Albumin determined by bromocresol green leads to erroneous results in routine evaluation of patients with chronic kidney disease.

OBJECTIVES: Measurement of plasma albumin is pivotal for clinical decision-making in patients with chronic kidney disease (CKD). Routinely used methods as bromocresol green (BCG) and bromocresol purple (BCP) can suffer from aselectivity, but the impact of aselectivity on the accuracy of plasma albumin results of CKD-patients is still unknown. Therefore, we evaluated the performance of BCG-, BCP- and JCTLM-endorsed immunological methods in patients with various stages of CKD. METHODS: We evaluated the performance of commonly used albumin methods in patients with CKD stages G1 through G5, the latter divided in two groups based on whether they received hemodialysis treatment. In total, 163 patient plasma samples were measured at 14 laboratories, on six different BCG and BCP-platforms, and four different immunological platforms. The results were compared with an ERM-DA-470k-corrected nephelometric assay. The implications on outcome is evaluated by the proportion of patient results <38 g/L for the diagnosis of protein energy wasting. RESULTS: Albumin results determined with BCP- and immunological methods showed the best agreement with the target value (92.7 and 86.2 %, respectively vs. 66.7 % for BCG, namely due to overestimation). The relative agreement of each method with the target value was platform-dependent, with larger variability in agreement between platforms noted for BCG and immunological methods (3.2-4.6 and 2.6-5.3 %) as opposed to BCP (0.7-1.5 %). The stage of CKD had similar effects on the variability in agreement for the three method-groups (0.6-1.8 % vs. 0.7-1.5 % vs. 0.4-1.6 %). The differences between methods cause discrepancies in clinical decision-making, as structurally fewer patients were diagnosed with protein energy wasting upon using BCG-based albumin results. CONCLUSIONS: Our study shows that BCP is fit for the intended use to measure plasma albumin levels in CKD patients from all stages, including patients on hemodialysis. In contrast, most BCG-based platforms falsely overestimate the plasma albumin concentration.

An Unusual Compound Heterozygosity for Hb O-Arab (HBB: c.364G>A) and Hb D-Los Angeles (HBB: c.364G>C).

We report a newborn with a compound heterozygosity for Hb O-Arab (HBB: 364G>A) and Hb D-Los Angeles (HBB: 364G>C). To the best of our knowledge, the combination of these two hemoglobin (Hb) variants has not been identified and reported before. The variants of the proband and parents were identified by high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE). DNA analysis was performed to confirm the variants. The levels of Hb variants of the proband were determined post-partum, at 3 months and 1 year after birth. Blood count analysis after 1 year revealed that the proband had a mild microcytic anemia. Furthermore, HPLC and CE analysis revealed an equal distribution of Hb D-Los Angeles compared to Hb O-Arab at the age of 1 year. The follow-up of the patient, suggested that the Hb combination is clinically silent or mild.

Validation and verification of examination procedures in medical laboratories: opinion of the EFLM Working Group Accreditation and ISO/CEN standards (WG-A/ISO) on dealing with ISO 15189:2012 demands for method verification and validation.

This paper reflects the opinion of the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) Working Group Accreditation and ISO/CEN standards (WG-A/ISO). It aims to provide guidance for drawing up local/national documents about validation and verification of laboratory methods. We demonstrate how risk evaluation can be used to optimize laboratory policies to meet intended use requirements as well as requirements of standards. This is translated in a number of recommendations on how to introduce risk evaluation in various stages of the implementation of new methods ultimately covering the whole process cycle.

When results matter: reliable creatinine concentrations in hyperbilirubinemia patients.

Background Failure to report a creatinine concentration, especially in icteric patients who are eligible for a liver transplant, can result in a life-threatening situation. We assessed the influence of bilirubin interference on several creatinine assays and investigated ways to circumvent icteric interference without interfering with our normal automated sample logistics. Methods Using icteric patient sera (total bilirubin >255 µmol/L) we determined creatinine concentrations using an enzymatic and Jaffé assay (Roche Diagnostics) in both normal (i.e. undiluted) and decreased mode (i.e. diluted) as well as an enzyme-coupled amperometric assay on a Radiometer ABL837 FLEX analyzer. Creatinine concentrations from the five methods were compared with an in-house developed LC-MS/MS method. Passing and Bablok (proportional and constant bias) as well as difference plot parameters (bias and 95% limits of agreement [LoA]) were calculated. Interferograph-based regression analysis of the enzymatic and Jaffé results was used to investigate if such an approach could be used to report corrected creatinine concentrations in icteric patient sera. Results In icteric patient sera the enzyme-coupled amperometric assay was hardly influenced by icteric interference as shown by a difference plot bias of -1.5% (95% LoA -11.6 to +8.5%). The undiluted Jaffé method had a bias of -1.4% with a very broad 95% LoA (-35.1 to +32.2%) emphasizing the poor specificity of this method. The undiluted enzymatic method had the largest bias (-13.4%, 95% LoA -35.8 to +9.0%). Diluting sera in the enzymatic method did not improve the bias (-10.5%, 95% LoA -25.4 to 4.4%), while diluting the Jaffé method resulted in a bias increase (+11.4%, 95% LoA -14.7 to 37.5%). Using interferograph-based regression analysis we were able to reliably correct enzymatic creatinine concentrations in 97 out of 100 icteric patient sera. Conclusions Analytically, quantifying creatinine in icteric sera using the Radiometer ABL837 FLEX analyzer is the method of choice within our laboratory. However, not all laboratories are equipped with this method and even if available, the limited number of highly icteric patient sera makes this method costly. For those laboratories using the Roche enzymatic method, mathematically correcting an icteric creatinine concentration using an interferograph based on an LC-MS/MS reference method is a suitable alternative to report reliable creatinine results in icteric patients.

A multicenter comparison of whole blood vitamin B6 assays.

BACKGROUND: The aim of this study was to compare different analytical methods that are currently in use in the Netherlands for the measurement of whole blood vitamin B6. METHODS: This method comparison study consisted of two separate parts. (1) Four laboratories participated in a pilot study in which the commercial Chromsystems and INstruchemie method, and a laboratory developed liquid chromatography-tandem mass spectrometry (LC-MS/MS) method and HPLC method were compared. Sixty-nine frozen whole blood samples and six lyophilized whole blood samples were used for comparison. (2) In the nationwide part of the study, 49 laboratories participated in the analysis of three identical sets of two whole blood samples of which one set was freshly analyzed, one set was analyzed after storage at -20 °C and one set was analyzed after lyophilization. RESULTS: In both parts of the study, the HPLC and LC-MS/MS methods showed equivalent results for all sample types tested. The Chromsystems method showed a positive bias of 45% (pilot study) and 30% (nationwide study) towards the LC-MS/MS method when fresh or frozen samples were used. The measurement of lyophilized samples showed no differences between the methods. The results of the INstruchemie method were inconclusive due to the low number of participants. CONCLUSIONS: The different analytical methods for measuring vitamin B6 produce different results when whole blood patient samples are measured. The recognition of a reference method or the development of suitable reference materials and quality control materials might serve as a first step towards improved standardization or harmonization of the whole blood vitamin B6 assay.

Enzyme-specific activation versus leaving group ability.

Enzyme-specific activation and the substrate mimetics strategy are effective ways to circumvent the limited substrate recognition often encountered in protease-catalyzed peptide synthesis. A key structural element in both approaches is the guanidinophenyl (OGp) ester, which enables important interactions for affinity and recognition by the enzyme--at least, this is usually the explanation given for its successful application. In this study we show that leaving group ability is of equal or even greater importance. To this end we used both experimental and computational methods: 1) synthesis of close analogues of OGp, and their evaluation in a dipeptide synthesis assay with trypsin, 2) molecular docking studies to provide insights into the binding mode, and 3) ab initio calculations to evaluate their electronic properties.

Improving the carboxyamidomethyl ester for subtilisin A-catalysed peptide synthesis.

A series of novel glycine esters was evaluated for efficiency in subtilisin A-CLEA-catalysed peptide synthesis. The reactivity of the easily accessible carboxyamidomethyl (Cam) ester was further enhanced by elongating it with an amino acid residue, thereby creating more recognition space for subtilisin A.

Papain-specific activating esters in aqueous dipeptide synthesis.

Enzymatic peptide synthesis has the potential to be a viable alternative for chemical peptide synthesis. Because of the increasing commercial interest in peptides, new and improved enzymatic synthesis methods are desirable. In recently developed enzymatic strategies such as substrate mimetic approaches and enzyme-specific activation, use of the guanidinophenyl ester (OGp) group has been shown to suffer from some drawbacks. OGp esters are sensitive to spontaneous chemical hydrolysis and the group is expensive to synthesize and therefore not suitable for large-scale applications. On the basis of earlier computational studies, we hypothesized that OGp might be replaceable by simpler ester groups to make the enzyme-specific activation approach to peptide bond formation more accessible. To this end, a set of potential activating esters (Z-Gly-Act) was designed, synthesized, and evaluated. Both the benzyl (OBn) and the dimethylaminophenyl (ODmap) esters gave promising results. For these esters, the scope of a model dipeptide synthesis reaction under aqueous conditions was investigated by varying the amino acid donor. The results were compared with those obtained from a previous study of Z-X(AA) -OGp esters. Computational docking analysis of the set of esters was performed in order to provide insight into the differences in the reactivities of all the potential activating esters. Finally, selected ODmap- and OBn-activated amino acids were applied in the synthesis of two biologically active dipeptides on preparative scales.

Production of Galacto-oligosaccharides by the ß-Galactosidase from Kluyveromyces lactis : comparative analysis of permeabilized cells versus soluble enzyme.

The transgalactosylation activity of Kluyveromyces lactis cells was studied in detail. Cells were permeabilized with ethanol and further lyophilized to facilitate the transit of substrates and products. The resulting biocatalyst was assayed for the synthesis of galacto-oligosaccharides (GOS) and compared with two soluble ß-galactosidases from K. lactis (Lactozym 3000 L HP G and Maxilact LGX 5000). Using 400 g/L lactose, the maximum GOS yield, measured by HPAEC-PAD analysis, was 177 g/L (44% w/w of total carbohydrates). The major products synthesized were the disaccharides 6-galactobiose [Gal-ß(1→6)-Gal] and allolactose [Gal-ß(1→6)-Glc], as well as the trisaccharide 6-galactosyl-lactose [Gal-ß(1→6)-Gal-ß(1→4)-Glc], which was characterized by MS and 2D NMR. Structural characterization of another synthesized disaccharide, Gal-ß(1→3)-Glc, was carried out. GOS yield obtained with soluble ß-galactosidases was slightly lower (160 g/L for Lactozym 3000 L HP G and 154 g/L for Maxilact LGX 5000); however, the typical profile with a maximum GOS concentration followed by partial hydrolysis of the newly formed oligosaccharides was not observed with the soluble enzymes. Results were correlated with the higher stability of ß-galactosidase when permeabilized whole cells were used.

Papain-catalyzed peptide bond formation: enzyme-specific activation with guanidinophenyl esters.

The substrate mimetics approach is a versatile method for small-scale enzymatic peptide-bond synthesis in aqueous systems. The protease-recognized amino acid side chain is incorporated in an ester leaving group, the substrate mimetic. This shift of the specific moiety enables the acceptance of amino acids and peptide sequences that are normally not recognized by the enzyme. The guanidinophenyl group (OGp), a known substrate mimetic for the serine proteases trypsin and chymotrypsin, has now been applied for the first time in combination with papain, a cheap and commercially available cysteine protease. To provide insight in the binding mode of various Z-X(AA)-OGp esters, computational docking studies were performed. The results strongly point at enzyme-specific activation of the OGp esters in papain through a novel mode of action, rather than their functioning as mimetics. Furthermore, the scope of a model dipeptide synthesis was investigated with respect to both the amino acid donor and the nucleophile. Molecular dynamics simulations were carried out to prioritize 22 natural and unnatural amino acid donors for synthesis. Experimental results correlate well with the predicted ranking and show that nearly all amino acids are accepted by papain.