Comparison and Harmonization of Different Semi-Automated and Automated qRT-PCR Assays in the Assessment of SARS-CoV-2.

In SARS-CoV-2 diagnostics, cycle threshold (Ct) values from qRT-PCRs semi-quantitatively estimate a patient's viral load. However, relevant analytical differences between qRT-PCR assays are often neglected. This study was designed (i) to identify such differences between five commonly used assays and (ii) to demonstrate a straightforward strategy to harmonize them. QRT-PCRs for SARS-CoV-2 were carried out in 85 oropharyngeal swab samples using three fully automated (Alinity m, cobas®6800 and GeneXpert) and two semi-automated (genesig® and RIDA®GENE) assays. Qualitative results (positive/negative) showed excellent comparability between the fully automated assays, but not between the Alinity m and semi-automated methods. Ct values significantly varied between all the methods, with the median values ranging from 22.76 (Alinity m) to 30.89 (RIDA®GENE) and 31.50 (genesig®), indicating the lowest sensitivity for semi-automated methods. Passing-Bablok analysis further revealed systemic biases. Assay-specific viral load concentration calculations-based on generated individual standard curves-resulted in much better comparability between the assays. Applying these calculations, significant differences were no longer detectable. This study highlights relevant analytical differences between SARS-CoV-2 qRT-PCR assays, leading to divergent decisions about the mandatory isolation of infected individuals. Secondly, we propose a strategy to harmonize qRT-PCR assays to achieve better comparability. Our findings are of particular interest for laboratories utilizing different assays.

Macro-CK type 2 in metastatic prostate cancer.

The isoenzyme creatine kinase muscle/brain (CK-MB) still plays an important role for the differential diagnosis of CK elevations and the clarification of their origin from heart or skeletal muscle. Therefore, it is necessary to know the diagnostic pitfalls in interpreting CK-MB results. We demonstrate a case of macro-CK type 2 in a 75-year-old patient with metastatic castration-resistant prostate cancer and its identification by isoenzyme electrophoresis, which can be typical for cancer diseases.

Validation of a high-performance liquid chromatography method for thiopurine S-methyltransferase activity in whole blood using 6-mercaptopurine as substrate.

BACKGROUND: Variation in metabolism, toxicity and therapeutic efficacy of thiopurine drugs is largely influenced by genetic polymorphisms in the thiopurine S-methyltransferase (TPMT) gene. Determination of TPMT activity is routinely performed in patients to adjust drug therapy. METHODS: We further optimized a previously established high-performance liquid chromatography (HPLC) method by measuring TPMT activity in whole blood instead of isolated erythrocytes, which is based on conversion of 6-mercaptopurine to 6-methylmercaptopurine using S-adenosyl-methionine as methyl donor. RESULTS: The simplified TPMT whole-blood method showed similar or better analytical and diagnostic performance compared with the former erythrocyte assay. The whole-blood method was linear for TPMT activities between 0 and 40 nmol/(mL·h) with a quantification limit of 0.1 nmol/(mL·h). Within-day imprecision and between-day imprecision were ≤5.1% and ≤8.5%, respectively. The optimized method determining TPMT activity in whole blood (y) showed agreement with the former method determining TPMT activity in erythrocytes (x) (n=45, y=1.218+0.882x; p>0.05). Phenotype-genotype concordance (n=300) of the whole-blood method was better when TPMT activity was expressed per volume of whole blood (specificity 92.2%), whereas correction for hematocrit resulted in lower genotype concordance (specificity 86.9%). A new cutoff for the whole-blood method to distinguish normal from reduced TPMT activity was determined at ≤6.7 nmol/(mL·h). CONCLUSIONS: This optimized TPMT phenotyping assay from whole blood using 6-MP as substrate is suitable for research and routine clinical analysis.

Single-Nucleotide Polymorphism of CYP3A5 Impacts the Exposure to Tacrolimus in Pediatric Renal Transplant Recipients: A Pharmacogenetic Substudy of the TWIST Trial.

BACKGROUND: The pharmacokinetics of tacrolimus (TAC) and mycophenolic acid (MPA) are highly variable. An impact of single-nucleotide polymorphisms (SNPs) of the genes coding for enzymes and transporters involved in the pharmacokinetics of TAC and/or MPA is intuitively conceivable. Accordingly, we sought to analyze the influence of different SNPs on TAC and MPA exposure in pediatric renal transplant recipients. METHODS: A subpopulation of 37 patients (median age: 12.8 years, range 2.2-18.3 years) participating in the TWIST study was included in the analysis of SNPs of CYP3A5, ABCB1 (MDR1), ABCG2, SLCO1B3 (coding for OATP2), ABCC2 (coding for cMOAT), and UGT1/2. TAC trough concentrations and abbreviated area under the concentration-time curves (AUC) of MPA were measured on days 7, 28, 91, and 183 after transplant. Both of these were adjusted to the respective dose the patient received. RESULTS: The allele frequencies of analyzed SNP's were comparable to those reported previously for white populations. Dose-adjusted trough concentrations of TAC were approximately 60% lower in patients with the CYP3A5*1/*3 allele as compared with the CYP3A5*3/*3 allele (P = 0.004). Steroid-free patients in CYP3A5*3/*3 and CYP3A5*1/*3 carrier subgroups had comparable dose-adjusted TAC concentrations to the subgroup on steroids (P = 0.13). Patients younger than 10 years had a significantly lower median dose-adjusted TAC C0 concentration than patients older than 10 years; this age effect was comparable in heterozygous and homozygous CYP3A5 carriers as well as in patients on and off steroid medication. As for MPA, the genetic variability of transporters or enzymes had no impact on dose-adjusted MPA-AUC due to the low allele frequencies. Patients off steroids had a higher dose-adjusted MPA-AUC (0.18 mg·h/L per mg/m, 0.012-0.27) compared with patients on steroids (0.12 mg·h·L·mg, 0.09-0.19; P = 0.04). CONCLUSIONS: Genetic variability of CYP3A5 has an impact on TAC metabolism in pediatric renal transplant recipients, contributing partly to the variability of TAC exposure. Therefore, adjusting initial TAC dosing to the genotype of CYP3A5 might be of clinical benefit.

Analytical evaluation of a real-time PCR-based DNA demethylation assay to assess the frequency of naturally occurring regulatory T cells in peripheral blood.

OBJECTIVES: Regulatory T cells (Tregs) which may indicate operational tolerance provide a promising biomarker for individualization of immunosuppression. Naturally thymus-derived Tregs (nTregs) represent the major suppressive phenotype and can be identified by their demethylation status in the Tregs Specific Demethylated Region (TSDR) of the Forkhead-Box-P3 (FOXP3) gene using quantitative PCR (qPCR). DESIGN AND METHODS: The analytical performance of a TSDR demethylation qPCR assay was assessed in whole blood of healthy individuals (HI) and kidney transplant recipients (KTR). The assay was compared to conventional flow cytometry and the agreement of results between two laboratories using a comparable qPCR protocol was assessed. In addition, the effect of gender, age, and medications was investigated. RESULTS: Within and between series imprecision was <20% (n=6). Whole blood samples are suitable for analysis within 3days when stored at room temperature; both whole blood and DNA samples - within 12months when frozen at -80°C. A significant correlation between the qPCR results and flow cytometry was lacking both with samples from HI and KTR. qPCR results between laboratories showed a bias of 76% but correlated well (r=0.645; p=0.0002, n=29). nTregs determined by qPCR were significantly (p<0.05) higher in HI (0.73%±0.23%, n=60) than in KTR (0.45%±0.21%, n=60) and in female HI (1.0%±0.27%, n=30) than in male HI (0.45%±0.23%, n=27). No effect of drugs or age was observed. CONCLUSIONS: The qPCR assay for nTregs provides reproducible results and is of sufficient quality for working with patient samples although inter-laboratory differences can be encountered due to a lack of method standardization. It was confirmed that gender-specific reference ranges are required.

Thiopurines induce oxidative stress in T-lymphocytes: a proteomic approach.

Thiopurines are extensively used immunosuppressants for the treatment of inflammatory bowel disease (IBD). The polymorphism of thiopurine S-methyltransferase (TPMT) influences thiopurine metabolism and therapy outcome. We used a TPMT knockdown (kd) model of human Jurkat T-lymphocytes cells to study the effects of treatment with 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) on proteome and phosphoproteome. We identified thirteen proteins with altered expression and nine proteins with altered phosphorylation signals. Three proteins (THIO, TXD17, and GSTM3) with putative functions in cellular oxidative stress responses were altered by 6-TG treatment and another protein PRDX3 was differentially phosphorylated in TPMT kd cells. Furthermore, reactive oxygen species (ROS) assay results were consistent with a significant induction of oxidative stress by both TPMT knockdown and thiopurine treatments. Immunoblot analyses showed treatment altered expression of key antioxidant enzymes (i.e., SOD2 and catalase) in both wt and kd groups, while SOD1 was downregulated by 6-TG treatment and TPMT knockdown. Collectively, increased oxidative stress might be a mechanism involved in thiopurine induced cytotoxicity and adverse effects (i.e., hepatotoxicity) and an antioxidant cotherapy might help to combat this. Results highlight the significance of oxidative stress in thiopurines' actions and could have important implications for the treatment of IBD patients.

Lack of correlation between Treg quantification assays in inflammatory bowel disease patients.

AIM: To compare the number of regulatory T-cells (Tregs) measured by flow cytometry with those obtained using a real-time quantitative PCR (qPCR) method in patients suffering from inflammatory bowel disease (IBD). METHODS: Tregs percentages obtained by both flow cytometry and qPCR methods in 35 adult IBD patients, 18 out of them with Crohn´s disease (CD) and 17 with ulcerative colitis (UC) were compared to each other as well as to scores on two IBD activity questionnaires using the Harvey Bradshaw Index (HBI) for CD patients and the Simple Colitis Clinical Activity Index (SCCAI) for UC patients. The Treg percentages by flow cytometry were defined as CD4(+)CD25(high)CD127(low)FOXP3(+) cells in peripheral blood mononuclear cells, whereas the Treg percentages by qPCR method were determined as FOXP3 promoter demethylation in genomic DNA. RESULTS: We found an average of 1.56% ± 0.78% Tregs by using flow cytometry, compared to 1.07% ± 0.53% Tregs by using qPCR in adult IBD patients. There were no significant correlations between either the percentages of Tregs measured by flow cytometry or qPCR and the HBI or SCCAI questionnaire scores in CD or UC patients, respectively. In addition, there was no correlation between Treg percentages measured by qPCR and those measured by flow cytometry (r = -0.06, P = 0.73; Spearman Rho). These data suggest that, either Treg-related immune function or the clinical scores in these IBD patients did not accurately reflect actual disease activity. Until the cause(s) for these differences are more clearly defined, the results suggest caution in interpreting studies of Tregs in various inflammatory disorders. CONCLUSION: The two methods did not produce equivalent measures of the percentage of total Tregs in the IBD patients studied which is consistent with the conclusion that Tregs subtypes are not equally detected by these two assays.

CR1 is potentially associated with rate of decline in sporadic Alzheimer's disease.

The objective of this study was to investigate potential associations of Alzheimer's disease risk single nucleotide polymorphisms (SNP) with disease progression. SNP in ACE, ApoE, BIN1, CLU, CR1, CST3, EXOC3L2, GWA14q32.13, IL8, LDLR, PICALM, and TNK1 were determined in 40 Alzheimer's disease patients who were observed for 2 to 3 years. Annual Mini Mental State Examination (MMSE) loss was used as the outcome parameter in multiple regression analyses. Regarding a CR1 SNP (rs3818361) G-allele carriers featured faster declines (approximately 3 MMSE points per year). To summarize, in addition to being a risk factor for Alzheimer's disease development, a CR1 SNP appears to be associated with higher rates of medium-term disease progression. Therefore, it may serve as a prognostic marker (among others) and may aid in differentiating slow from fast progressors early in the disease course.

FoxP3 demethylation is increased in human colorectal cancer and rat cholangiocarcinoma tissue.

OBJECTIVES: FoxP3 expression is a marker for Tregs which are known to be involved in tumor immunity. We aimed to evaluate FoxP3 promoter demethylation in human colorectal cancer (CRC) and rat intrahepatic cholangiocarcinoma (ICC). DESIGN AND METHODS: Bisulfite-treated genomic DNA templates of shock frozen paired samples were studied from 13 anonymous CRC patients and from 10 male rats (n=6 ICC induced by thioacetamide and n=4 age-matched controls). Real-time PCR was carried out using a LightCycler 480 system. Human FoxP3 and CD3 promoter demethylations were estimated using previously described assays; and rat FoxP3 promoter demethylation using a newly developed assay. RESULTS: A significant 3.5-fold increase of the demethylation in FoxP3 promoter region was found in human CRC and rat ICC (P<0.05). The average frequency of cells with FoxP3 demethylation in patients suffering from CRC was 0.26% in normal tissue and 0.92% in tumor tissue (n=11 paired samples). Although, no significant difference was found between the mean frequency of CD3 demethylation in normal tissue (4.80%, n=6) and in tumor tissue (4.14%, n=6) from CRC patients, the ratio of demethylated CD3/FoxP3 promoter areas was significantly lower in tumor specimens (P<0.05). Using our novel assay, we found a significant increase in mean frequencies of cells with FoxP3 demethylation in rats with ICC (7.42%, n=6) in comparison to controls (2.14%, n=4). CONCLUSION: FoxP3 seems to be an interesting biomarker for immune response to epithelial tumors. Functional consequences from the increase of Tregs remain to be demonstrated. Further studies with outcome data are necessary.

Improved method for therapeutic drug monitoring of 6-thioguanine nucleotides and 6-methylmercaptopurine in whole-blood by LC/MSMS using isotope-labeled internal standards.

OBJECTIVES: Thiopurine drugs (azathioprine, 6-mercaptopurine) show wide interindividual variability and a narrow therapeutic range thus making therapeutic monitoring of their active metabolite 6-thioguanine nucleotides (6-TGN) desirable. We improved the currently available laborious and complex methodology of therapeutic drug monitoring of 6-TGN and the metabolite 6-methylmercaptopurine (6-MMP) in washed erythrocytes (ery) based on a whole-blood method. METHODS: The analytes were hydrolyzed and extracted from 25-µL ethylenediaminetetraacetic acid-anticoagulated whole-blood spiked with isotope labeled 6-TG-C2N and 6-MMP-d3 internal standards. Chromatography was performed in 5.1 minutes on a C18 reverse phase column followed by detection via electrospray interface-coupled API 4000 mass spectrometer set up in the positive multiple reaction monitoring mode. The hemoglobin concentration was measured in 20 µL of the original sample (AHD575 method), and the results were standardized to 120 g/L of hemoglobin. RESULTS: Calibration curves were linear with r > 0.999 (6-TGN and 6-MMP up to 10,000 pmol/0.2 mL). The limit of quantification was 30 pmol/0.2 mL for 6-TGN and 6-MMP. Intraassay and interassay imprecision was <7.5% at 3 tested levels for 6-TGN and 6-MMP, respectively. Method comparisons were as follows: Ery 6-TGN: y = 1.3x - 11 and ery 6-MMP y = 1.1x - 124. CONCLUSIONS: The new method compares favorably with established ones, allowing for rapid single run determination of 6-TGN and 6-MMP from <50 µL of fresh or frozen whole blood. Linearity and limits of quantification cover the clinically relevant range. Variability during sample preparation and matrix effects are compensated by the use of isotope-labeled internal standards. The whole-blood method is hemoglobin standardized to avoid falsely low results in the case of anemia. The method correlates well with 6-TGN measured in washed erythrocytes, but it requires significantly less hands-on time. Preliminary therapeutic ranges for the most common indications of azathioprine and 6-MP are provided.

cobas 8000 Modular analyzer series evaluated under routine-like conditions at 14 sites in Australia, Europe, and the United States.

Clinical laboratories need to test patient samples precisely, accurately, and efficiently. The latest member of the Roche cobas modular platform family, the cobas 8000 modular analyzer series allows compact and convenient consolidation of clinical chemistry and immunochemistry assays in high-workload laboratories with a throughput of 3 to 15 million tests annually. Here we present the results of studies designed to test the overall system performance under routine-like conditions that were conducted at 14 laboratories over 2 y. Experiments that test analytical performance of the new module were integrated with overall system functionality testing of all modules in different configurations. More than two million results were generated and evaluated for ~100 applications using serum/plasma, urine, or EDTA blood samples. During the workflow studies, eight configurations of the possible 38 combinations were used, covering all available analytical modules. The versatility of the module combinations makes the system customizable to fit the needs of diverse laboratories, allowing precise and accurate analysis of a broad spectrum of clinical chemistry and immunochemistry parameters with short turnaround times. This new system will contribute to the ability of clinical laboratories to offer better service to their customers and support vital clinical decision making.

Establishment of thiopurine S-methyltransferase gene knockdown in jurkat T-lymphocytes: an in vitro model of TPMT polymorphism.

BACKGROUND: Thiopurine S-methyltransferase (TPMT) is an excellent example of an enzyme whose pharmacogenetic polymorphisms affect efficacy and toxicity of a drug. The association between TPMT activity and thiopurine-related myelosuppression is well recognized. To study the significance of TPMT deficiency in thiopurine metabolism and immunosuppressive activity in vitro, we established RNA interference-based TPMT knockdown (kd) in a Jurkat cell line. RESULTS: In Jurkat TPMT kd cells, TPMT expression was reduced to 73% at the RNA level and 83% at the protein level. TPMT kd cells were more sensitive to 6-mercaptopurine (6-MP) (10 µmol/L) and 6-thioguanine (6-TG) (8 µmol/L) than wild-type (wt) cells, (32% versus 20%) and (18% versus 9%), respectively. Both Jurkat wt and kd cells were more sensitive to 6-TG-induced apoptosis than to 6-MP. 6-TG activity was also more affected by TPMT levels than was 6-MP as reflected by IC60, concentrations that is, 6-MP [4.6 µmol/L (wt) and 4.7 µmol/L (kd)], 6-TG [2.7 µmol/L (wt) and 0.8 µmol/L (kd)]. IC60 concentrations induced significant apoptosis in both Jurkat wt and kd cells (257%, versus 314%) with 6-MP and (323% versus 306%) with 6-TG, respectively. At IC60 (6-MP) 6-thioguanine nucleotides (6-TGN) accumulation in cells was 518 versus 447 pmol/million cells in wt and kd cells, respectively. On the other hand 6-TGN accumulation at IC60 (6-TG) was 477 versus 570 pmol/million cells in wt and kd cells, respectively. 6-Methylated mercaptopurine (6-MeMP) concentrations were more affected than 6-TGN by TPMT kd (194 versus 10 pmol/million cells) in wt and kd cells, respectively. CONCLUSION: We conclude that TPMT kd cells are an appropriate in vitro model to investigate the significance of TPMT deficiency with thiopurine therapy and could be helpful in understanding possible clinical consequences of TPMT polymorphism.

Low vitamin D and elevated immunoreactivity against Epstein-Barr virus before first clinical manifestation of multiple sclerosis.

OBJECTIVE: Vitamin D deficiency and Epstein-Barr virus (EBV) infection may be associated with the development of multiple sclerosis (MS). We investigated serum 25-hydroxyvitamin D (25-OH-D) levels and anti-EBV immunoreactivity in 25 individuals before the first clinical manifestation of MS. PATIENTS AND METHODS: 56 serum samples of 25 individuals who had donated blood prior to the first clinical MS manifestation (clinically isolated syndrome (CIS)) (four male subjects, 21 female subjects, mean age 31.5 years at time of pre-CIS blood sampling; mean age at disease onset 33.4 years) were available, covering an interval of 7.3 years-2 months (mean 31.5 months) before CIS. In 18 of 25 patients serum samples were also obtained after established diagnosis of MS. Longitudinal age- and gender-matched healthy blood donors (four male subjects, 21 female subjects, 39 samples, mean age 32.5 years) served as controls. Serum 25-OH-D was measured by isotope dilution-liquid chromatography-tandem mass spectrometry. 25-OH-D levels were deconvoluted using published seasonal coefficients from a German population. Immunoglobulin G (IgG) against Epstein-Barr virus nuclear antigen-1 (EBNA1) were assessed using commercially available ELISA. RESULTS: Low 25-OH-D levels were observed during the 24-month pre-CIS interval (47.8 (32.5-77.2) nmol/l, median (IQR); healthy controls: 81.6 (57.7-98.5), p=0.004, however, still higher than after established diagnosis (24.5 (13.7-47.7), p<0.0001 compared with controls). IgG against EBNA1 during the 36-month pre-CIS interval was increased (185.9 (91.2-460.0) IU/ml, median (IQR); healthy controls 63.7 (29.5-121.6), p=0.002). CONCLUSIONS: Low vitamin D and remote EBV infection may be associated with clinical MS breakthrough within 2-3 years.

Therapeutic monitoring of immunotherapies in autoimmune diseases.

Immunosuppressive treatment still is an important element in the management of autoimmune mediated diseases. However, immunosuppressive therapy is often complicated by a narrow therapeutic index and high variability of treatment response. This review discusses the clinical management and monitoring strategies for the use of ciclosporin A, tacrolimus, azathioprine, mycophenolat mofetil, mitoxantrone and some monoclonal antibodies with focus on natalizumab.

Alzheimer's disease: genetic polymorphisms and rate of decline.

BACKGROUND/AIM: To investigate the influence of established genetic risk factors for Alzheimer's disease on the speed of disease progression. METHODS: Polymorphisms (in ACE, ApoE, BIN1, CLU, CR1, CST3, EXOC3L2, GWA14q32.13, IL8, LDLR, PICALM, TNK1) of 40 Alzheimer's disease patients from a longitudinal study were analyzed. A standardized loss of Mini-Mental State Examination points was used as the progression parameter. RESULTS: Polymorphisms in CST3 and EXOC3L2 as well as the absence of APOE4 were associated with more aggressive disease courses. A trend was observed for BIN1. CONCLUSION: In addition to being a risk factor for disease development, some of the polymorphisms investigated here are associated with higher rates of decline and disease progression and thus might act as prognostic disease markers. This effect needs to be considered in future treatment strategies.

Pharmacogenetic screening for drug therapy: from single gene markers to decision making in the next generation sequencing era.

Pharmacogenetics has substantially added to our understanding of the variability of drug response. A number of single gene markers have been established and are ready to use in clinical practice. Here we review the validity and utility of markers in a number of genes (CYP2D6, CYP2C19, CYP2C9, VKORC1, TPMT, UGT1A1, OATP1B1, KRAS and HLA locus) for therapy decisions. As drug response is a complex trait in the majority of cases, most of the identified functional variants will only explain a limited part of the variability of drug response. In this sense, a phenotype is the product of many low-penetrance variations. Technical progress has not only improved the cost-effectiveness of screening for single gene markers, but offers the possibility of generating vast amounts of genome-wide single nucleotide polymorphism (SNP) or sequence data for each patient. The latest challenge is to incorporate these amounts of data into pharmacogenetic decision support. We discuss here the challenges associated with choosing the correct therapy for patients who present to their physicians with personal genome data.

Monovalent and divalent salt correction algorithms for Tm prediction--recommendations for Primer3 usage.

Primer3 is a widely used program for selection of oligonucleotide primers for PCR. The websites used for implementation of Primer3 have recently been updated. PCR requires Mg(2+)(,) which has a significant dsDNA stabilizing effect that must be taken into account when designing PCR primers. The data sets and formulas used to correct for salt concentrations have been updated in Primer3 to give better prediction of melting temperature (T(m)). The liberal combination of different formulas for monovalent and divalent salt correction can lead to different results, depending on the formula chosen by the user. Using published T(m) for 475 different oligonucleotides, it is shown that the combination of the implemented conversion of divalent to monovalent cation concentration works well with one salt correction formula but not with an alternative one. Use of a more recently described alternative formula would lead to equally good T(m) predictions if divalent cations are present. The proper selection of compatible primer pairs depends on the choice of a good combination of salt correction formulas. Currently the SantaLucia salt correction formula should be used if Mg(2+) is present. The alternative formula should be updated to its recent form for future releases.

Pre-analytical effects of different lithium heparin plasma separation tubes in the routine clinical chemistry laboratory.

BACKGROUND: In this study the pre-analytical effects of sample storage on frequently used routine clinical chemistry assays were evaluated by comparing four different lithium heparin plasma separation tubes to a reference collection procedure. METHODS: Blood was collected from 20 healthy volunteers using plasma separation tubes from four different manufacturers together with manually separated plasma as reference. In total, 15 clinical chemistry parameters were determined at 0 h, 24 h, and 72 h. Samples were stored at 4°C. Statistical differences were evaluated using a generalized estimating equation regression model. RESULTS: Significant differences could be demonstrated for almost every parameter when comparing the separation tubes to the reference collection system. The estimated maximum allowable storage time in the primary tube was considerably reduced using separation tubes, e.g., for glucose the maximum storage time was reduced from >72 h to 7-15 h, and for potassium from 60 h to 10-13 h, respectively. CONCLUSIONS: These data indicate that sample storage in the primary tube using plasma separation tubes is associated with clinically relevant changes for certain parameters. Therefore, storing samples for retesting should be avoided when using plasma separation tubes, in particular for parameters susceptible to interference by erythrocyte or platelet contamination.

Proteomics approach to identify the interacting partners of cellular prion protein and characterization of Rab7a interaction in neuronal cells.

The present study was undertaken to identify proteins interacting with PrP(C) that could provide new insights into its physiological functions and pathological role. Human PrP(C) was expressed in prion protein-deficient murine hippocampus (HpL3-4) neuronal cells. The PrP(C) along with its interacting proteins were affinity purified using STrEP-Tactin-chromatography, in-gel digested, and identified by Q-TOF MS/MS analysis. Forty-three proteins appeared to interact with PrP(C) in this neuronal cell line. Of these, 15 were already known for their interaction with PrP(C) or PrP(Sc), while 28 new proteins were identified. Interaction of a novel interacting partner of GTPase family-Rab7a, having a suggested role in vesicle trafficking, was further investigated using confocal laser scanning microscopy and reverse coimmunoprecipitation. Both reverse coimmunoprecipitation and immunofluorescence results confirmed potential interaction of Rab7a with the PrP(C). siRNA against the Rab7a gene decreased expression of Rab7a protein, in PrP(C) expressing HpL3-4 and SH-SY5Y cells. This depleted Rab7a expression led to the enhanced accumulation of PrP(C) in Rab9 positive endosomal compartments and consequently an increased colocalization between PrP(C)/Rab9. However, the Rab9 accumulated PrP(C) remained sensitive to proteinase-K digestion. The work described demonstrated for the first time that Rab7a interacts with PrP(C) and highlighted the involvement of endosomal compartments in the trafficking and regulation of PrP(C).