To the Editor,
Vitamin B6 is a cofactor in many biological processes. Both a deficiency and an excess of this molecule can give rise to health problems, e.g. polyneuropathy [1, 2]. Recently, a multicenter comparison of various vitamin B6 assays in whole blood observed a large bias between users of the Chromsystems kit (Munich, Germany) and other users, when measuring vitamin B6 as pyridoxal-5′-phosphate (PLP) in both fresh and frozen blood . Also, the variation within the group of Chromsystems users was substantial. In patient samples, the Chromsystems method showed a positive bias of 30% when compared with a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. More so, a similar bias was observed between Chromsystems vs. INstruchemie (Delfzijl, The Netherlands) and laboratory-developed high-performance liquid chromatography (HPLC) methods. Remarkably, in lyophilized quality control (QC) material, this difference was absent. This is of great concern because QC material is used for monitoring the performance of the assay in internal and external QC procedures to achieve clinically reliable results [4, 5]. As a reaction on these observations, Chromsystems adjusted their HPLC and UHPLC kits for the combined determination of vitamin B6 and B1 in whole blood. Two new internal standards were introduced to give better recovery during the derivatization procedure, thereby improving matrix robustness. The addition of stabilization buffer is not required anymore and the mobile phase and gradient were adapted. The effect of these adjustment on the measurement of vitamin B6 was evaluated and the consequences for hospital laboratories are outlined in this letter.
One hundred whole-blood EDTA patient samples (remaining from routine analysis) with a PLP concentration in the range of 40–285 nmol/L were collected, anonymized and blinded by giving them a number, aliquoted and stored at −20°C. The Erasmus MC guidelines state that for the use of anonymous leftover samples, no permission from the Ethical Committee is needed. In addition, 16 external QC samples from the Dutch and German proficiency schemes (12× SKML, Nijmegen, The Netherlands and 4× Instand e.V., Düsseldorf, Germany) were selected. These samples contain lyophilized whole blood without any additives. Although these samples are not proven to be commutable, all participating laboratories obtain similar results, independent of the method used. All 116 samples were measured by a hospital laboratory (Maasstad Hospital, Rotterdam, The Netherlands) using the adjusted Chromsystems HPLC method (kit 52052), and a hospital laboratory (Medisch Spectrum Twente, Enschede, The Netherlands) using the adjusted Chromsystems UHPLC method (kit 52952). Both kits contain a calibrator (lyophilized material), which is traceable to a certified standard and HPLC reference testing. All results were compared with a laboratory-developed LC-MS/MS method using a stable isotope as internal standard and a calibration line in whole blood for measuring PLP (ErasmusMC, Rotterdam, The Netherlands) . Blinding was implemented to keep the known values from all participating laboratories until all measurements were performed and results were obtained. The results were analyzed with Analyse-It software package v2.30 for Excel (Leeds, UK) using Passing and Bablok regression analysis with a 95% confidence interval for comparison of the above-mentioned methods and a %-difference plot to determine the total bias.
The adjusted Chromsystems HPLC and UHPLC methods showed a negative total bias of −11.4% and −19.5%, respectively, compared to the LC-MS/MS method (Figure 1). The difference between both Chromsystems methods was +9.1%, where the UHPLC method gives lower results than the HPLC method. To validate these observations, Chromsystems duplicated the measurement of all samples with HPLC and UHPLC in their own laboratory using the exact same blinded sample set. This led to similar results in comparison with the hospital laboratory-derived HPLC and UHPLC results (data not shown). Before the kits were adjusted, Chromsystems users (mostly HPLC users in 2013) measured on average 30% higher in patient samples . Therefore, adjustment of the kits leads to convergence of the results obtained with the LC-MS/MS method; however, the difference between results of patient samples before and after the adjustment is substantial. Considering that 30% is only an average, it may well be that the values of patient samples drop >50% after the switch to the adjusted kit based on the laboratory’s individual variation (Figure 1). It is therefore important for hospital laboratories to determine the effect of the adjusted kit on their results and adapt their reference intervals accordingly.
In lyophilized material, the bias between both Chromsystems methods was much smaller, only +3.0%. Also, in these samples the duplicated results of Chromsystems did not differ (data not shown). This is in line with earlier observations that the Chromsystems assays are more robust using lyophilized samples than using fresh or frozen material.
In our previous multicenter study, equivalent results were obtained for lyophilized samples with the Chromsystems method and other methods, in contrast to patient samples . Thus, the QC material did not reflect the performance of the assay in patient samples. In this light, it is interesting to plot the values of the QC material obtained with the adjusted HPLC and UHPLC kits against the overall consensus values determined at that time with the same material in the respective ring trials. SKML provides an overall consensus for all method groups, including our LC-MS/MS method.
For the Instand e.V. samples, the overall consensus was calculated as the weighted average of the median of each method group and the number of users of that method. Currently, it is only possible to work with consensus values for vitamin B6 in whole blood, because a reference method and suitable certified reference material (CRM) are lacking. Although a NIST standard reference material for plasma PLP does exist, this material has two major drawbacks for application in whole blood measurements: (1) the matrix is different, which can obviously influence the outcome of method comparability and (2) the concentration of PLP in this plasma material is much lower than is commonly found in whole blood samples. As can be seen in Figure 2, the adjusted Chromsystems HPLC and UHPLC kit show a negative bias of −14.5% and −17.1%, respectively. Comparable biases were obtained when the results were plotted against LC-MS/MS instead of consensus (data not shown). This corresponds well with the biases of −11.4% and −19.5% found in patient samples, which seems to indicate that the adjusted Chromsystems test kits now show identical results for patient samples and lyophilized samples. It is to be expected that Chromsystems users will start to deviate from the overall consensus with the introduction of the adjusted kits on the market. Initially, also within their method group, larger differences are to be expected, until the transition of all participating laboratories to the adjusted kits is completed.
In this study, the effect of the adjustment of the Chromsystems kits on vitamin B1 was not evaluated, due to the unavailability of an LC-MS/MS method to measure this compound. In the meantime, such a method is described by Puts et al.  and is also under development in one of our own laboratories, enabling an evaluation of the different methods measuring vitamin B1 in the near future.
In conclusion, the adjustment of the HPLC and UHPLC kits for the combined analysis of vitamin B6 and B1 in whole blood appears to be an improvement in quality. First, the acquisition of clinically reliable results can be monitored better, because QC material and patient samples behave similarly in the adjusted assays, which was previously not the case. Second, it is a step forward in the comparability of vitamin B6 results between different laboratories, because the bias compared to the LC-MS/MS has reduced. As bias is not completely resolved and may still be large in individual cases, hospital laboratories should be critical when implementing the adjusted kits. For standardization, obviously, a reference method or CRM is required, which comes in view with the development of other LC-MS/MS methods for the determination of PLP [7, 8].
We thank Marjolein Neele and Eugenie Schrama (Maasstad Hospital) and Harry Wanschers (Medisch Spectrum Twente) for their analytical support. Chromsystems Instruments & Chemicals GmbH (Munich, Germany) is kindly acknowledged for the donation of the HPLC and UHPLC kits used in this study.
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About the article
Published Online: 2017-07-21
Published in Print: 2017-11-27
Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
Research funding: None declared.
Employment or leadership: None declared.
Honorarium: None declared.
Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.