M. Blaha

Ultra high performance liquid chromatography tandem mass spectrometric detection in clinical analysis of simvastatin and atorvastatin.

Simvastatin and atorvastatin belong to the group of hypolipidemic drugs, more exactly to the second generation of inhibitors of microsomal 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. They induce a significant reduction in total cholesterol, low-density lipoprotein cholesterol and plasma triglycerides, therefore they are widely used in the treatment of hypercholesterolemia even of its severe form-familiar hypercholesterolemia. Simvastatin and atorvastatin as the most widely used statins in clinical treatment and their hydroxy-acid/lactone forms were determined by means of UPLC in connection with triple quadrupole mass spectrometer. Deuterium labeled reference standard compounds were used as internal standards for the quantitation. Separation was performed on Acquity BEH C18 (100 mm x 2.1 mm, 1.7 microm) using gradient elution by mobile phase containing acetonitrile and ammonium acetate pH 4.0, which is convenient in order to prevent interconversion of analytes. ESI in positive mode was used for the ionization of all compounds. Two SRM (selected reaction monitoring) transitions were carefully optimized for each analyte in order to get high sensitivity and selectivity. SPE on Discovery DSC-18 was used as a sample preparation step. Intra-day precision was generally within 10% RSD, while inter-day precision within 15% RSD. Method accuracy expressed as recovery ranged from 75 to 100%. The method was validated with the sensitivity reaching LOQ 0.08-5.46 nmol/l and LOD 0.01-1.80 nmol/l in biological samples. Atorvastatin, simvastatin, its metabolites and hydroxy-acid/lactone forms were monitored in human serum and in lipoprotein fractions (LDL, HDL and VLDL) at patients with end stage renal diseases.

World apheresis registry 2003-2007 data

OBJECTIVES Seventy-five centers from many countries have applied for a login code to the WAA apheresis registry. Fifteen centers from 7 countries have been actively entering data at the internet site from 2003 until 2007. We report on data from the registry so far. METHODS This is a web-based registry. A link is available from the WAA homepage (www.worldapheresis.org). So far data from 2013 patients (12,448 procedures) have been included. A median of 6 treatments have been performed (range 1-140). Mean age 51 years (range 1-94 years; 45% women). Seven percent of the patients were < or = 21 years and 4% were < or = 16 years. RESULTS The purpose of the apheresis procedure was therapeutic in 67% and retrieval of blood components in 33%. Main indications: neurological and hematological diseases, lipid apheresis and stemcell collection (autologous, and some allogeneic). Blood access: peripheral vessels (71%), central dialysis catheter through jugular (6.5%) or subclavian veins (6.7%), femoral vein (8%) and AV fistula (4%). ACD was used for anticoagulation in 73% of the procedures. Albumin was mainly used as replacement fluid. Adverse events (AE) were registered in 5.7% of the procedures. AE was graded as mild (2.5%), moderate (2.7%) or severe (0.5%). No death occurred due to treatment. The procedures were interrupted in 2.6%. Most frequent AEs were blood access problems (29%), tingling around the mouth (20%), hypotension (18%), and urticaria (9%). There were significant differences between the centers regarding mild and moderate AEs. Data indicate that centers using continuous infusion of calcium had fewer AEs. CONCLUSION There was a limited number of severe AEs. Centers use various standard procedures for apheresis. By learning from the experience of others the treatment quality will improve further. In the near future, an update of the registry will enable more extensive evaluation of the data.

Microextraction by packed sorbent as sample preparation step for atorvastatin and its metabolites in biological samples—Critical evaluation

Atorvastatin belongs to the group of lipid-lowering drugs known as statins. They significantly reduce the levels of total cholesterol, low-density cholesterol and plasma triglycerides therefore they are widely used in the treatment of hypercholesterolemia. Recently developed methods for the determination of atorvastatin and its metabolites in plasma used SPE (solid phase extraction) or LLE (liquid-liquid extraction) as the sample preparation step. However, both procedures are quite time-consuming and need relatively high volume of solvent/sample, which is impractical for the routine analyses of many biological samples. The aim of this work was to develop and validate more suitable sample preparation method for the determination of atorvastatin and its metabolites in biological samples using MEPS (microextraction by packed sorbent). The optimal conditions of MEPS extraction were using C8 sorbent and only 50 μl of the sample. The analytes were eluted by 100 μl of the mixture of acetonitrile:0.1 M ammonium acetate pH 4.5 (95:5, v:v). The analytical method was validated and demonstrated good linearity (r(2)>0.9990), recovery (89-115%) and intra-day precision (RSD<10%). Total time of the sample preparation was three times shorter (7 min) compared to SPE. The volume of sample was twenty times lower and the volume of solvents about ten times lower compared to SPE. Combination of fast MEPS method together with quick UHPLC-MS/MS was used for the determination of atorvastatin and its two metabolites in serum obtained from patients with familiar hypercholesterolemia.

World apheresis registry data from 2003 to 2007, the pediatric and adolescent side of the registry

OBJECTIVES Paediatric patients are a special group in apheresis. It is general accepted to use adult indications in paediatric patients, but data in this age group are rare. In order to provide more information of apheresis practise in children and young adults (<21a) we will report of knowledge learnt by data from the registry from 2003 until 2007. METHODS This is a web-based registry. A link is available from the WAA homepage (www.worldapheresis.org). So far data from 12,448 procedures have been included. Six hundred and twelve procedures were performed in 135 children and young adults (308 procedures<16a, 237 from 17 to 20a, and 67 with 21a) representing 5% of the total population. The median age was 14 years (range 1-21 years), 74 male and 61 female. These data were entered by 15 centres with a frequency of in median 18 aphereses in young patients per centre (range 1-287) from 2003 to 2007. RESULTS Main indications: haematological diseases and also nephrological, and neurological. The type of aphereses was mainly Leukapheresis (196, 33%), plasma exchange (149, 25%), photopheresis (127, 21%), and lipid aphereses (79, 13%). Blood access: peripheral vessels in 305 procedures (50%, compared to 73% in adults), central venous catheter in 239 (38%), and AV-fistula in 2% and 0.3%, and in 8 (1.31%) procedures an arterial line was used. Anticoagulation was mostly by ACD (71%), heparin (18% or the combination of both (3%). 39 adverse events (AE) were registered in 22 (=3.59%) of the procedures, mostly graded as mild. Treatment was interrupted in 14 procedures (2.29%). AEs were abdominal pain, anaphylactic shock, flush, hyper- and hypotension, nausea, vertigo, cephalea and need for sedation and technical problems with the device and problems with the venous access. The rate of AEs was similar for stem cell harvesting and for plasma exchange (4% and 4.7%, respectively). CONCLUSION The paediatric data compared to the whole registry data set are showing that aphereses are performed as safe in paediatrics as in adults. Centres are mostly handling only a few cases younger than 21. Therefore more exchange of information and experience in paediatric apheresis is warranted.

Beneficial effect of plasma exchange in the treatment of toxic epidermal necrolysis: A series of four cases

Introduction: Toxic epidermal necrolysis (TEN) is a rare, life‐threatening disease with a high mortality rate that is linked to drug toxicity. There is a lack of data about the underlying pathophysiologic mechanisms and treatment options. The only widely accepted treatment of TEN is withdrawal of the offending drug followed by supportive care. The potential roles of corticosteroids, intravenous immunoglobulin (IVIG) and plasmapheresis (TPE) remain controversial. Aims: We present four patients with severe TEN (all with >80% involvement of body surface) who were treated with TPE following unsuccessful treatment with corticosteroids/IVIG. Methods: TPE was performed using a COBE Spectra blood cell separator. ACD‐A was used as anticoagulant fluid and the target‐washed plasma volume was one body volume. Plasma was replaced by a 5% solution of human albumin + Ringers lactate. Results: The mean number of TPE sessions was 5.25 ± 2.22 (range 3–8). Drugs were implicated as an etiologic agent in each case. TPE led to prompt improvement of acute condition and general health as well as halting of disease progression. Additionally, the restoration of the epithelium began in all four patients. Conclusion: Plasmapheresis should be considered as an alternative treatment modality for patients with the most severe form of TEN if initial treatment with other agents, including corticosteroids and/or IVIG, fails. Drugs were suspected to be the cause of TEN in all four cases. J. Clin. Apheresis, 2012.

Safety and Tolerability of Long Lasting LDL-apheresis in Familial Hyperlipoproteinemia

Abstract:  The aim of this work is to arbitrate the incidence of side effects and tolerability of long lasting LDL‐apheresis in familial hyperlipoproteinemia. 1200 procedures were performed and the last 463 of them were evaluated. An immunoadsorption method of LDL‐apheresis was used (continuous blood cell separator Cobe Spectra; secondary device: automated adsorption‐desorption ADA, Medicap; absorption columns: Lipopak). As a whole, 6.26% adverse events were found and subsequently resolved by standard symptomatic therapy. Vaso‐vagal reactions (symptoms of neurovegetative lability) were the most common adverse effects, presented as malaise, weakness, slight and short‐term drop in blood pressure or other general signs. They were all well controlled by symptomatic therapy. We conclude that LDL‐apheresis in the hands of experienced personnel is a safe procedure. An acceptable procedure duration limit, balancing the possibility to achieve a targeted cholesterol level while still maintaining an acceptable patient tolerance, was confirmed to be 4 hours.

Prognostic impact of hemoglobin level prior to radiotherapy on survival in patients with glioblastoma.

Purpose:To evaluate prognostic factors in patients with glioblastoma treated with postoperative or primary radiotherapy.Patients and Methods:From 1989 to 2000, a total of 100 patients underwent irradiation as part of their initial treatment for glioblastoma. All patients had undergone surgery or biopsy followed by conventional external-beam radiotherapy. 85 patients who received the planned dose of irradiation (60 Gy in 30 fractions) were analyzed for the influence of prognostic factors. 73/85 (86%) of patients were given postoperative irradiation, while 12/85 (14%) of patients were primarily treated with radiotherapy after biopsy.Results:The median overall survival was 10.1 months (range, 3.7–49.8 months), the 1- and 2-year survival rates were 41% and 5%, respectively. Univariate analysis revealed age ≤ 55 years (p < 0.001), pre-radiotherapy hemoglobin (Hb) level > 12 g/dl (p = 0.009), and pre-radiotherapy dose of dexamethasone ≤ 2 mg/day (p = 0.005) to be associated with prolonged survival. At multivariate analysis, younger age (p < 0.001), higher Hb level (p = 0.002), lower dose of dexamethasone (p = 0.026), and a hemispheric tumor location (p = 0.019) were identified as independent prognostic factors for longer survival. The median survival for patients with an Hb level > 12 g/dl was 12.1 months compared to 7.9 months for those with a lower Hb level. Contingency-table statistics showed no significant differences for the two Hb groups in the distribution of other prognostic factors.Conclusion:The results indicate that lower Hb level prior to radiotherapy for glioblastoma can adversely influence prognosis. This finding deserves further evaluation.Ziel:Evaluation prognostischer Faktoren bei Patienten mit Glioblastom, die mit postoperativer oder primärer Strahlentherapie behandelt wurden.Patienten und Methodik:Bei 100 Patienten mit Glioblastom wurde in den Jahren 1989–2000 die Strahlentherapie im Rahmen der Primärbehandlung angewandt. Bei allen Patienten wurde eine Operation oder Biopsie mit nachfolgender konventioneller perkutaner Bestrahlung durchgeführt. Der Einfluss der prognostischen Faktoren wurde bei 85 Patienten, die die geplante Strahlendosis (60 Gy in 30 Fraktionen) erhielten, evaluiert. 73/85 Patienten (86%) wurden mit postoperativer Bestrahlung, 12/85 Patienten (14%) mit primärer Strahlentherapie und Biopsie behandelt.Ergebnisse:Die mittlere Überlebenszeit betrug 10,1 (3,7–49,8) Monate, die Überlebenszeit nach 1 und 2 Jahren lag bei 41% bzw. 5%. Mittels der univariaten Analyse stellten sich folgende Faktoren dar, die mit einer längeren Überlebenszeit verbunden sind: ein Alter ≤ 55 Jahre (p < 0,001), eine Hämoglobin-(Hb-)Konzentration zu Beginn der Strahlentherapie > 12 g/dl (p = 0,009) und eine prätherapeutische Dexamethasondosis ≤ 2 mg/Tag (p = 0,005). Die multivariate Analyse ermittelte ein jüngeres Alter (p < 0,001), eine höhere Hb-Konzentration (p = 0,002), eine niedrigere Dexamethasondosis (p = 0,026) und eine hemisphärische Tumorlokalisation (p = 0.019) als unabhängige prognostische Faktoren für eine längere Überlebenszeit. Die mittlere Überlebenszeit bei Patienten mit einer Hb-Konzentration > 12 g/dl betrug 12,1 Monate, bei Patienten mit einem niedrigeren Blut-Hb-Wert dagegen nur 7,9 Monate. Die Kontingenztabellenstatistik zeigte keine signifikanten Unterschiede in der Distribution der anderen prognostischen Faktoren bei beiden Hb-Gruppen.Schlussfolgerung:Die Ergebnisse weisen darauf hin, dass eine niedrigere Hb-Konzentration vor Beginn der Strahlentherapie wegen Glioblastoms einen negativen Einfluss auf die Prognose haben kann. Diese Beobachtung verdient weitere Aufmerksamkeit.

Haemorheopheresis could block the progression of the dry form of age‐related macular degeneration with soft drusen to the neovascular form

Purpose:  To evaluate the influence of haemorheopheresis on anatomical and functional findings in patients with soft‐drusen maculopathy.

Urinary Neopterin and Microalbuminuria in Patients Treated by Low-density Lipoprotein Apheresis

Abstract Low-density lipoprotein (LDL)-apheresis is a method of extracorporeal elimination of LDL-cholesterol in patients with severe primary lipoprotein disorders. LDL-cholesterol activates macrophages, which play an important role in atheromatous plaque formation. In the present study, we have investigated urinary neopterin, a specific marker of macrophage activation and microalbuminuria, an indicator of generalized vascular dysfunction, after a single LDL-apheresis procedure in 10 patients with severe primary lipoprotein disorder. The urinary neopterin/creatinine ratio was increased in patients compared to controls. No significant changes of the neopterin/creatinine and albumin/creatinine ratios were observed after LDL-apheresis, except a significant (p < 0.006) decrease of urinary neopterin/creatinine ratio in the evening after the apheresis. This decrease showed significant negative correlation with the pre-apheretic levels of atherogenic cholesterol fractions (p < 0.05) and with cholesterol decrease during the apheresis (p < 0.05). Urinary albumin/creatinine ratio correlated positively with total and LDL-cholesterol levels before the apheresis and with the evening urinary neopterin/creatinine ratio after the apheresis, but did not correlate with glycemia and triacylglycerides. Elevated urinary neopterin in the patients with severe primary lipoprotein disorders reflects the presence of atherosclerosis. A single LDL-apheresis procedure did not significantly affect microalbuminuria. The decrease of urinary neopterin in the evening after the apheresis corresponds with the diurnal rhythm of neopterin excretion and was less pronounced in patients with more severe hypercholesterolemia. The correlations between microalbuminuria, neopterin and pre-apheretic cholesterol concentrations indicate a possible connection between microvascular dysfunction, macrophage activity and severity of hyperlipidemia, but these results should be interpreted with caution because of small number of subjects evaluated.

Plasma filtration in the treatment of graves' ophthalmopathy: A randomized study

The aim of this study was to perform a randomized study to evaluate the role of plasma filtration in the treatment of severe thyroid‐associated ophthalmopathy (TAO). 20 patients were enrolled, and all patients were treated with methylprednisolone IV pulses. 10 randomly chosen patients were also subjected to plasma filtration (twice weekly in Weeks 1, 2, 4, 7, and 10). The procedure proved to be safe. All immunoglobulin classes as well as autoantibodies directed against thyroglobulin, thyroid peroxidase, and TSH receptor exhibited statistically significantly decreases. Some markers of cell‐mediated immunity such as soluble antigen CD30 and monocyte chemotactic protein 1 decreased, but serum levels of other markers such as CD40 ligand and soluble protein Fas/Apo‐1 did not change significantly. The decrease of immunoglobulins was long lasting, whereas cytokine levels returned to basal values before the next apheresis. Although the clinical activity score (CAS) dropped in all patients, it occurred more rapidly in patients treated with plasma filtration. The CAS difference between the two groups was statistically significant (p = 0.027). The amplitude of visual evoked potentials improved after 3 months in the plasma filtration group. At the end of the study, there was no difference between patients treated with aphereses and the control group. Eye muscle width and proptosis measured by CT scan did not differ between the two groups. We conclude that apheresis can decrease disease activity more rapidly than standard high‐dose IV glucocorticoid therapy. Whether this superior treatment effect could potentially avoid surgical procedures remains to be determined. J. Clin. Apheresis 25:209–215, 2010.