Analytical Performance of the New Siemens Affinity Chrome-Mediated Immunoassay Everolimus Assay and Its Interchangeability With the Thermo Quantitative Microsphere System for Routine Therapeutic Drug Monitoring of Patients After Solid Organ Transplantation.

BACKGROUND: A new homogeneous affinity chrome-mediated immunoassay (ACMIA) "EVRO" from Siemens Healthcare was evaluated for therapeutic drug monitoring of everolimus (EVL) with automated sample pretreatment and compared with quantitative microsphere system (QMS) "EVER" from Thermo Fisher Scientific. METHODS: Imprecision, inaccuracy, and limit of quantitation (LoQ) of ACMIA/EVRO were verified using both hemolysate quality control (QC) samples and pooled whole blood specimens. The interchangeability of methods and the agreement of results were analyzed using 72 specimens (from 38, 30, and 4 kidney, liver, and lung transplant recipients, respectively). RESULTS: Within-run imprecision ranged within %CV = 2.81-2.53 with pooled whole blood specimens and within %CV = 2.88-2.53 with QCs; total imprecision with QCs was within %CV = 2.14-1.51. Inaccuracy with value assigned QC was %△ = 5.36 at the 5.6 ng/mL level and %△ = 5.56 at the 11.7 ng/mL level. LoQ was 0.93 ng/mL (%CV = 10). Passing-Bablok regression showed a constant bias of 0.679 ng/mL (95% CI: 0.216-1.026) and a proportional bias of 1.326 (95% CI: 1.240-1.425). Bland-Altman analysis showed 5/72 (6.9%) paired differences exceeding the limits of agreement and 1/72 (1.4%) paired differences exceeding 1.96 SD to a combined bias of 39.9% after detrending. CONCLUSIONS: ACMIA/EVRO shows satisfactory analytical performances that comply with recommendations, but it does not fulfill requirements for interchangeability with QMS/EVER. Particularly, this new assay using sirolimus-specific antibody shows a sizable proportional bias versus the more specific comparator, which may be because of EVL metabolites. This is supported by the lack of agreement for individual differences in most samples collected at the peak concentration (C2). Therefore, further evidence is needed to support the transition of EVL level monitoring from QMS/EVER to ACMIA/EVRO without making extensive changes to both reference interval and patient's baseline.

Evaluation of the interchangeability between the new fully-automated affinity chrome-mediated immunoassay (ACMIA) and the Quantitative Microsphere System (QMS) with a CE-IVD-certified LC-MS/MS assay for therapeutic drug monitoring of everolimus after solid organ transplantation.

OBJECTIVES: This study aims to evaluate the interchangeability between the Siemens Healthineers' "EVRO" new affinity chrome-mediated immunoassay (ACMIA/EVRO) and Thermo Fisher Scientific's "EVER" Quantitative Microsphere System (QMS/EVER) with Chromsystems' CE-IVD-certified "MassTox" liquid-chromatography/tandem-mass spectrometry (LC-MS/MS) assay for the therapeutic drug monitoring of everolimus. METHODS: A single lot of reagent, calibrators and controls were used for each assay. A total of 67 whole blood samples (n=67) from patients receiving solid organ transplant were analyzed (n=31 with kidney transplant and n=36 with liver transplant); Passing-Bablok regression and Bland-Altman difference plot were used to evaluate bias and individual agreement; LC-MS/MS analysis was used to measure the actual concentrations of calibrators and controls compared to the assigned value. RESULTS: ACMIA/EVRO did not show any systematic bias compared to LC-MS/MS (intercept=0.244 ng/mL, 95% CI: -0.254 to 0.651 ng/mL). Nevertheless, significant proportional bias (slope=1.511, 95% CI: 1.420 to 1.619) associated to a combined bias of 44.8% (95% CI: 41.2-48.3%) was observed. Conversely, QMS/EVER did not show any bias at both systematic (intercept=-0.151 ng/mL, 95% CI: -0.671 to 0.256 ng/mL) and proportional level (slope=0.971, 95% CI: 0.895 to 1.074) with a non-statistically significant combined bias of -3.6% (95% CI: -8.4-1.1%). Based on a concentration of calibrators and controls above the assigned value for both the analytical methods, in the ACMIA/EVRO a correction which was approximately one-third of the correction for the QMS/EVER was observed. CONCLUSIONS: ACMIA/EVRO but not QMS/EVER shows a lack of interchangeability with the CE-IVD-certified LC-MS/MS assay. We hypothesize that, as the ACMIA/EVRO uses an anti-sirolimus antibody, the under-corrected assigned value in the assay calibrators was not sufficient to reproduce the everolimus metabolites cross-reactivity occurring in real samples.

Clinical and pharmacokinetics equivalence of multiple doses of levodopa benserazide generic formulation vs the originator (Madopar).

AIMS: While several generic preparations of levodopa/carbidopa and levodopa/benserazide (LBD) are currently available, pharmacokinetic (PK) equivalence and therapeutic equivalence studies with levodopa generics are not available in Italy. Lack of data on generic formulations is a critical factor for their limited use in this country and often lead patients to refuse the generic version of the branded drug. METHODS: An experimental, 2-centre, randomized, double-blind, 2-sequence, noninferiority cross-over study was designed to evaluate both the PK equivalence and clinical equivalence of multiple doses of the generic preparation of LDB, Teva Italia, compared to the originator (Madopar). Forty-three out-patients with a diagnosis of idiopathic Parkinson's disease on LDB, were recruited and randomly assigned to 1 of 2 study sequences: generic-originator or originator-generic. Clinical evaluations were performed at the end of each study period. A PK study with an LDB fixed dose (100 + 25 mg) was performed in a subpopulation of 14 subjects. RESULTS: Clinical data showed a reduction of 0.49 and 1.54 in the mean UPDRS III scores for the LDB and the originator, respectively. The 95% CIs [-2.21: 0.11] of the mean difference original vs LDB are smaller than the clinically significant difference of 3 UPDRS III points, supporting the conclusion that the treatment with LDB is not inferior to the originator. No statistically significant differences were found with respect to area under the curve to last dose, half-life, maximum concentration, time to maximum concentration and last observed concentration. CONCLUSION: These findings prove the therapeutic clinical equivalence as well the PK equivalence of the generic LDB and the originator (Madopar).

Dopamine-beta-hydroxylase 19-bp insertion/deletion polymorphism affects medication overuse in patients with chronic migraine.

Dopamine-beta-hydroxylase (DBH) enzyme activity is modulated at the genetic level by the presence of several polymorphisms. Among these, the 19-bp insertion/deletion (I/D) polymorphism (rs72393728/rs141116007) was investigated in several genetic association studies for its correlation with the susceptibility to develop episodic migraine, but conflicting results were achieved. In the present study we analyzed this genetic variant in a carefully characterized population of migraineurs encompassing both episodic and chronic migraine (with and without medication overuse) with the aim to perform a replication study and verify any possible correlation with migraine endophenotypes. Genotyping of the DBH 19-bp I/D polymorphism was performed on 400 migraine patients and 204 healthy individuals. The associations between genotypic frequencies and the clinical and sociodemographic features of migraineurs were then investigated. The DBH 19-bp I/D polymorphism did not correlate with migraine susceptibility or most clinical variables, with the exception of a statistically significant correlation within the subgroup of patients affected by chronic migraine were the individuals carrying the deleted (D) allele were significantly more prone to abuse in analgesics. As a result of this finding, the DBH 19-bp I/D polymorphism does not influence migraine susceptibility, but it might contribute to the development of medication overuse in patient with chronic migraine.

Validation of the Six Sigma Z-score for the quality assessment of clinical laboratory timeliness.

BACKGROUND: The International Federation of Clinical Chemistry and Laboratory Medicine has introduced in recent times the turnaround time (TAT) as mandatory quality indicator for the postanalytical phase. Classic TAT indicators, namely, average, median, 90th percentile and proportion of acceptable test (PAT), are in use since almost 40 years and to date represent the mainstay for gauging the laboratory timeliness. In this study, we investigated the performance of the Six Sigma Z-score, which was previously introduced as a device for the quantitative assessment of timeliness. METHODS: A numerical simulation was obtained modeling the actual TAT data set using the log-logistic probability density function. Five thousand replicates for each size of the artificial TAT random sample (n=20, 50, 250 and 1000) were generated, and different laboratory conditions were simulated manipulating the PDF in order to generate more or less variable data. The Z-score and the classic TAT indicators were assessed for precision (%CV), robustness toward right-tailing (precision at different sample variability), sensitivity and specificity. RESULTS: Z-score showed sensitivity and specificity comparable to PAT (≈80% with n≥250), but superior precision that ranged within 20% by moderately small sized samples (n≥50); furthermore, Z-score was less affected by the value of the cutoff used for setting the acceptable TAT, as well as by the sample variability that reflected into the magnitude of right-tailing. CONCLUSIONS: The Z-score was a valid indicator of laboratory timeliness and a suitable device to improve as well as to maintain the achieved quality level.

Long story short: an introduction to the short-term and longterm Six Sigma quality and its importance in laboratory medicine for the management of extra-analytical processes.

There is a compelling need for quality tools that enable effective control of the extra-analytical phase. In this regard, Six Sigma seems to offer a valid methodological and conceptual opportunity, and in recent times, the International Federation of Clinical Chemistry and Laboratory Medicine has adopted it for indicating the performance requirements for non-analytical laboratory processes. However, the Six Sigma implies a distinction between short-term and long-term quality that is based on the dynamics of the processes. These concepts are still not widespread and applied in the field of laboratory medicine although they are of fundamental importance to exploit the full potential of this methodology. This paper reviews the Six Sigma quality concepts and shows how they originated from Shewhart's control charts, in respect of which they are not an alternative but a completion. It also discusses the dynamic nature of process and how it arises, concerning particularly the long-term dynamic mean variation, and explains why this leads to the fundamental distinction of quality we previously mentioned.

Smart management of sample dilution using an artificial neural network to achieve streamlined processes and saving resources: the automated nephelometric testing of serum free light chain as case study.

BACKGROUND: Saving resources is a paramount issue for the modern laboratory, and new trainable as well as smart technologies can be used to allow the automated instrumentation to manage samples more efficiently in order to achieve streamlined processes. In this regard the serum free light chain (sFLC) testing represents an interesting challenge, as it usually causes using a number of assays before achieving an acceptable result within the analytical range. METHODS: An artificial neural network based on the multi-layer perceptron (MLP-ANN) was used to infer the starting dilution status of sFLC samples based on the information available through the laboratory information system (LIS). After the learning phase, the MLP-ANN simulation was applied to the nephelometric testing routinely performed in our laboratory on a BN ProSpec® System analyzer (Siemens Helathcare) using the N Latex FLC kit. RESULTS: The MLP-ANN reduced the serum kappa free light chain (κ-FLC) and serum lambda free light chain (λ-FLC) wasted tests by 69.4% and 70.8% with respect to the naïve stepwise dilution scheme used by the automated analyzer, and by 64.9% and 66.9% compared to a "rational" dilution scheme based on a 4-step dilution. CONCLUSIONS: Although it was restricted to follow-up samples, the MLP-ANN showed good predictive performance, which alongside the possibility to implement it in any automated system, made it a suitable solution for achieving streamlined laboratory processes and saving resources.

New insights into the pharmacokinetics of intravenous busulfan in children with sickle cell anemia undergoing bone marrow transplantation.

BACKGROUND: Busulfan (Bu) is an integral part of conditioning regimens for patients with sickle cell anemia (SCA) undergoing transplantation. Patients with SCA might predispose to transplant-related neurological and pulmonary toxicities due to pre-existing disease-related cerebrovascular and lung injury. Bu therapy appears to be an important contributing factor in this context. PROCEDURE: We studied the pharmacokinetics of intravenous Bu and clinical outcomes of 36 children with SCA undergoing bone marrow transplantation. Most patients had pre-existing organ system damage. Busulfan was administered every 6 hr for 4 days with pharmacokinetic-guided dose adjustment to target a conservative area under the concentration versus time curve (AUC) range of 900-1,350 µMol*min. RESULTS: We found that the first-dose Bu clearance was significantly higher (P < 0.0005) than the subsequent daily clearance, which remained unchanged during the following days. After the first-dose, 69% of patients achieved the target range. We adapted a new dose-adjustment strategy targeting exposures to the lower end (900 µMol*min) of the AUC range after the first dose of Bu to avoid unnecessary dose increases on subsequent days due to differences in clearance. This strategy enabled most patients to maintain the AUC within therapeutic range following dose adjustments. CONCLUSIONS: Differences in Bu clearance after the first-dose and subsequent daily doses in patients with SCA should be considered for pharmacokinetic-guided dose adjustment. Conservative AUC range and targeting exposures to the lower end of the range after the first dose was associated with negligible toxicity, and high engraftment and sickle cell-free survival rates.

Look beyond Catechol-O-Methyltransferase genotype for cathecolamines derangement in migraine: the BioBIM rs4818 and rs4680 polymorphisms study.

BACKGROUND: The study of COMT gene polymorphisms in migraine could be of particular interest since impaired catecholaminergic neurotransmission, namely chronic dopaminergic and noradrenergic hypofunction, is a peculiar migraine trait. In this study, for the first time, we focused on the role of COMT rs4818 genetic variant, the polymorphism most strongly affecting COMT activity, in migraine. This study was conducted in a cohort of carefully clinical characterized Caucasian migraineurs recruited in a specifically dedicated migraine biobank, providing also a replication study on rs4680 polymorphism. FINDINGS: Genotyping of rs4680 and rs4818 Catechol-O-Methyltransferase gene polymorphisms was performed on 380 unrelated migraine patients, and 132 healthy subjects matched for age, gender and race-ethnicity, with no clinical evidence or family history of migraine or other neurological diseases. The rs4680 and rs4818 genotypic frequencies did not deviate from those expected for a population in Hardy-Weinberg equilibrium and did not correlate with demographics or clinical migraine features, even when considering migraine subtypes such as dopaminergic migraine, menstrual migraine, and menstrually related migraine . CONCLUSIONS: COMT genotype does not influence migraine susceptibility or phenotype, even considering rs4818 polymorphism and peculiar clinical subtypes. This finding prompts to go over COMT to explain catecholamine derangement in migraine, exploring enzymes involved in catecholamines synthesis and catabolism, such as monoamine-oxidase, dopamine beta-hydroxylase, tyrosine-hydroxylase or tyrosine-decarboxylase, among others.

Blood alcohol concentration in the clinical laboratory: a narrative review of the preanalytical phase in diagnostic and forensic testing.

The analysis of blood alcohol concentration (BAC), a pivotal toxicological test, concerns acute alcohol intoxication (AAI) and driving under the influence (DUI). As such, BAC presents an organizational challenge for clinical laboratories, with unique complexities due to the need for forensic defensibility as part of the diagnostic process. Unfortunately, a significant number of scientific investigations dealing with the subject present discrepancies that make it difficult to identify optimal practices in sample collection, transportation, handling, and preparation. This review provides a systematic analysis of the preanalytical phase of BAC that aims to identify and explain the chemical, physiological, and pharmacological mechanisms underlying controllable operational factors. Nevertheless, it seeks evidence for the necessity to separate preanalytical processes for diagnostic and forensic BAC testing. In this regard, the main finding of this review is that no literature evidence supports the necessity to differentiate preanalytical procedures for AAI and DUI, except for the traceability throughout the chain of custody. In fact, adhering to correct preanalytical procedures provided by official bodies such as European federation of clinical chemistry and laboratory medicine for routine phlebotomy ensures both diagnostic accuracy and forensic defensibility of BAC. This is shown to depend on the capability of modern pre-evacuated sterile collection tubes to control major factors influencing BAC, namely non-enzymatic oxidation and microbial contamination. While certain restrictions become obsolete with such devices, as the use of sodium fluoride (NaF) for specific preservation of forensic BAC, this review reinforces the recommendation to use non-alcoholic disinfectants as a means to achieve "error-proof" procedures in challenging operational environments like the emergency department.

Systems Biology and Inborn Error of Metabolism: Analytical Strategy in Investigating Different Biochemical/Genetic Parameters.

Inborn errors of metabolism (IEM) are a group of about 500 rare genetic diseases with large diversity and complexity due to number of metabolic pathways involved in. Establishing a correct diagnosis and identifying the specific clinical phenotype is consequently a difficult task. However, an inclusive diagnosis able in capturing the different clinical phenotypes is mandatory for successful treatment. However, in contrast with Garrod's basic assumption "one-gene one-disease," no "simple" correlation between genotype-phenotype can be vindicated in IEMs. An illustrative example of IEM is Phenylketonuria (PKU), an autosomal recessive inborn error of L-phenylalanine (Phe) metabolism, ascribed to variants of the phenylalanine hydroxylase (PAH) gene encoding for the enzyme complex phenylalanine-hydroxylase. Blood values of Phe allow classifying PKU into different clinical phenotypes, albeit the participation of other genetic/biochemical pathways in the pathogenetic mechanisms remains elusive. Indeed, it has been shown that the most serious complications, such as cognitive impairment, are not only related to the gene dysfunction but also to the patient's background and the participation of several nongenetic factors.Therefore, a Systems Biology-based strategy is required in addressing IEM complexity, and in identifying the interplay between different pathways in shaping the clinical phenotype. Such an approach should entail the concerted investigation of genomic, transcriptomics, proteomics, metabolomics profiles altogether with phenylalanine and amino acids metabolism. Noticeably, this "omic" perspective could be instrumental in planning personalized treatment, tailored accordingly to the disease profile and prognosis.

Prion protein gene M129V polymorphism and variability in age at migraine onset.

Prion protein, a sialoglycoprotein with neuroprotective properties on oxidative stress damage, has been related with the mechanisms leading to migraine. In the present case-control study, we investigated the correlation between the common methionine/valine polymorphism at codon 129 within the prion protein gene (PRNP) and migraine. Genotyping of PRNP V129M variant was performed in 384 migraine patients and 185 age-, sex-, and race-ethnicity-matched healthy controls. The frequencies of the PRNP V129M genotype did not differ significantly between migraineurs and controls. The frequencies of 129VV genotype were significantly higher in patients with earlier age at migraine onset. No correlation was found between PRNP 129 genotype and demographics, and other clinical migraine features. Our data suggest that the PRNP 129VV polymorphism is not a direct migraine risk factor but is significantly associated with an earlier onset of the disease.

Characterization of Gut Microbiota Composition in Type 2 Diabetes Patients: A Population-Based Study.

(1) Background: A clinical laboratory index to assess gut dysbiosis is the F/B ratio < 0.8. In fact, an elevated proportion of Firmicutes and a reduced population of Bacteroides in diabetes type 2 (T2D) subjects has been observed. This study aimed to detail the dysbiosis status in the Italian population, focusing on some pathogenic spectra (T2D) or metabolic disorders. (2) Material and methods: A quantity of 334 fecal samples was analyzed in order to perform genetic testing and sequencing. (3) Results: A trend in over imbalance was observed in the percentage of Proteobacteria (median value: 6.75%; interquartile range (IQR): 3.57−17.29%). A statistically significant association (χ2p = 0.033) was observed between type of dysbiosis and T2D, corresponding to an Odds Ratio (OR) of 1.86. It was noted that females with cystitis/candidiasis are significantly prevalent in T2D patients (p < 0.01; OR: 3.59; 95% CI: 1.43−8.99). Although, in non-diabetic males, a sugar craving is significantly associated with the rate of dysbiosis in non-diabetic males (p < 0.05; OR 1.07; 95% CI 1.00−1.16). (4) Conclusion: In T2D patients, the Bacteroidetes/Firmicutes ratio was biased in favor of Proteobacteria, to be expected due to the nutritional habits of the patients. Thus, T2D females had altered gut permeability favoring the development of infections in the vaginal tract.

Vitamin D, SARS-CoV-2 and Causal Associations in Transversal Studies: The Time-Series Analysis to Reveal Potential Confounders. Comment on Gaudio et al. Vitamin D Levels Are Reduced at the Time of Hospital Admission in Sicilian SARS-CoV-2-Positive Patients. Int. J. Environ. Res. Public Health 2021, 18, 3491.

We read with great interest the paper by Gaudio and colleagues on vitamin D and on the state of patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the time of admission [...].

Six Sigma revisited: We need evidence to include a 1.5 SD shift in the extraanalytical phase of the total testing process.

The Six Sigma methodology has been widely implemented in industry, healthcare, and laboratory medicine since the mid-1980s. The performance of a process is evaluated by the sigma metric (SM), and 6 sigma represents world class performance, which implies that only 3.4 or less defects (or errors) per million opportunities (DPMO) are expected to occur. However, statistically, 6 sigma corresponds to 0.002 DPMO rather than 3.4 DPMO. The reason for this difference is the introduction of a 1.5 standard deviation (SD) shift to account for the random variation of the process around its target. In contrast, a 1.5 SD shift should be taken into account for normally distributed data, such as the analytical phase of the total testing process; in practice, this shift has been included in all type of calculations related to SM including non-normally distributed data. This causes great deviation of the SM from the actual level. To ensure that the SM value accurately reflects process performance, we concluded that a 1.5 SD shift should be used where it is necessary and formally appropriate. Additionally, 1.5 SD shift should not be considered as a constant parameter automatically included in all calculations related to SM.

A liquid chromatography-tandem mass spectrometry method for the determination of 5-Fluorouracil degradation rate by intact peripheral blood mononuclear cells.

5-Fluorouracil (5-FU) is a major chemotherapy drug used for the treatment of tumors. It is catabolized mainly by dihydropyrimidine dehydrogenase, and patients with a complete or partial deficiency of dihydropyrimidine dehydrogenase activity are at risk of developing severe 5-FU-associated toxicity. The aim of this study was to demonstrate that intact peripheral blood mononuclear cells (PBMCs) can be an effective model to evaluate the degradation rate of 5-FU. We developed a sensitive and specific liquid chromatography-tandem mass spectrometry method to measure in vitro the rate of 5-FU degradation by intact PBMC. 5-FU degradation rate was determined by measuring the decrease of a fixed amount of 5-FU (10 microg/mL) added to a solution of PBMC, after 2 hours incubation, expressed as nanogram per milliliter of 5-FU degraded per minute x 10(6) cells. Freshly prepared intact PBMC can degrade efficiently in vitro-added 5-FU. The assay consists of 3 steps: (1) PBMC isolation from peripheral blood, (2) PBMC incubation with 5-FU in vitro for different times, and (3) determination of 5-FU amount to calculate the degradation rate. 5-FU was analyzed by a Q Trap 2000 triple quadrupole/ion trap mass spectrometer in the multiple-reaction-monitoring modes. The chromatographic separation was accomplished using a C18 column with a run time of 16 minutes. By analyzing samples from 39 patients with no 5-FU toxicity, the mean 5-FU degradation rate was 1.85 +/- 0.50 ng x mL(-1) x min(-1) x 10(6) cells. The assessment of a test to measure 5-FU degradation rate in PBMC of patients before 5-FU administration could represent a prescreening method for evaluating the possible toxicity of this drug as an aid to set up a personalized medicine approach for each patient.