Analytical Quality by Design-based development and validation of ultra pressure liquid chromatography/MS/MS method for glycopeptide antibiotics determination in human plasma.

AIM: An ultra pressure liquid chromatography (UPLC)/MS/MS method for vancomycin and teicoplanin determination in human plasma was developed in accordance with analytical quality by design (AQbD) concept and fully validated. MATERIALS & METHODS: Chromatographic separation was performed on ACQUITY UPLC C18 charge surface hybrid (CSH) column (2.1 mm × 50 mm, 1.7 µm particle size) in gradient mode and the mobile phase consisted of 0.1% formic acid in water and pure acetonitrile. The experimental design methodology was used for the definition of optimal chromatographic and protein precipitation conditions. RESULTS: The linearity ranges were 0.05-10 µg ml-1 for vancomycin and 0.5-200 µg ml-1 for total teicoplanin. The relative standard deviations for precision estimation were below 15% and the accuracy was within 85-115% for all quality control levels. CONCLUSION: The method was utilized for glycopeptide antibiotics bioanalysis.

Living unrelated donor kidney transplantation--a fourteen-year experience.

BACKGROUND: In countries without a national organization for retrieval and distribution of organs of the deceased donors, problem of organ shortage is still not resolved. In order to increase the number of kidney transplantations we started with the program of living unrelated - spousal donors. The aim of this study was to compare treatment outcome and renal graft function in patients receiving the graft from spousal and those receiving ghe graft from living related donors. METHOD: We retrospectively identified 14 patients who received renal allograft from spousal donors between 1996 and 2009 (group I). The control group consisted of 14 patients who got graft from related donor retrieved from the database and matched than with respect to sex, age, kidney disease, immunological and viral pretransplant status, the initial method of the end stage renal disease treatment and ABO compatibility. In the follow-up period of 41 +/- 38 months we recorded immunosuppressive therapy, surgical complications, episodes of acute rejection, CMV infection and graft function, assessed by serum creatinine levels at the beginning and in the end of the follow-up period. All patients had pretransplant negative cross-match. In ABO incompatible patients pretransplant isoagglutinine titer was zero. RESULTS: The patients with a spousal donor had worse HLA matching. There were no significant differences between the groups in surgical, infective, immunological complications and graft function. Two patients from the group I returned to hemodialysis after 82 and 22 months due to serious comorbidities. CONCLUSION: In spite of the worse HLA matching, graft survival and function of renal grafts from spousal donors were as good as those retrieved from related donors.

Systemic and intraperitoneal proinflammatory cytokine profiles in patients on continuous ambulatory peritoneal dialysis.

Our cross-sectional study included 44 patients (27 men, 17 women; mean age: 57.12 +/- 16.66 years; mean dialysis treatment period: 3.59 +/- 2.67 years) on continuous ambulatory peritoneal dialysis (CAPD). Of the 44 patients, 21 were using standard solutions (Stay*Safe, ANDY-disc: Fresenius Medical Care, Bad Homburg, Germany), and 23 were using biocompatible solutions (Gambrosol Trio: Gambro Lundia AB, Lund, Sweden; Stay*Safe Balance: Fresenius Medical Care). In all CAPD patients dialyzed longer than 6 months, we analyzed levels of interleukin 1beta (IL-1beta), tumor necrosis factor alpha (TNFalpha), and interleukin 6 (IL-6) in serum and dialysis effluent when patients were free of acute infection-related (CAPD peritonitis, exit-site infection, other acute infections) complications. In a control group of 20 patients with chronic renal failure [CRF (stages IV and V)], we also determined serum levels of the same cytokines. Levels of the inflammatory cytokines were measured using specific commercial ELISA kits (BioSource, Camarillo, CA, U.S.A.). Statistical analysis of the results was performed using commercial statistics software for the PC (Statistica for Windows, rev. 4.5: StatSoft, Tulsa, OK, U.S.A.). Serum levels of LL-1beta and IL-6 were not statistically significantly different between the patients on CAPD, regardless of the type of dialysis the used, and between the patients and the control group with CRF. Serum levels of TNFalpha, unlike those for IL-1beta and IL-6, were statistically significantly higher in patients on CAPD than in the control group with CRF (13.20 +/- 3.23 pg/mL vs. 5.59 +/- 4.54 pg/mL, p < 0.001, Mann-Whitney test). Serum and effluent IL-1beta levels in patients on CAPD for less than 1 year and more than 1 year did not significantly differ, but effluent IL-6 levels were significantly higher than serum IL-6 levels in both groups of patients, and effluent IL-6 levels were significantly higher in CAPD patients dialyzed for more than 1 year than in patients dialyzed for less than 1 year. Serum and intraperitoneal (IP) levels of the examined cytokines did not significantly differ in patients on standard and biocompatible solutions, but a trend toward lower IP levels of IL-6 was seen in patients on biocompatible solutions. Residual renal function and number of episodes of CAPD peritonitis had no important effect on serum and IP levels of the examined cytokines. Elevated serum levels of TNFalpha and significant local IL-6 production in our CAPD patients indirectly confirmed the importance of peritoneal dialysis (PD) in amplifying the chronic inflammation that substantially depends on duration of dialysis treatment.

[Correlation between dialysis solution type and cardiovascular morbidity rate in patients undergoing continuous ambulatory peritoneal dialysis].

BACKGROUND/AIM: Peritoneal dialysis (PD) patients have an increased risk for cardiovascular diseases. The aim of the study was to evaluate the cardiovascular changes in patients undergoing chronic PD and the eventual existing differences depending on biocompatibility of dialysis solutions. METHODS: After 3 +/- 2 years of starting PD, 21 PD patients on the treatment with bioincompatible dialysis solutions (conventional glucose- based solutions: PDP-1), average age 47.43 +/- 12.87 years, and 21 PD patients on the treatment with biocompatible dialysis solutions (neutral solutions with lower level of glucose degradation products, lower concentration of Ca2+ and neutral pH: PDP-2), average age 68.62 +/- 13.98 years, participated in the longitudinal study. The average number of episodes of peritonitis was similar in both groups: 1 episode per 36 months of the treatment. The control group included 21 patients with preterminal phase of chronic renal failure (Glomerular Filtration Rate: 22.19 +/- 10.73 ml/min), average age 65.29 +/- 13.74 years. All the patients underwent transthoracal echocardiography (in order to detect: eject fraction (EF), left ventricular hypertrophy (LVH), and valvular calcification (VC) and B-mode ultrasonography of common carotid artery (CCA): IMT, lumen narrowing, and plaque detection. RESULTS: The values of EF were: in PDP-1 group 62.05 +/- 5.65%, in PDP-2 group 53.43 +/- 7.47%, and in the control group 56.71 +/- 8.12% (Bonferroni test, p = 0.001). The recorded LVH was: in PDP-1 group in 47.6% of the patients; in PDP-2 group in 61.9% of the patients; and in control the group in 52.4% (chi2 test; p = 0.639). The detected VC was: in PDP-1 in 52.4% of the patients, in PDP-2 group in 42.9% of the patients, and in the control group in 23.8% of the patients (chi2 test; p = 0.776). The IMT was: in PDP-1 group 1.26 +/- 0.54 mm, in PDP-2 group 1.23 +/- 0.32, and in the control group 1.25 +/- 0.27 mm (Bonferroni test; p = 0.981). An average lumen narrowing was: in PDP-1 group 13.78 +/- 18.26%, in PDP-2 group 18.57 +/- 22.98%, and in the control group 25.00 +/- 28.02% (Kruskal Wallis test; p = 0.413). Calcified plaques of CCA were detected in PDP-1 group in 61.9% of the patients, in PDP-2 group in 85.7%, of the patients and p = 0.159). CONCLUSION: Generally, PD had a significant influence on cardiovascular morbidity in the treated patients, especially on the left ventricular function and peripheral atherosclerosis. The age of the patients had more influence on acceleration of atherosclerosis than the length of dialysis or biocompatibility of dialysis solutions.

Systemic and intraperitoneal proinflammatory cytokines profiles in patients on chronic peritoneal dialysis.

Cytokines are essential mediators of immune response and inflammatory reactions. Patients with chronic renal failure and on Continuous Ambulatory Peritoneal Dialysis commonly present abnormalities of immune function related to impaired kidney function, accumulation of uremic toxins and bioincompatibility of peritoneal dialysis solutions. Aim of this study was to examine effects of the CAPD solutions (standard v.s. biocompatible), as well as dialysis duration upon the local and systemic profile of the pro-inflammatory cytokines (IL-1, TNF and IL-6) in patients on CAPD. The cross-sectional study included 44 CAPD patients (27 M and 17 F, average mean age 57.12+/-16.66), of whom 21 patients were on the standard solutions (A.N.D.Y.Disc) for peritoneal dialysis and 23 on the biocompatible solutions (Gambrosol bio trio, Stay Safe balance). The average dialysis treatment period was 3.59+/-2.67 years. In all CAPD patients dialysed longer than 6 months, levels of IL-1. TNF and IL-6 in the serum and dialysis effluent were analysed in the phase without acute infection-related complications (CAPD peritonitis, infection of the catheter exit-site, other acute infections). The control group included 20 patients with the CRF (stage IV and V) whose serum levels of the examined cytokines were also determined. Levels of the inflammatory cytokines were measured by commercial specific ELISA kits (BioSource, Camarillo, California, USA). Statistical analysis of the obtained results was performed by commercial statistics PC software (Stat for Windows, R.4.5. SAD). The serum IL-1 and IL-6 levels were not statistically significantly different in patients on CAPD, irrespective of the type of the used dialysis solutions and in the control group of patients with CRF. The serum TNF levels, unlike IL-1 and IL-6, were statistically significantly higher in patients on CAPD in comparison with the control group of patients (13.203.23 v.s. 5.594.54, p< 0.001, Mann Whitney test). The serum and effluent IL-1 levels in patients on CAPD within one and longer than one year of dialysation did not significantly differ, but the effluent IL-6 levels were significantly higher than in the serum of both groups of patients, that is, effluent IL-6 levels in CAPD patients dialysed more than one year was significantly higher in comparison with those in patients dialysed within a year. Both serum and intraperitoneal levels of the examined cytokines did not significantly differ in patients on the standard and biocompatible solutions, regardless of the present trend toward decrease of intraperitoneal IL-6 levels in patients on biocompatible solutions. Residual renal funcion and number of CAPD peritonitis did not have any important impact upon the serum and IP levels of the examined ctokynes. Elevated serum TNF levels and significant local IL-6 production in our CAPD patients indirectly confirm importance of peritoneal dialysis in amplification of the chronic inflammation substantially depend on the duration of dialysis treatment.

[Ten year experience in kidney transplantation in patients undergoing peritoneal dialysis (1996-2006)].

INTRODUCTION: Kidney transplantation is nowadays considered the most sophisticated method of treatment of chronic terminal renal insufficiency. The advancement in surgical technique and the use of new immunosuppressive medications provide a longer survival period of both the patient and the graft. MATERIAL AND METHODS: The objective of this paper is a retrospective presentation of the ten-year monitoring of kidney transplantation in patients undergoing peritoneal dialysis performed at the Military Medical Academy. RESULTS AND DISCUSSION: During that period, 32 patients underwent the transplantation (18 men and 14 women of the average age of 32.48+/-2.1). The total of 34 transplantations were performed, out of which 2 were retransplantations. Patient monitoring period was 7-92 months. The immunosuppressive therapy was quadrupled in 17 (50%) patients and tripled in 17 (50%) of them. The loss of the graft in the early period following the transplantation occurred in 5 (15.6%) patients due to trombosis a. renalis, out of whom two were retransplantation cases. Registered surgical vascular complications included 5 (15.6%) bleeding, 4 (12.5%) hematomes and 6 (18.7%) lymfocells. Delayed graft function occurred in 5 (14.7%) and acute rejection in 7 (20.5%) patients while the disease reccured in 2 patients. As for the complications, rejection without the loss of graft was registered in 4 (12.5%) pts, ureter stenosis in 3 (8.82%) pts, bacterial infections in 4 (12.5%) pts and reactivation of CMV infections in 9 (26.4%) pts. Our patients are observed for the maintenance of stable medium volume of serum creatinine (which is 123.6 +/-10.2 umol/l in the early stage and 133.24+/-11.1 umol/l in the end of the monitoring period as well as the medium volume of clearence creatinine (which is 64.80+/-3.5 ml/min at the early phase of monitoring period and 69.47+/-3.3 ml/min. in the end). CONCLUSION: Our group of renal transplantation patients, undergoing peritoneal dialysis was with stable renal function registered through the monitoring period.

Lupus nephritis: histopathologic features, classification and histologic scoring in renal biopsy.

Lupus nephritis (LN) is one of the most significant manifestations in systemic lupus erythematosus (SLE), although in some cases there is no direct correlation among clinical, serologic and histologic findings. Therefore, renal biopsy and histopathologic classification by the activity and chronicity assessment of LN are considered necessary before the initiation of the treatment. In this paper 311 renal biopsies in patients with LN were analyzed and classified according to the WHO Classification published in 1974. Renal biopsy specimens were routinely processed for standard analysis by light microscopy (LM), immunofluorescent (IF), and electron microscopy (EM). Biopsy findings were compared with the common clinical symptoms. It is important to recognize that histopathologic lesions in the analyzed tissue might precede the symptoms and clinical manifestations, and therefore are an important factor in the appropriate therapeutic approach. Histopathological assessment of the class of LN represents an important predictor in most patients because the lesions of LN class I and class II most often have no progression and consequently do not require an aggressive treatment. Diffuse proliferative glomerulonephritis (class IV) as a severe form of renal lesion requires the application of high doses of corticosteroids and cytotoxic medicaments. The established quantitive indexes have significant predictive value. Activity index (AI) evaluates the presence of fresh inflammatory and potentially reversible lesions and chronicity index (CI) evaluate the presence of irreversible glomerular lesions such as sclerosis, tubular atrophy, interstitial fibrosis, etc. High AI and low CI, particularly in the class IV of LN, suggest the necessity of an aggressive treatment of the inflammatory process aiming to preserve the renal function. High CI and low AI characterize irreversible sclerosing lesions, where an aggressive treatment is considered unnecessary. Our experience suggests the necessity of introducing the new morphologic elements into the activity and chronicity scoring system which would correlate better the clinical symptoms. This should provide higher reliability of scoring, since it evaluates important elements in clinical-morphologic diagnosis of LN.