A simple and green capillary electrophoresis-mass spectrometry method for therapeutic drug monitoring of colistin in clinical plasma samples.

Colistin and other polymyxin antibiotics have become increasingly used in clinical settings as a result of treating multidrug-resistant infections in critically ill patients. The highly variable pharmacokinetics of colistin in these patients is accompanied by a high risk of toxicity or underdosing. An effective tool that allows rational optimization of the drug dosage regimen is therapeutic drug monitoring. Therefore, there is a need to dispose with appropriate, sensitive, and accurate analytical methods. Here, a simple, specific, and accurate on-line capillary electrophoresis - tandem mass spectrometry method was developed and applied for the first time to determine colistin in human plasma. Protein precipitation using acidified acetonitrile was the solitary procedure used to achieve sample pretreatment. A bare fused silica capillary was employed for the separation process, and the background electrolyte used was 50 mM formic acid (pH 2.54). The FDA's bioanalytical method validation guidelines were followed in the validation of the proposed method. For colistin A and colistin B, favorable performance and validation parameters were obtained (such as linearity, limit of detection, lower limit of quantitation, intra-day and inter-day precision, accuracy, and stability).The validated method was then effectively used to analyze real clinical samples taken from patients who were in critical condition. Our newly developed method is comparable with previously published liquid chromatography approaches and has the potential to be applied in the therapeutic monitoring of colistin in routine clinical laboratories. Moreover, according to the greenness assessment, the developed capillary electrophoresis - mass spectrometry method represents a very interesting green and sustainable tool in the field of bioanalysis.

An Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry Method for Simultaneous Determination of 4 ß-Lactam Antibiotics, Tazobactam, and Linezolid in Human Plasma Samples.

BACKGROUND: Optimization of antimicrobial therapy is a challenge in critically ill patients who develop extreme interindividual and intraindividual pharmacokinetic variability. Therapeutic drug monitoring is a valuable tool for maximizing the effect of a drug and minimizing its adverse and unwanted effects. The aim of the current work was to develop and validate an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method to determine multiple antibiotics in clinical plasma samples from critically ill patients; low sample volume and rapid processing of samples were considered the main criteria. METHODS: A separation method based on an online combination of UHPLC-MS/MS was developed for the simultaneous determination of 4 ß-lactam antibiotics (cefepime, meropenem, cefotaxime, and piperacillin), tazobactam, and linezolid in human plasma samples. The volume of plasma sample used for analysis was 20 µL. The developed method was validated according to Food and Drug Administration guidelines. RESULTS: The chromatographic run time was 8 minutes. Calibration curves were linear for concentration ranges of 0.1-100 mcg/mL (r 2 > 0.99) for tazobactam, meropenem, cefotaxime, linezolid, and piperacillin and 1-100 mcg/mL (r 2 > 0.99) for cefepime. The intraday and interday accuracy of the method ranged from 92.4% to 110.7% and 93.6% to 113.3%, respectively. The intraday and interday precision values were ≤17.3% and ≤17.4%, respectively. No interfering and carryover analytes were observed. CONCLUSIONS: The developed UHPLC-MS/MS method is an appropriate and practical tool for therapeutic drug monitoring of the selected antibiotics. Owing to its rapidity, requirement of low sample volume, and high selectivity, sensitivity, and reliability, it can be effectively implemented in routine clinical laboratory tests for critically ill patients.

Profiling of Amino Acids in Urine Samples of Patients Suffering from Inflammatory Bowel Disease by Capillary Electrophoresis-Mass Spectrometry.

Urine represents a convenient biofluid for metabolomic studies due to its noninvasive collection and richness in metabolites. Here, amino acids are valuable biomarkers for their ability to reflect imbalances of different biochemical pathways. An impact of amino acids on pathology, prognosis and therapy of various diseases, including inflammatory bowel disease (IBD), is therefore the subject of current clinical research. This work is aimed to develop a capillary electrophoresis-tandem mass spectrometry (CE-MS/MS) method for the quantification of the 20 proteinogenic amino acids in human urine samples obtained from patients suffering from IBD and treated with thiopurines. The optimized CE-MS/MS method, with minimum sample preparation (just "dilute and shoot"), exhibited excellent linearity for all the analytes (coefficients of determination were higher than 0.99), with inter-day and intra-day precision yielding relative standard deviations in the range of 0.91-15.12% and with accuracy yielding relative errors in the range of 85.47-112.46%. Total analysis time, an important parameter for the sample throughput demanded in routine practice, was shorter in ca. 17% when compared to established CE-MS methods. Favorable performance of the proposed CE-MS/MS method was also confirmed by the comparison with corresponding ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) method. Consistent data for the investigated amino acid metabolome were obtained using both methods. For the first time, the amino acid profiling by CE-MS approach was applied on the clinical IBD samples. Here, significant differences observed in the concentration levels of some amino acids between IBD patients undergoing thiopurine treatment and healthy volunteers could result from the simultaneous action of the disease and the corresponding therapy. These findings indicate that amino acids analysis could be a valuable tool for the study of mechanism of the IBD treatment by thiopurines.

Immunoglobulin abnormalities in 1677 solid organ transplant recipients. Implications for posttransplantation follow-up.

BACKGROUND: Posttransplant lymphoproliferative disorder (PTLD) is a severe complication of solid organ transplantation (SOT). However, there is no consensus on PTLD screening methods. Gammopathies (GP), which occur in 10-25% of SOT recipients, have been linked to subsequent development of PTLD. Therefore, GP detection methods, such as serum protein electrophoresis (SPE), serum protein immunofixation (SIFE), urine protein immunofixation (UIFE) and the quantitative measurement of serum free light chains (SFLC) are candidate methods for PTLD screening. OBJECTIVE: We aimed to assess the frequency of PTLD and GP, association of GP with subsequent PTLD, allograft loss or death and the diagnostic performance of SPE/SIFE in PTLD screening. The main objective was to explore, whether GP detection methods can be used to enhance the efficiency of PTLD screening and to formulate a concise algorithm for posttransplantation (post-Tx) follow-up. METHODS: We performed a cohort study on 1677 SOT recipients with SPE/SIFE data who underwent kidney, liver, heart, pancreas, Langerhans islets or multiple organ transplantation at the Institute of Clinical and Experimental Medicine between 1966 and 2015. The median (IQR) of follow-up time was 8.0 (4.0-12.0) years. RESULTS: The frequencies of PTLD and GP in SOT recipients were 2.8% and 6.4%, respectively. The frequencies of transient GP, GP of undetermined significance and malignant GP were 33%, 63% and 4% respectively. The median time between SOT and GP detection was 2.0 (interquartile range 1.0-7.0) years. GP was associated with a significantly higher risk of PTLD, allograft loss and death, with hazard ratios (95% confidence intervals) of a 6.06 (2.51-14.64), 2.61 (1.49-4.6) and 1.99 (1.2-3.3), respectively. Additionally, GP was associated with 2.98-fold increased risk of allograft loss in kidney transplant patients. SPE diagnostic sensitivity and specificity for PTLD were 14.8% and 93.9%, respectively. PTLD was diagnosed more often and earlier if SPE/SIFE was included in the post-Tx follow-up. CONCLUSIONS: GP after SOT is associated with a high risk of PTLD, allograft loss and poor survival. The combination of SPE, SIFE, SFLC and UIFE is optimal for GP detection. These methods aid in identifying patients who are at risk for PTLD or allograft damage and should be included in regular post-Tx follow-up.

Tacrolimus has immunosuppressive effects on heavy/light chain pairs and free light chains in patients after heart transplantation: A relationship with infection.

The aim of the study was to investigate the relationship between tacrolimus (TAC) immunosuppressive treatment and serum concentrations of immunoglobulin heavy/light chain pairs (sHLC) and free light chains (sFLC) in patients after heart transplantation (HTX) and to use these biomarkers to predict the risk of infection in these patients. A total of 88 patients with an immunosuppressive regimen involving tacrolimus who underwent HTX were analyzed over 24 months of follow-up. sFLC and sHLC levels were determined before and at three time points after HTX. TAC concentrations were determined at several time points after HTX, and mean TAC concentrations and areas under the curve (AUCs) of TAC concentration were calculated. Relevant clinical data were obtained from patients' medical records. A larger AUC of TAC was associated with decreases in the concentrations of IgG total (p < 0.05); similarly, cumulative AUC of TAC during 18 post-transplant months correlated inversely with sHLC IgG kappa (r = -0.228, p < 0.05) and IgG total (r = -0.352, p < 0.05). Concentrations of sFLC kappa, sFLC lambda, sHLC IgG kappa, and sHLC IgG total were significantly lower in infected patients (in the 9th month after HTX, all p < 0.05). Combined criteria for increased AUC (greater than the median of 12.9 mg·d/l) and decreased sFLC kappa (less than the median of 12.5 mg/l) correlated with the presence of infection (p < 0.03) in the 9th month after HTX. Ratio of concentration of TAC to sFLC kappa or lambda was significantly higher in infected patients (both p < 0.05). Intensive treatment with tacrolimus after HTX is possibly reflected by decreases in sFLC and sHLC (mainly sHLC IgG). Patients with decreased concentrations of these biomarkers are at increased risk for infection, primarily in the 9th month after HTX, when the concentrations of tacrolimus were the highest.

Falsely elevated human epididymis protein 4 results and Risk of Ovarian Malignancy Algorithm in polymorbid women after solid organ transplantation: A pilot and case-control study.

BACKGROUND: Cancer prevention is essential after transplantation (Tx). The use of HE4 and Risk of Ovarian Malignancy Algorithm (ROMA) is recommended as a tool for selective ovarian cancer screening; however, creatinine is a known confounder. This study assessed the reliability of HE4, CA125, and ROMA after Tx. METHODS: We matched a total of 202 women without gynecological malignancies and 236 men by age and serum creatinine. Each pair consisted of a patient after Tx (kidney, liver, heart, and pancreas) and a diseased but non-Tx consecutive patient. Serum HE4, CA125 (Roche Cobas 6000), and creatinine (enzymatic, Abbott Architect) were measured in all patients. RESULTS: Creatinine correlated with HE4 (women: r = .864, P < .0001; men: r = .848, P < .0001). Age correlated slightly with HE4 in women (r = .250, P < .005) and men (r = .240, P < .0005). HE4 in women after Tx (median of 84.8 pmol/L) was significantly higher than non-Tx women (53.7 pmol/L, P < .0001) in the reference range of serum creatinine. Neither HE4 nor CA125 correlated with tacrolimus concentration, but anemia, hyperparathyroidism, kidney, liver, and lung diseases were possible confounders for HE4 after transplantation (P < .05). CONCLUSION: Human epididymis protein 4 (HE4) was significantly increased in women after solid organ transplantation compared to levels without transplants matched by age and serum creatinine. HE4 results may be misleading in these patients.

Free light chain and intact immunoglobulin abnormalities in heart transplant recipients: Two year follow-up timelines and clinical correlations.

OBJECTIVES: To assess the timelines of serum free light chain (sFLC) concentrations and the kappa/lambda light chain (K/L) ratio in heart transplant (HTX) recipients. To analyze the performance of serum protein electrophoresis (SPE), serum immunofixation (sIFE) and sFLC measurements for gammopathy detection following a HTX. METHODS: A total of 96 patients who underwent a HTX were analyzed during a two-year follow-up period. The relevant clinical data were obtained from patient medical records. SPE, sIFE and sFLC methods were used for the detection of free light chain and intact immunoglobulin gammopathies at 4 time points after HTX. RESULTS: A statistically significant decrease in sFLC K and L (a decrease of 39.1% and 27.6%, respectively, when compared to pretransplant values) was found 9months after the HTX (p<0.001, Friedman test). We detected SPE or sIFE abnormalities in 23 (8.4%) samples, and sFLC K/L ratio abnormalities in 34 (12.4%) samples. All of the K/L ratio abnormalities had normal SPE/sIFE values, and 19% of the findings were persistent. CONCLUSIONS: A significant and consistent dynamics in the sFLC concentration was found in the HTX patients during a 2-year follow-up period, which reflected changes in the immunosuppressant dosage. A remarkable number of monoclonal and polyclonal gammopathies was identified with some persistent abnormalities, using the SPE/sIFE and sFLC methods. Some of the detected abnormalities, which might possess a higher risk for PTLD if interpreted according to common practice in nonTX patients can only be detected by sFLC methods.

Serial measurement of presepsin, procalcitonin, and C-reactive protein in the early postoperative period and the response to antithymocyte globulin administration after heart transplantation.

Differentiation between systemic inflammatory response syndrome and sepsis in surgical patients is of crucial significance. Procalcitonin (PCT) and C-reactive protein (CRP) are widely used biomarkers, but PCT becomes compromised after antithymocyte globulin (ATG) administration, and CRP exhibits limited specificity. Presepsin has been suggested as an alternative biomarker of sepsis. This study aimed to demonstrate the role of presepsin in patients after heart transplantation (HTx). Plasma presepsin, PCT, and CRP were measured in 107 patients serially for up to 10 days following HTx. Time responses of biomarkers were evaluated for both noninfected (n=91) and infected (n=16) patients. Areas under the concentration curve differed in the two groups of patients for presepsin (P<.001), PCT (P<.005), and CRP (P<.001). The effect of time and infection was significant for all three biomarkers (P<.05 all). In contrast to PCT, presepsin was not influenced by ATG administration. More than 25% of noninfected patients had PCT above 42 µg/L on the first day, and the peak concentration of CRP in infected patients was reached on the third post-transplant day (median 135 mg/L). Presepsin seems to be as valuable a biomarker as PCT or CRP in the evaluation of infectious complications in patients after HTx.

Multiple qualitative and quantitative methods for free light chain analysis are necessary as first line tests for AL amyloidosis.

The objective of this study was to demonstrate the necessity of using different methods for amyloidogenic light chain detection. Serum and urine agarose gel electrophoresis and immunofixation, as well as serum free light chain (FLC) immunoassay measurements, were evaluated in a patient with verified multiple myeloma and consequent AL amyloidosis confirmed by Congo red staining and immunofluorescence techniques. Conventional chemistry tests [serum and urine electrophoresis (SPE and UPE); serum and urine immunofixation (SIFE and UIFE)] were inconclusive. Only quantitative FLC immunoassay (serum free light chain immunoanalysis, SFLC) provided correct diagnostic information. A combination of gel-based SIFE and UIFE with more novel quantitative FLC immunoassays appears necessary when searching for monoclonal immunoglobulin light chain-related diseases.

The role of timely measurement of galectin-3, NT-proBNP, cystatin C and hsTnT in predicting prognosis and heart function after heart transplantation.

BACKGROUND: Changes of biomarkers measured soon after heart transplantation (HTx) can reflect different processes: cardiomyocyte necrosis (troponins, high-sensitivity cardiac TnT and TnI), heart function (natriuretic peptides, BNP and NT-proBNP), fibrosis (galectin-3 and ST2), and global cardiorenal risk (cystatin C). We assessed the prognostic role of hsTnT, NT-proBNP, galectin-3 and cystatin C during the early post-transplant period. METHODS: A total of 121 consecutive post-HTx patients were assessed. The main outcomes were survival, left ventricular ejection fraction (LVEF) and rejection periods. Survival was assessed after intermediate (12 months) and long periods (total follow-up during study, median of survival 763 days, IR 527-1038 days). LVEF was assessed 12 months after HTx. Rejection was evaluated during follow-up. We report biomarker concentrations measured 10 days and 12 months after HTx. RESULTS: Ten days after HTx, cystatin C and hsTnT predicted death both under univariable and multivariable analysis. These two biomarkers along with galectin-3 were increased in patients with decreased LVEF measured 1 year after HTx. NT-proBNP did not show early prognostic power. None of the measured biomarkers predicted rejection, but hsTnT and NT-proBNP were increased significantly 12 months after HTx in patients with at least one rejection. CONCLUSIONS: Cystatin C and hsTnT measured 10 days after HTx can provide prognostic information on survival and galectin-3 measured at the same time may display a relationship to heart function assessed 1 year after HTx. Further study should be carried out in a large cohort of patients.

A clinical and laboratory approach used to elucidate discordant results of high-sensitivity troponin T and troponin I.

BACKGROUND: Careful interpretation of discordant results in high-sensitivity troponin measurements is necessary in cases of suspect immunoassay interferences. We describe several procedures taken in a case of a polymorbid patient with chest pain, without clear evidence of myocardial necrosis and with increased high-sensitivity cardiac troponin T (hs-cTnT). We checked the Vafaie's algorithm for the evaluation of suspect interference in troponin measurements. METHODS: We conducted a case report analysis, additional measurements, a dilution test and pretreatment of plasma with blocking agents. RESULTS: Concentration of hs-cTnT (99 th percentile of "healthy" population 14 ng/L) increased from 120.1 ng/L to 280.4 ng/L during an 8-month period and decreased to 216.3 ng/L during the following month with repeatedly negative troponin I (TnI), hs-cTnI, myoglobin and creatine kinase MB (CK-MB). Suspected false positivity of hs-cTnT was further confirmed by treatment of plasma with an antiheterophile blocking agent (hs-cTnT before treatment 280.4 ng/L, after 16.53/16.23 ng/L). This outcome was further confirmed by the manufacturer's experiments. CONCLUSIONS: The false-positive results of hs-cTnT were caused by the presence of extremely rare high molecular weight protein, presumably IgM, most likely HAMA (human anti-mouse antibody). Only the pre-treatment of plasma with a blocking agent provided a reliable indication of the interference. Cooperation among clinicians, laboratory personnel and the manufacturer is essential.

Long-term biological variability of galectin-3 after heart transplantation.

BACKGROUND: Galectin-3 is an emerging biomarker of heart failure and of myocardial fibrosis risk. Monitoring of galectin-3 is essential during treatment with galectin-3 inhibitors. The aim of our study was to assess long-term biological variability in a specific group of unhealthy subjects. METHODS: The biological variability of galectin-3 was measured in a group of 44 patients after heart transplantation (HTx). Six samples were taken from each patient during a 12-month period. Galectin-3 was measured with an Abbott Architect automated immunoassay. RESULTS: Intraindividual (CVi) and interindividual (CVg) variabilities were calculated together with the reference change value (RCV), the log-normal RCV for increase (RCV+), and the log-normal RCV for decrease (RCV-). The CVi, CVg, RCV, RCV+, and RCV- were 28.2%, 35.6%, 78.6%, 116%, and -53.7%, respectively. The index of individuality was 0.79. CONCLUSIONS: The concentrations of galectin-3 in patients followed 12 months after HTx fluctuated around the homeostatic point, with CVi of approximately 28%. RCVs of +116% (log-normal increase) and -54% (log-normal decrease) mean that the concentration of galectin-3 would need to approximately double or decrease by half to indicate a new process.