Goal-directed Perioperative Albumin Substitution Versus Standard of Care to Reduce Postoperative Complications: A Randomized Clinical Trial (SuperAdd Trial).

OBJECTIVE: To investigate whether goal-directed albumin substitution during surgery and postanesthesia care to maintain a serum albumin concentration >30 g/L can reduce postoperative complications. BACKGROUND: Hypoalbuminemia is associated with numerous postoperative complications. Since albumin has important physiological functions, substitution of patients with hypoalbuminemia is worth considering. METHODS: We conducted a single-center, randomized, controlled, outcome assessor-blinded clinical trial in adult patients, American Society of Anesthesiologists physical status classification 3 to 4 or undergoing high-risk surgery. Patients, whose serum albumin concentration dropped <30 g/L were randomly assigned to goal-directed albumin substitution maintaining serum concentration >30 g/L or to standard care until discharge from the postanesthesia intermediate care unit. Standard of care allowed albumin substitution in hemodynamic instable patients with serum concentration <20 g/L, only. Primary outcome was the incidence of postoperative complications ≥2 according to the Clavien-Dindo Classification in at least 1 of 9 domains (pulmonary, infectious, cardiovascular, neurological, renal, gastrointestinal, wound, pain, and hematological) until postoperative day 15. RESULTS: Of 2509 included patients, 600 (23.9%) developed serum albumin concentrations <30 g/L. Human albumin 60 g (40-80 g) was substituted to 299 (99.7%) patients in the intervention group and to 54 (18.0%) in the standard care group. At least 1 postoperative complication classified as Clavien-Dindo Classification ≥2 occurred in 254 of 300 patients (84.7%) in the intervention group and in 262 of 300 (87.3%) in the standard treatment group (risk difference -2.7%, 95% CI, -8.3% to 2.9%). CONCLUSION: Maintaining serum albumin concentration of >30 g/L perioperatively cannot generally be recommended in high-risk noncardiac surgery patients.

Evaluation of a host-protein signature score for differentiating between bacterial and viral infections: real-life evidence from a German tertiary hospital.

PURPOSE: A host-protein signature score, consisting of serum-concentrations of C-reactive protein, tumour necrosis factor-related apoptosis-inducing ligand, and interferon gamma-induced protein 10, was validated for distinguishing between bacterial and viral infections as an antimicrobial stewardship measure for routine clinical practice among adult patients in a German tertiary hospital. METHODS: This single-centre, explorative study prospectively assessed the host-protein signature score, comparing it with serum procalcitonin (PCT) in patients with blood stream infections (BSI) and evaluating its efficacy in patients with viral infections against the standard of care (SOC) to assess the need for antibiotics due to suspected bacterial super/coinfection. Manufacturer-specified threshold scores were used to differentiate viral (< 35) and bacterial (> 65) infections. RESULTS: Ninety-seven patients (BSI [n = 56]; viral infections [n = 41]) were included. The score (cut-off score > 65) tended to detect BSI with higher sensitivity than did PCT (cut-off > 0.5 ng/mL) (87.5% vs. 76.6%). Three patients (5.4%) with BSI had a score < 35. One patient with BSI did not receive antibiotic treatment following SOC prior to positive blood culture results. Among patients with viral infections, 29 (70.7%) had scores > 65, indicating bacterial superinfections. Additionally, 11 patients (26.8%) had scores < 35, indicating no bacterial superinfections. In total, the antibiotic treatment discrepancy in the viral group between the SOC and a host-protein signature score guided approach was 2/41 patients (4.9%). CONCLUSION: The score tended towards a higher sensitivity in detecting BSI than that with PCT. However, its impact on reducing antibiotic use in viral infections was minor compared with that of SOC.

The influence of undetected hemolysis on POCT potassium results in the emergency department.

OBJECTIVES: This study aimed to evaluate discrepancies in potassium measurements between point-of-care testing (POCT) and central laboratory (CL) methods, focusing on the impact of hemolysis on these measurements and its impact in the clinical practice in the emergency department (ED). METHODS: A retrospective analysis was conducted using data from three European university hospitals: Technische Universitat München (Germany), Hospital Universitario La Paz (Spain), and Erasmus University Medical Center (The Netherlands). The study compared POCT potassium measurements in EDs with CL measurements. Data normalization was performed in categories for potassium levels (kalemia) and hemolysis. The severity of discrepancies between POCT and CL potassium measurements was assessed using the reference change value (RCV). RESULTS: The study identified significant discrepancies in potassium between POCT and CL methods. In comparing POCT normo- and mild hypokalemia against CL results, differences of -4.20 % and +4.88 % were noted respectively. The largest variance in the CL was a +4.14 % difference in the mild hyperkalemia category. Additionally, the RCV was calculated to quantify the severity of discrepancies between paired potassium measurements from POCT and CL methods. The overall hemolysis characteristics, as defined by the hemolysis gradient, showed considerable variation between the testing sites, significantly affecting the reliability of potassium measurements in POCT. CONCLUSIONS: The study highlighted the challenges in achieving consistent potassium measurement results between POCT and CL methods, particularly in the presence of hemolysis. It emphasised the need for integrated hemolysis detection systems in future blood gas analysis devices to minimise discrepancies and ensure accurate POCT results.

Point-of-care testing: state-of-the art and perspectives.

Point-of-care testing (POCT) is becoming an increasingly popular way to perform laboratory tests closer to the patient. This option has several recognized advantages, such as accessibility, portability, speed, convenience, ease of use, ever-growing test panels, lower cumulative healthcare costs when used within appropriate clinical pathways, better patient empowerment and engagement, and reduction of certain pre-analytical errors, especially those related to specimen transportation. On the other hand, POCT also poses some limitations and risks, namely the risk of lower accuracy and reliability compared to traditional laboratory tests, quality control and connectivity issues, high dependence on operators (with varying levels of expertise or training), challenges related to patient data management, higher costs per individual test, regulatory and compliance issues such as the need for appropriate validation prior to clinical use (especially for rapid diagnostic tests; RDTs), as well as additional preanalytical sources of error that may remain undetected in this type of testing, which is usually based on whole blood samples (i.e., presence of interfering substances, clotting, hemolysis, etc.). There is no doubt that POCT is a breakthrough innovation in laboratory medicine, but the discussion on its appropriate use requires further debate and initiatives. This collective opinion paper, composed of abstracts of the lectures presented at the two-day expert meeting "Point-Of-Care-Testing: State of the Art and Perspective" (Venice, April 4-5, 2024), aims to provide a thoughtful overview of the state-of-the-art in POCT, its current applications, advantages and potential limitations, as well as some interesting reflections on the future perspectives of this particular field of laboratory medicine.

Surface plasmon resonance assays for the therapeutic drug monitoring of infliximab indicate clinical relevance of anti-infliximab antibody binding properties.

OBJECTIVES: The therapeutic antibody infliximab (IFX) has improved the life quality of numerous autoinflammatory disease patients. However, IFX can trigger the generation of anti-drug antibodies (ADA), whose optimal evaluation and management are currently subject of controversial discussions. We present two novel surface plasmon resonance (SPR) biosensor assays for therapeutic drug monitoring of IFX and characterization of ADA and investigated the diagnostic value of ADA binding properties. METHODS: IFX and ADA were quantified via developed SPR biosensor assays (IFXmon and ADAmon, respectively) and diagnostics-approved ELISA in sera from inflammatory bowel disease patients. Pre-analytic ADA enrichment with magnetic beads enabled analytical drug tolerance of the ADAmon assay. The dissociation ratio (DissR) as an index for ADA:IFX binding stability was calculated from the SPR sensorgrams of ADA quantification runs. RESULTS: IFX levels determined by IFXmon assay and ELISA showed high agreement, whereas ADA quantification concordance between ADAmon assay and ELISA was poor. In patients, DissR was predominantly constant over time and differed significantly between therapy outcomes. A DissR cut-off of 1.524 indicated undetectable IFX levels with 71.4% sensitivity and 88.9% specificity. Additionally, the SPR reference surface was exploited as serum-individual negative control to check result plausibility within multi-sample run sequences. CONCLUSIONS: Overall, both SPR biosensor assays exhibited reliable quantitative performance with accuracies superior to their ELISA counterparts and precision inferior to ELISA only for ADAmon. DissR presented itself as promising ADA binding parameter and could contribute to both earlier and more tailored therapeutic decisions.

Failure Mode and Effects Analysis (FMEA) at the preanalytical phase for POCT blood gas analysis: proposal for a shared proactive risk analysis model.

OBJECTIVES: Proposal of a risk analysis model to diminish negative impact on patient care by preanalytical errors in blood gas analysis (BGA). METHODS: Here we designed a Failure Mode and Effects Analysis (FMEA) risk assessment template for BGA, based on literature references and expertise of an international team of laboratory and clinical health care professionals. RESULTS: The FMEA identifies pre-analytical process steps, errors that may occur whilst performing BGA (potential failure mode), possible consequences (potential failure effect) and preventive/corrective actions (current controls). Probability of failure occurrence (OCC), severity of failure (SEV) and probability of failure detection (DET) are scored per potential failure mode. OCC and DET depend on test setting and patient population e.g., they differ in primary community health centres as compared to secondary community hospitals and third line university or specialized hospitals. OCC and DET also differ between stand-alone and networked instruments, manual and automated patient identification, and whether results are automatically transmitted to the patient's electronic health record. The risk priority number (RPN = SEV × OCC × DET) can be applied to determine the sequence in which risks are addressed. RPN can be recalculated after implementing changes to decrease OCC and/or increase DET. Key performance indicators are also proposed to evaluate changes. CONCLUSIONS: This FMEA model will help health care professionals manage and minimize the risk of preanalytical errors in BGA.

An integrated device for fast and sensitive immunosuppressant detection.

The present paper describes a compact point of care (POC) optical device for therapeutic drug monitoring (TDM). The core of the device is a disposable plastic chip where an immunoassay for the determination of immunosuppressants takes place. The chip is designed in order to have ten parallel microchannels allowing the simultaneous detection of more than one analyte with replicate measurements. The device is equipped with a microfluidic system, which provides sample mixing with the necessary chemicals and pumping samples, reagents and buffers into the measurement chip, and with integrated thin film amorphous silicon photodiodes for the fluorescence detection. Submicrometric fluorescent magnetic particles are used as support in the immunoassay in order to improve the efficiency of the assay. In particular, the magnetic feature is used to concentrate the antibody onto the sensing layer leading to a much faster implementation of the assay, while the fluorescent feature is used to increase the optical signal leading to a larger optical dynamic change and consequently a better sensitivity and a lower limit of detection. The design and development of the whole integrated optical device are here illustrated. In addition, detection of mycophenolic acid and cyclosporine A in spiked solutions and in microdialysate samples from patient blood with the implemented device are reported.

Immunosuppressant quantification in intravenous microdialysate - towards novel quasi-continuous therapeutic drug monitoring in transplanted patients.

OBJECTIVES: Therapeutic drug monitoring (TDM) plays a crucial role in personalized medicine. It helps clinicians to tailor drug dosage for optimized therapy through understanding the underlying complex pharmacokinetics and pharmacodynamics. Conventional, non-continuous TDM fails to provide real-time information, which is particularly important for the initial phase of immunosuppressant therapy, e.g., with cyclosporine (CsA) and mycophenolic acid (MPA). METHODS: We analyzed the time course over 8 h of total and free of immunosuppressive drug (CsA and MPA) concentrations measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in 16 kidney transplant patients. Besides repeated blood sampling, intravenous microdialysis was used for continuous sampling. Free drug concentrations were determined from ultracentrifuged EDTA-plasma (UC) and compared with the drug concentrations in the respective microdialysate (µD). µDs were additionally analyzed for free CsA using a novel immunosensor chip integrated into a fluorescence detection platform. The potential of microdialysis coupled with an optical immunosensor for the TDM of immunosuppressants was assessed. RESULTS: Using LC-MS/MS, the free concentrations of CsA (fCsA) and MPA (fMPA) were detectable and the time courses of total and free CsA comparable. fCsA and fMPA and area-under-the-curves (AUCs) in µDs correlated well with those determined in UCs (r≥0.79 and r≥0.88, respectively). Moreover, fCsA in µDs measured with the immunosensor correlated clearly with those determined by LC-MS/MS (r=0.82). CONCLUSIONS: The new microdialysis-supported immunosensor allows real-time analysis of immunosuppressants and tailor-made dosing according to the AUC concept. It readily lends itself to future applications as minimally invasive and continuous near-patient TDM.

Convalescent plasma therapy in B-cell-depleted and B-cell sufficient patients with life-threatening COVID-19 - A case series.

OBJECTIVE: To investigate the effect of convalescent plasma therapy (CPT) on clinical courses of B-cell-sufficient and B-cell-depleted patients with life-threatening COVID-19. PATIENTS AND METHODS: In this case series, we retrospectively analysed clinical, laboratory and cardiopulmonary parameters of six patients with life-threatening COVID-19 receiving convalescent plasma (CP) as rescue therapy between April 11, 2020 to October 10, 2020. Clinical and laboratory parameters before and after transfusion were compared in two B-cell-depleted patients and four B-cell sufficient patients (control group). RESULTS: Both B-cell-depleted patients cleared SARS-CoV-2 virus and survived, while all other patients died within 14 days from intervention despite maximal therapeutic efforts. D-dimer levels increased in both cohorts subsequent to CPT. In control patients, mean Interleukin-6 increased and platelet levels decreased as opposed to decreasing and stable levels in B-cell-depleted patients, respectively. Control patients required increased doses of vasopressor compared to decreasing doses in B-cell depleted patients subsequent to CPT. PO2/FiO2 decrease was more pronounced and respiratory deterioration required postinterventional extracorporeal membrane oxygenation in two control patients. Transpulmonary thermodilution revealed a further increase of the Extravascular Lung Water Index upon CPT in control patients. CONCLUSION: Use of CP in late stages of life-threatening COVID-19 should be used with caution but may be beneficial in B-cell-depleted patients. Further studies are necessary to assess factors predicting potential therapeutic benefits as well as possible hazards.

Influence of diagnosis of venous thromboembolism on immature platelets, absolute platelet count and platelet aggregation over time.

The extent of the involvement of platelets in venous thromboembolisms (VTE) is still not fully understood. Immature platelets are large, RNA-rich, prothrombotic platelets. They are involved in arterial thromboembolisms and are associated with adverse cardiovascular events. Their role in VTE has not been investigated before. The aim of this study was to assess different platelet parameters including immature platelet fraction (IPF), immature platelet count (IPC), absolute platelet count and platelet aggregation (PA) over time in patients with VTE at time of diagnosis, as well as at 3-10 days and at 90-110 days after diagnosis. 50 healthy volunteers similar in age and sex to patients served as controls at diagnosis. IPF was measured by the Sysmex XE-5000 analyzer, PA was assessed using the Multiplate analyzer. Diagnosis of VTE had no relevant effect on IPF and IPC whereas absolute platelet count and PA were significantly decreased compared to controls. In the course of VTE, IPF decreased significantly, whereas IPC, absolute platelet count and PA increased. In conclusion, VTE was associated with relevant changes of the absolute platelet count and PA at diagnosis, as well as changes in IPF and IPC over time reflecting a relevant and measurable platelet consumption in VTEs.

A lipid-binding loop of botulinum neurotoxin serotypes B, DC and G is an essential feature to confer their exquisite potency.

The exceptional toxicity of botulinum neurotoxins (BoNTs) is mediated by high avidity binding to complex polysialogangliosides and intraluminal segments of synaptic vesicle proteins embedded in the presynaptic membrane. One peculiarity is an exposed hydrophobic loop in the toxin's cell binding domain HC, which is located between the ganglioside- and protein receptor-binding sites, and that is particularly pronounced in the serotypes BoNT/B, DC, and G sharing synaptotagmin as protein receptor. Here, we provide evidence that this HC loop is a critical component of their tripartite receptor recognition complex. Binding to nanodisc-embedded receptors and toxicity were virtually abolished in BoNT mutants lacking residues at the tip of the HC loop. Surface plasmon resonance experiments revealed that only insertion of the HC loop into the lipid-bilayer compensates for the entropic penalty inflicted by the dual-receptor binding. Our results represent a new paradigm of how BoNT/B, DC, and G employ ternary interactions with a protein, ganglioside, and lipids to mediate their extraordinary neurotoxicity.

Users Regard Operator Training as Critical for Successful POCT Applications.

BACKGROUND: Point-of-care tests (POCT) measure analytes close to the patients and are a complementary supplement to the test menu of medical laboratories. However, the involvement of many different stakeholders makes it challenging to ensure reliable results. METHODS: In a survey, we asked experienced POCT users how they control their total POCT process and what factors they consider essential for success. Results were verified in four in-depth interviews. RESULTS: Overall, 73 German participants from various medical disciplines completed the survey. All but one participant regarded operator training as important but only half of the participants' institutions conducted operator training on a regular basis. Participants often requested e-learning, but face-to-face teaching is still preferred. Twenty-one percent of participants already used e-learning and reported mixed satisfaction. Fifty-five percent of the participants never refer to the quality management manual. Instead, 94% stated that if a POCT error arises a contact person for POCT is always available at their workplace. The majority of participants think that external and, in particular, internal quality controls are important for POCT. Only a few difficulties for performing quality control such as "temporal expenditure" and "lack of information about the importance of internal quality control" were commonly mentioned. For future developments, participants expect evolution and improvements especially with regard to "measurement quality and reliability". The answers of the experts in the in-depth interviews largely corresponded with the participants of the survey. CONCLUSIONS: The importance of operator training is well established and confirmed in this work. How to conduct this training is less certain, but the answers in this survey suggest some form of blended learning with e-learning and practical elements. The discrepancy between the high importance that guidelines and other normative documents place on written information and their low practical usage was striking.

Highly sensitive immunodiagnostics at the point of care employing alternative recognition elements and smartphones: hype, trend, or revolution?

Immunodiagnostic tests performed at the point of care (POC) today usually employ antibodies for biorecognition and are read out either visually or with specialized equipment. Availability of alternative biorecognition elements with promising features as well as smartphone-based approaches for signal readout, however, challenge the described established configuration in terms of analytical performance and practicability. Assessing these developments' clinical relevance and their impact on POC immunodiagnostics is demanding. The first part of this review will therefore give an overview on suitable diagnostic biosensors based on alternative recognition elements (such as nucleic acid-based aptamers or engineered binding proteins) and exemplify advantages and drawbacks of these biomolecules on the base of selected assays. The second part of the review then focuses on smartphone-connected diagnostics and discusses the indispensable considerations required for successful future clinical POCT implementation. Together, the joint depiction of two of the most innovative and exciting developments in the field will enable the reader to cast a glance into the distant future of POC immunodiagnostics.

Recent advances in immunodiagnostics based on biosensor technologies-from central laboratory to the point of care.

Immunological methods are widely applied in medical diagnostics for the detection and quantification of a plethora of analytes. Associated analytical challenges usually require these assays to be performed in a central laboratory. During the last several years, however, the clinical demand for rapid immunodiagnostics to be performed in the immediate proximity of the patient has been constantly increasing. Biosensors constitute one of the key technologies enabling the necessary, yet challenging transition of immunodiagnostic tests from the central laboratory to the point of care. This review is intended to provide insights into the current state of this transition process with a focus on the role of biosensor-based systems. To begin with, an overview on standard immunodiagnostic tests presently employed in the central laboratory and at the point of care is given. The review then moves on to demonstrate how biosensor technologies are reshaping this landscape. Single analyte as well as multiplexed immunosensors applicable to point of care scenarios are presented. A section on the areas of clinical application then creates the bridge to day-to-day diagnostic practice. Finally, the depicted developments are critically weighed and future perspectives discussed in order to give the reader a firm idea on the forthcoming trends to be expected in this diagnostic field.

Mortality prediction in stable hemodialysis patients is refined by YKL-40, a 40-kDa glycoprotein associated with inflammation.

Chronic inflammation contributes to increased mortality in hemodialysis (HD) patients. YKL-40 is a novel marker of inflammation, tissue remodeling, and highly expressed in macrophages inside vascular lesions. Elevated levels of YKL-40 have been reported for HD patients but how it integrates into the proinflammatory mediator network as a predictor of mortality remains elusive. We studied serum YKL-40, Interleukin-6 (IL-6), high-sensitivity C-reactive protein, monocyte chemotactic protein-1 (MCP-1), and interferon-gamma induced protein-10 (IP-10) in 475 chronic hemodialysis patients. Patients were followed for mortality for a median of 37 [interquartile range: 25-49] months and checked for interrelation of the measured mediators. To plot cumulative incidence functions, patients were stratified into terciles per YKL-40, IL-6, MCP-1, and IP-10 levels. Multivariable Cox regression models were built to examine associations of YKL-40, IP-10, and MCP-1 with all-cause and cause-specific mortality. Net reclassification improvement was calculated for the final models containing YKL-40 and IL-6. Increased YKL-40 was independently associated with age, IP-10, and IL-6 serum levels. After adjustment for demographic and laboratory parameters, comorbidities, and IL-6, only YKL-40 significantly improved risk prediction for all-cause (hazard ratio 1.4; 95% confidence interval 1.1-1.8) and cardiovascular mortality (hazard ratio 1.5; 95% confidence interval 1.03-2.2). Thus, in contrast to other biomarkers of aberrant macrophage activation, YKL-40 reflects inflammatory activity, which is not covered by IL-6. Mechanistic and prospective studies are needed to test for causal involvement of YKL-40 and whether it might qualify as a therapeutic target.

External quality assessment schemes for glucose measurements in Germany: factors for successful participation, analytical performance and medical impact.

BACKGROUND: Determination of blood glucose concentration is one of the most important measurements in clinical chemistry worldwide. Analyzers in central laboratories (CL) and point-of-care tests (POCT) are both frequently used. In Germany, regular participation in external quality assessment (EQA) schemes is mandatory for laboratories performing glucose testing. METHODS: Glucose testing data from the two German EQAs "Reference Institute for Bioanalytics" (RfB) and "INSTAND - Gesellschaft zur Förderung der Qualitätssicherung in medizinischen Laboratorien" (Instand) were analyzed from 2012 to 2016. Multivariable odds ratios (OR) for the probability to reach a "good" result were calculated. Imprecision and bias were determined and clinical risk of measurement errors estimated. RESULTS: The device employed was the most important variable required for a "good" performance in all EQAs. Additional participation in an EQA for CL automated analyzers improved performance in POCT EQAs. The reciprocal effect was less pronounced. New participants performed worse than experienced participants especially in CL EQAs. Imprecision was generally smaller for CL, but some POCT devices reached a comparable performance. Large lot-to-lot differences occurred in over 10% of analyzed cases. We propose the "bias budget" as a new metric to express the maximum allowable bias that still carries acceptable medical risk. Bias budgets were smallest and clinical risks of errors greatest in the low range of measurement 60-115 mg/dL (3.3-6.4 mmol/L) for most devices. CONCLUSIONS: EQAs help to maintain high analytical performances. They generate important data that serve as the foundation for learning and improvement in the laboratory healthcare system.

Express incorporation of membrane proteins from various human cell types into phospholipid bilayer nanodiscs.

Analysis of membrane proteins is still inadequately represented in diagnostics despite their importance as drug targets and biomarkers. One main reason is the difficult handling caused by their insolubility in aqueous buffer solutions. The nanodisc technology was developed to circumvent this challenge and enables analysis of membrane proteins with standard research methods. However, existing nanodisc generation protocols rely on time-consuming membrane isolation and protein purification from overexpression systems. In the present study, we present a novel, simplified procedure for the rapid generation of nanodiscs directly from intact cells. Workflow and duration of the nanodisc preparation were shortened without reducing the reconstitution efficiency, and all the steps were modified for the use of only standard laboratory equipment. This protocol was successfully applied to various human cell types, such as cultivated human embryonic kidney 293 (HEK-293) cells, as well as freshly isolated human red blood cells and platelets. In addition, the reconstitution of membrane proteins from cell organelles was achieved. The use of endogenous lipids ensures a native-like environment, which promotes native protein (re)folding. Nanodisc generation was verified by size exclusion chromatography and EM, whereas incorporation of different membrane proteins was demonstrated by Western blot analysis. Our protocol enabled the rapid incorporation of endogenous membrane proteins from human cells into nanodiscs, which can be applied to analytical approaches.

Stronger Together: Aggregated Z-values of Traditional Quality Control Measurements and Patient Medians Improve Detection of Biases.

BACKGROUND: In clinical chemistry, quality control (QC) often relies on measurements of control samples, but limitations, such as a lack of commutability, compromise the ability of such measurements to detect out-of-control situations. Medians of patient results have also been used for QC purposes, but it may be difficult to distinguish changes observed in the patient population from analytical errors. This study aims to combine traditional control measurements and patient medians for facilitating detection of biases. METHODS: The software package "rSimLab" was developed to simulate measurements of 5 analytes. Internal QC measurements and patient medians were assessed for detecting impermissible biases. Various control rules combined these parameters. A Westgard-like algorithm was evaluated and new rules that aggregate Z-values of QC parameters were proposed. RESULTS: Mathematical approximations estimated the required sample size for calculating meaningful patient medians. The appropriate number was highly dependent on the ratio of the spread of sample values to their center. Instead of applying a threshold to each QC parameter separately like the Westgard algorithm, the proposed aggregation of Z-values averaged these parameters. This behavior was found beneficial, as a bias could affect QC parameters unequally, resulting in differences between their Z-transformed values. In our simulations, control rules tended to outperform the simple QC parameters they combined. The inclusion of patient medians substantially improved bias detection for some analytes. CONCLUSIONS: Patient result medians can supplement traditional QC, and aggregations of Z-values are novel and beneficial tools for QC strategies to detect biases.

Chromogranin A and neurone-specific enolase serum levels as predictors of treatment outcome in patients with metastatic castration-resistant prostate cancer undergoing abiraterone therapy.

OBJECTIVE: To determine the impact of elevated neuroendocrine serum markers on treatment outcome in patients with metastatic castration-resistant prostate cancer (mCRPC) undergoing treatment with abiraterone in a post-chemotherapy setting. PATIENTS AND METHOD: Chromogranin A (CGa) and neurone-specific enolase (NSE) were determined in serum drawn before treatment with abiraterone from 45 patients with mCRPC. Outcome measures were overall survival (OS), prostate-specific antigen (PSA) response defined by a PSA level decline of ≥50%, PSA progression-free survival (PSA-PFS), and clinical or radiographic PFS. RESULTS: The CGa and NSE serum levels did not correlate (P = 0.6). Patients were stratified in to low- (nine patients), intermediate- (18) or high-risk (18) groups according to elevation of none, one, or both neuroendocrine markers, respectively. The risk groups correlated with decreasing median OS (median OS not reached vs 15.3 vs 6.6 months; P < 0.001), decreasing median clinical or radiographic PFS (8.3 vs 4.4 vs 2.7 months; P = 0.001) and decreasing median PSA-PFS (12.0 vs 3.2 vs 2.7 months; P = 0.012). In multivariate Cox regression analysis the combination of CGa and NSE (≥1 marker positive vs both markers negative) remained significant predictors of OS, clinical or radiographic PFS, and PSA-PFS. We did not observe a correlation with PSA response (63% vs 35% vs 31%; P = 0.2). CONCLUSION: Chromogranin A and NSE did not predict PSA response in patients with mCRPC treated with abiraterone. However, we observed a correlation with shorter PSA-PFS, clinical or radiographic PFS, and OS. This might be due to an elevated risk of developing resistance under abiraterone treatment related to neuroendocrine differentiation.

Simultaneous Determination of Protein-Unbound Cyclosporine A and Mycophenolic Acid in Kidney Transplant Patients Using Liquid Chromatography-Tandem Mass Spectrometry.

BACKGROUND: Therapeutic drug monitoring (TDM) of immunosuppressants is essential to optimize patient care after organ transplantation. In blood, most immunosuppressive drugs are bound to plasma proteins or located inside blood cells. However, it is generally assumed that only protein-unbound (free) drug concentrations are pharmacologically active and could therefore better reflect the clinical outcome. Study data are still limited due to lacking rapid analytical methods. Therefore, a simple multiplex method for direct measurement of free cyclosporine A (CsA) and mycophenolic acid (MPA) has been developed. METHODS: The sample preparation included ultracentrifugation, followed by liquid-liquid extraction. Stable isotope labeled analogues of CsA and MPA were used as internal standards. The LC-MS/MS analysis was performed on a triple quadrupole mass spectrometer in the multiple reaction monitoring mode. The validated assay was used in a study of 40 blood samples from kidney transplant patients. RESULTS: The lower limits of quantification were 0.1 (CsA) and 0.5 ng/mL (MPA). Assay linearity was confirmed in the concentration ranges of 0.1-10.0 ng/mL (CsA) and 0.5-100 ng/mL (MPA). For both analytes, inaccuracy was ≤9.8% and imprecision was ≤7.8%. The extraction efficiency ranged between 91% and 96%. In the patient samples the average free CsA and MPA fractions were 5.8% (2.1%-16.8%) and 1.2% (0.5%-2.4%) respectively. CONCLUSIONS: A reliable and highly sensitive LC-MS/MS method as a new suitable tool for measuring protein-unbound CsA and MPA has been developed, validated and applied in kidney transplant patient samples. Now, larger studies can be conducted to investigate the benefit of free drug monitoring in transplant recipients.