problems in INR testing, with test variation exceeding 15 % at therapeutic INR levels [2].In this response to their reply [1], I can also provide an update from the same EQA provider (RCPAQAP; Royal College of Pathologists of Australasia Quality Assurance Program) I reported previously [2], but using 2025 data (Figure 1).I thank the authors of the reply [1] for their clarification of several points: (1) First, they clarify that the PT MTTM, and the process of ISI calibration using the PT MTTM, is only meant for experts in this process; essentially those laboratory personnel who have been appropriately trained, and who are otherwise involved in the process of calibration for the international standard (IS) thromboplastin.Although to further clarify, the process may be utilised to calibrate both the primary IS, as well as secondary standard thromboplastins, the latter potentially being prepared by manufacturers of thromboplastin (or commercial PT) reagents (see Figure 1 in the reply [1]).(2) Second, they clarify that "the new calibration hierarchy is ISO 17511:2020 compliant and makes use of a single human recombinant IRP [international reference preparation] in contrast to the two higher-order IRPs (rabbit brain and human recombinant origin) historically."Although reversion to a single IRP is inevitable, given production and maintenance of multiple IS thromboplastins of different origin may no longer be tenable, this may potentially create another disconnect between a single human IS thromboplastin and commercial PT reagents that are based on rabbit thromboplastin.(3) Third, the authors hope this new calibration hierarchy, once adopted by commercial manufacturers, will yield