Plasma ascorbic acid preparation and storage for epidemiological studies using TCA precipitation

Objectives:To introduce a procedure to validate an ascorbic acid method using trichloroacetic acid (TCA) for plasma stabilization at different storage temperatures.Methods:EDTA and heparin plasma were precipitated with TCA (1:5) containing 0.54 mol/L EDTA, or without. Samples were stored at ? 20 °C and ? 70 °C and their stability was tested at room temperature for 24 h.Results:A significant 40% loss (p < 0.001) of plasma ascorbic acid was found when EDTA samples with added EDTA were stored at ? 20 °C for 2–4 weeks compared with storage at ? 70 °C. Ascorbic acid in heparin plasma without added EDTA was most unstable and samples left at room temperature for 24 h lead to almost a total loss of ascorbic acid. Addition of EDTA to the TCA solution improved stability of samples of both plasma types at room temperature.Conclusion:The recommended procedure for ascorbic acid determination in plasma stabilized with TCA is immediate storage at ? 70 °C and inclusion of EDTA into the TCA solution.     

Clotting factor activity in thawed Octaplas® LG during storage at 2–6 °C for 6 days from a quality assurance point of view

IntroductionOctaplas® LG is a second-generation solvent/detergent-treated plasma that offers an additional safety benefit by prion elimination. The stability of clotting factors of the new S/D plasma after thawing has not been investigated yet. This study intended to measure the time course of fibrinogen, FII, FV, FVII, FVIII, FIX, PC, fPS and PI through storage at 2–6 °C over 6 days.Materials and methodsWe investigated 20 plasma bags (five bags per blood group) and measured fibrinogen, FII, FV, FVII, FVIII, FIX, PC, fPS and PI immediately after thawing and after 2, 4, 6, 24, 48, 72, 96, 120 and 144 h storage at 2–6 °C. Five separate plasma bags were thawed and stored at 2–6 °C for microbiological assessment. After 6 days samples were drawn for blood cultures that were incubated for six more days.ResultsAfter 6 days FII, FIX and PC showed no significant changes. FV (?16%, p < 0.001), FVII (?19%, p < 0.001), FVIII (?19%, p < 0.001), FXI (?13%, p < 0.0001) and fPS (?4%, p < 0.0007) decreased significantly. PI levels were stable at 56%. The microbiological investigation showed no bacterial contamination.ConclusionsIn Octaplas® LG plasma clotting factors decreased slightly through storage of 6 days. PI levels were remarkably higher and stable over time in the new Octaplas® LG. Stability of stored Octaplas® LG was limited by the decrease of FVIII to 53%, which may warrant storage up to 24 h from a quality assurance point of view. This could result in reduced plasma wastage and costs for healthcare givers.     

Plasma ceruloplasmin as a biomarker for obesity: a proteomic approach

ObjectivesThis study aimed to investigate new biomarkers of obesity particularly in relation with inflammation-associated proteins using protein differential display techniques.Design and methodsComparison of protein expression in plasma between non-obese (n = 109, body mass index, BMI < 25 kg/m²) and obese (n = 32, BMI ≥ 25 kg/m²) groups was carried out using two-dimensional gel electrophoresis (2-DE) analysis. ELISA was also performed for validation.ResultsAmong six differentially expressed protein spots, ceruloplasmin (Cp) and fibrinogen were over-expressed in obese group. Plasma Cp levels were significantly higher in obese group than non-obese group (34.0 ± 8.6 vs. 41.3 ± 12.7 mg/dL, p < 0.001) and positively correlated with age (r = 0.253, p < 0.005), BMI (r = 0.265, p < 0.001) and hsCRP (r = 0.385, p < 0.001). In stepwise multiple linear regression analysis, plasma Cp along with hsCRP were found predictors for obesity (adjusted β-coefficient = 0.266, p < 0.01).ConclusionElevated plasma Cp levels were significantly associated with obesity, which may be suggested to be a marker of obesity.     

Association of high sensitivity C-reactive protein (hsCRP) and tumour necrosis factor-alpha (TNF-alpha) with carotid intimal medial thickness in subjects with different grades of glucose intolerance--the Chennai Urban Rural Epidemiology Study (CURES-31)

ObjectiveTo assess the association of high sensitivity C-Reactive Protein [hsCRP] and Tumour Necrosis Factor-α [TNF-α] with IMT in Asian Indians with different grades of glucose intolerance.Design and methodsSubjects with normal glucose tolerance [NGT](n = 150), impaired glucose tolerance [IGT] (n = 150) and type 2 diabetes (DM) (n = 150) were recruited from the Chennai Urban Rural Epidemiology Study [CURES], in south India. hsCRP was estimated by nephelometry and TNF-α by enzyme linked immunosorbent assay. Carotid IMT was assessed by high resolution B-mode ultrasonography.ResultshsCRP and TNF-α levels were higher in those with DM [p < 0.001] and IGT [p < 0.001] compared to NGT. In linear regression analysis, both hsCRP [p = 0.003] and TNF-α [p =0.001] showed an association with IMT among NGT subjects even after adjusting for age and gender. Among IGT subjects, TNF-α was associated with IMT [p < 0.001], while no association was observed either with hsCRP or TNF-α in diabetic subjects. In NGT subjects, mean IMT was highest in those with high values [III tertile] of both TNF-α and hsCRP [0.83 ± 0.1 mm; p < 0.001] followed by those with high TNF-α + low hsCRP [0.74 ± 0.09 mm; p < 0.001], high hsCRP  low TNF-α [0.67 ± 0.09 mm; p < 0.001], and lowest in those with both low TNF-α and hsCRP [I tertile] [0.63 ± 0.05 mm.ConclusionWe conclude that in Asian Indians 1. Levels of hsCRP and TNF-α increase with increasing severity of glucose intolerance 2. Both hsCRP and TNF-α are associated with IMT in NGT subjects while TNF-α alone is associated with IMT in IGT subjects 3. hsCRP and TNF-α have a cumulative effect on mean IMT values in NGT subjects.     

Long-term stability of biochemical markers in pediatric serum specimens stored at -80 °C: a CALIPER Substudy

ObjectivesPediatric serum samples collected from healthy children in the CALIPER (Canadian Laboratory Initiative in Pediatric Reference Interval) project are stored at ? 80 °C for various periods of time. This study aimed to determine the stability of chemistry, protein, and hormone analytes under these conditions.Design and methodsSerum samples collected from children of 0–18 years of age attending outpatient clinics were pooled into a single pool or into age-group specific pools. Following baseline measurement, each pool was aliquoted and kept frozen at ? 80 °C until analysis. Samples were analyzed for 57 biochemical markers at monthly intervals over a 10–13 month period and each aliquot was subject to one freeze–thaw cycle before analysis. The analysis was performed on VITROS® Chemistry System, COBAS INTEGRA® 400 Plus and IMMULITE® 2500. Values obtained at monthly intervals were compared to baseline measurements and examined for trends over time.ResultsA majority of analytes measured in this study showed no significant time-dependent change relative to baseline or trend over time after up to 13 months of storage. PTH showed up to 27.2% decline after 10 months of storage with most of the decline evident after 2 months. Most analytes showed variability over time, which is thought to reflect assay variability rather than changes in analyte stability.ConclusionsThe study shows stability for a majority of analytes stored in serum at ? 80 °C after up to 13 months of storage. Samples do not require immediate testing for reference interval determination for the selected analytes with possible exception of PTH.     

Particle enhanced turbidimetric immunoassay for the determination of urine cystatin C on Cobas c501

ObjectiveUrinary cystatin C has been reported to be a good marker for tubular damage and acute kidney injury. The aim of this study was to develop a high throughput assay for the quantification of urine cystatin C.MethodsAntigen-excess, imprecision, interference, linearity, recovery, sample stability and reference values were evaluated on Cobas c501.ResultsThe assay was linear over the dynamic range of the study (R² = 0.9994). The total assay imprecision was below 5%. The assay recovery was estimated at 87–100%. No tendency to antigen-excess (up to 35 mg/L), nor interference with haemoglobin (1.25–10 g/L) was observed. Cystatin C was stable for 1 day at ambient temperature (19–23 °C) but for 2 days at + 4 °C. The reference interval for cystatin C in urine was < 0.414 mg/L.ConclusionsThe urinary cystatin C assay verified to be a reliable assay with convenient performance characteristics, enabling routine testing on clinical chemistry platforms.     

Atrial natriuretic peptide stability

ObjectiveAtrial natriuretic peptide (ANP) is a key regulator in the homeostasis of water excretion and has emerged as an important prognostic marker for symptomatic chronic heart failure (CHF). The stability of ANP represents a crucial factor in assessing its use as a cardiac biomarker. Accordingly, we assessed the stability of ANP in blood samples collected from healthy controls and CHF subjects for a 12 month period.MethodsBlood samples from 10 healthy controls and 12 symptomatic CHF subjects with left ventricular systolic dysfunction were drawn. Determination of plasma ANP was performed by a standardized radioimmunoassay protocol.ResultsThe ANP levels of healthy subjects were 68.5 ± 11.6 pg/mL at baseline and 69.9 ± 17.2 pg/mL at 12 months (p = 0.71). The ANP concentrations of CHF subjects were 199.25 ± 44.8 pg/mL at baseline and 197.83 ± 47.4 pg/mL at 12 months (p = 0.70) respectively.ConclusionANP is a stable molecule with no evidence of degradation when stored at ? 80 °C.     

Influence of diabetes on homocysteine-lowering therapy in chronic hemodialysis patients

IntroductionThe effect of homocysteine (Hcy)-lowering therapy may be different in hemodialysis (HD) patients with and without diabetes mellitus (DM).MethodsStable HD patients with uremia were administered folic acid and vitamin B for 3 months. The impact of treatment was compared in patients with and without DM.ResultsA total of 61 patients (31 men and 30 women) aged 56 ± 13 y completed the study. Among these, 44 patients (72%) did not have DM and 17 (28%) had DM. At baseline, total Hcy and high-sensitivity C-reactive protein (hsCRP) levels were similar. After treatment, the levels of total Hcy and hsCRP were significantly decreased in the nondiabetic group (total Hcy level decreased from 33.63 ± 14.13 μmol/l to 18.94 ± 8.46 μmol/l, p < 0.001; hsCRP level decreased from 0.58 mg/dl [range, 0.21–1.05 mg/dl] to 0.22 mg/dl [range, 0.11–0.53 mg/dl], p < 0.001) but not in the diabetic group (total Hcy level decreased from 34.97 ± 17.12 μmol/l to 29.53 ± 11.36 μmol/l, p = 0.057; hsCRP level decreased from 0.80 mg/dl [range, 0.24–1.47 mg/dl] to 0.49 mg/dl [range, 0.45–0.98 mg/dl], p = 0.28). Serial monitoring of total Hcy level showed a more sustained effect of therapy on patients without DM.ConclusionFolic acid and vitamin B administration significantly lower total Hcy and hsCRP levels in HD patients without DM but not in those with DM.     

Long-term stability of soluble ST2 in frozen plasma samples

ObjectiveThe aim of this study was to investigate the long-term in vitro stability of soluble ST2 (sST2).Design and methodsEDTA plasma samples were drawn from 15 individuals with various diseases. The Presage^TM ST2 assay was used for measurement of sST2 concentrations directly after blood collection and after storing plasma samples for 18 months at ?20 °C and ?80 °C. The default criterion for analyte stability was set at 95%.ResultssST2 concentrations in the 15 individuals ranged from 12 U/mL to 140 U/mL. Directly after blood collection, the mean (± SD) sST2 concentration was 51 ± 37 U/mL, and absolute analyte recoveries were 50 ± 35 U/mL and 51 ± 34 U/mL after storage of samples for 18 months at ?20 °C and ?80 °C, respectively. Relative analyte recoveries after 18 months of storage at ?20 °C and ?80 °C were 99 ± 5% and 101 ± 7%.ConclusionsST2 is stable for at least 1.5 years in plasma samples stored at ?20 °C and ?80 °C.     

The influence of rewarming after therapeutic hypothermia on outcome after cardiac arrest

IntroductionTreatment with hypothermia has been shown to improve outcome after cardiac arrest (CA). Current consensus is to rewarm at 0.25–0.5 °C/h and avoid fever. The aim of this study was to investigate whether active rewarming, the rate of rewarming or development of fever after treatment with hypothermia after CA was correlated with poor outcome.MethodsThis retrospective cohort study included adult patients treated with hypothermia after CA and admitted to the intensive care unit between January 2006 and January 2009. The average rewarming rate from end of hypothermia treatment (passive rewarming) or start active rewarming until 36 °C was dichotomized in a high (≥0.5 °C/h) or normal rate (<0.5 °C/h). Fever was defined as > 38 °C within 72 h after admission. Poor outcome was defined as death, vegetative state, or severe disability after 6 months.ResultsFrom 128 included patients, 56% had a poor outcome. Actively rewarmed patients (38%) had a higher risk for poor outcome, OR 2.14 (1.01–4.57), p < 0.05. However, this effect disappeared after adjustment for the confounders age and initial rhythm, OR 1.51 (0.64–3.58). A poor outcome was found in 15/21 patients (71%) with a high rewarming rate, compared to 54/103 patients (52%) with a normal rewarming rate, OR 2.61 (0.88–7.73), p = 0.08. Fever was not associated with outcome, OR 0.64 (0.31–1.30), p = 0.22.ConclusionsThis study showed that patients who needed active rewarming after therapeutic hypothermia after CA did not have a higher risk for a poor outcome. In addition, neither speed of rewarming, nor development of fever had an effect on outcome.     

Determination of creatine and guanidinoacetate by GC-MS: study of their stability in urine at different temperatures

ObjectivesEvaluation of a GC-MS method using N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA) as the silylating agent for GC-MS. Study of the stability of creatine and guanidinoacetate in urine.Design and methods22 urines were kept at RT, 4 °C and ? 30 °C for 15 days.ResultsMTBSTFA produces a single chromatographic peak in contrast with other derivatizing agents. Creatine concentration increases at room temperature (326% on average), and at 4 °C (75%). However, detection decreases after freezing (? 37%). Guanidinoacetate is stable, but decreases after freezing (? 37%). Sonication before analysis is crucial to obtain repetitive results.ConclusionsA modified GC-MS method has been validated and the conditions for preservation of the urine have been established.     

Effects of temperature and light on the stability of bilirubin in plasma samples

BackgroundAlthough it is known that bilirubin is photo-sensitive, detailed effects of both temperature and artificial light exposure on bilirubin stability in plasma have not been well investigated. We determined the impact of temperature and artificial light on bilirubin stability in plasma.MethodsPlasma total and direct bilirubin were analyzed using a diazo method. The aliquots of 38 samples were stored at 3 °C and 22 °C with light protection for 2, 4, 8, and 24 h respectively before analysis. The aliquots of 20 samples with normal bilirubin and additional 20 with elevated bilirubin were exposed to artificial light for 2, 4, 8, 24, and 48 h at 22 °C, and total and direct bilirubin were measured. The differences between the baselines and subsequent measurements were analyzed with analysis of variance.ResultsThe baseline total bilirubin was 9.6 ± 8.1 mg/dl (mean ± SD) and the concentrations were 9.6 ± 8.2, 9.0 ± 7.4, 9.0 ± 7.5, and 8.8 ± 7.5 mg/dl at 3 °C and 9.5 ± 8.1, 9.0 ± 7.4, 9.6 ± 8.1, and 9.5 ± 8.0 mg/dl at 22 °C after 2, 4, 8, and 24 h (p > 0.05, n = 38). The baseline direct bilirubin was 1.3 ± 1.2 mg/dl and the concentrations after 2, 4, 8, and 24 h were 1.4 ± 1.2, 1.4 ± 1.2, 1.5 ± 1.2, and 1.3 ± 1.1 mg/dl at 3 °C and 1.4 ± 1.1, 1.3 ± 1.1, 1.3 ± 1.1, and 1.3 ± 1.0 mg/dl at 22 °C (p > 0.05, n = 19). In samples with elevated bilirubin exposed to light at 22 °C, the baseline total and direct bilirubin concentrations were 10.2 ± 1.7 mg/dl and 5.0 ± 1.9 mg/dl, respectively. After 2, 4, 8, 24, and 48 h, total bilirubin concentrations were 10.1 ± 1.8, 10.0 ± 1.8, 10.0 ± 1.8, 9.3 ± 2.0 (p > 0.05, n = 20), and 8.4 ± 2.3 (p < 0.01, n = 20) mg/dl and direct bilirubin concentrations were 4.9 ± 1.8, 4.9 ± 1.9, 4.8 ± 1.8, 4.2 ± 1.6 (p > 0.05, n = 20), and 3.5 ± 1.5 (p < 0.01, n = 20) mg/dl. For samples with normal bilirubin levels under the same conditions, the average baseline total and direct bilirubin concentrations were 0.7 ± 0.1 mg/dl and below the lower limit of quantification (LLOQ), respectively. After 2, 4, 8, 24, and 48 h, the average total bilirubin concentrations were 0.7 ± 0.1, 0.6 ± 0.1, 0.6 ± 0.1 (p > 0.05, n = 20), 0.5 ± 0.1, and 0.4 ± 0.1 mg/dl (p < 0.01, n = 20) and direct bilirubin concentrations were still below LLOQ.ConclusionsBilirubin in plasma is stable in refrigerator or at room temperature without light exposure for at least 24 h. In normal laboratory environment, a delay of up to 8 h in the measurement of bilirubin left unprotected from light at room temperature does not significantly affect the results. Under these conditions, the changes in bilirubin concentration are not clinically significant until 24 h (direct bilirubin) and after 48 h (total bilirubin).     

BDNF Val66Met polymorphism is associated with unstable angina

BackgroundBrain-derived neurotrophic factor (BDNF) is involved in the pathophysiology of coronary artery disease (CAD). The human BDNF Val66Met polymorphism has been shown to be associated with altered susceptibility to neuropsychiatric disorders. However it is unknown whether this polymorphism plays a role in cardiovascular disease.MethodsGenotyping of BDNF Val66Met polymorphism was carried out in 513 controls, 628 unstable angina pectoris (UAP) and 276 stable angina pectoris (SAP) patients. The plasma concentrations of BDNF and high-sensitivity C-reactive protein (hsCRP) were measured by ELISA. The general clinical data in patients and controls were obtained.ResultsThere was a significant association between genotype and allele frequency of the BDNF Val66Met polymorphism and UAP (all P < 0.05). Multivariate logistic regression analysis revealed that the BDNF_Met/Met genotype had a protective effect on the occurrence of UAP after controlling for known risk factors of CAD (OR 0.53, P = 0.005). Subjects with BDNF_Met/Met genotype also had decreased plasma hsCRP levels compared with the Val carriers (P < 0.01).ConclusionThe BDNF_Met/Met genotype has a protective effect on the occurrence of UAP, which might in part be due to the decreased plasma hsCRP level in BDNF_Met/Met carriers. To our knowledge, this is the first study that demonstrates the link between BDNF Val66Met polymorphism and CAD.     

Cox proportional hazard model analysis of survival in end-stage renal disease patients with small-sized high-density lipoprotein particles

ObjectiveDyslipidemia is commonly seen in patients with end-stage renal disease (ESRD). This prospective study investigates whether small-sized high-density lipoprotein (HDL) particles alone or in combination with high sensitivity C-reactive protein (hsCRP) are independent determinants of ESRD mortality.Design and methodsWe performed 36 months follow-up study in 122 haemodialysis (HD) patients. HDL size and subclass distribution were determined by gradient gel electrophoresis. Baseline characteristics of the patients were evaluated for the prediction of mortality.ResultsCox regressions analysis showed that patients with small-sized HDL particles had 2.8-fold higher risk of lethal outcome (P < 0.05). Concomitant presence of small-sized HDL particles and increased hsCRP concentration were significantly associated with reduced survival rate (HR = 3.907; P < 0.05). Observed relationships persisted after adjustment for serum lipid and lipoprotein concentrations.ConclusionsOur results indicate that small-sized HDL particles alone and combined with elevated hsCRP concentrations are independent predictors of reduced survival in HD patients.     

Effects of a single dose of oral iron on hepcidin concentrations in human urine and serum analyzed by a robust LC-MS/MS method

BackgroundThe measurement of serum hepcidin, a peptide hormone that regulates iron metabolism, is clinically important to the understanding of iron homeostasis in health and disease. To date, the quantification of serum hepcidin levels by conventional immunological detection methods has proven problematic due to challenges in obtaining high quality antibodies which demonstrate good reproducibility. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) has been employed recently for more sensitive quantification of hepcidin; however, this method has high background levels and therefore less than optimal specificity.MethodsIn order to increase the specificity of the mass spectrometry based assay, we developed a robust, ultra-performance liquid-chromatography-tandem mass spectrometry (UPLC-MS/MS) protocol using multiple selected reaction monitoring (mSRM) for quantification of hepcidin levels in urine and serum of human subjects. With this assay, we assessed levels of hepcidin before and for up to 8 h after oral ingestion of ferrous sulfate in ten adult human subjects without known disease.ResultsThe linear response of hepcidin quantitation on each instrument was measured, and the correlation coefficients of these calibrations were r² = 0.9512 ± 0.0202 (n = 5) for urine and r² = 0.9709 ± 0.0291 (n = 5) for serum [r² = mean ± SD]. Compared to baseline, the levels of urinary hepcidin between 2–4 h and 4–8 h of both women and men showed significant increases with p < 0.05 and p < 0.001, respectively. The levels of serum hepcidin between 4 h and 8 h in both women and men showed significant increases, compared with baseline values, with both p < 0.01. Interestingly, we also observed some degree of oscillation of levels, occurring at later time points.ConclusionsWe have developed and validated a new method for measuring hepcidin concentrations in human serum and urine and used it to demonstrate early increases with iron supplement in both urinary and serum levels of hepcidin, which return to baseline levels, except in urine samples from men.     

Quantitative analysis of chest compression interruptions during in-hospital resuscitation of older children and adolescents

AimTo quantitatively describe pauses in chest compression (CC) delivery during resuscitation from in-hospital pediatric and adolescent cardiac arrest. We hypothesized that CPR error will be more likely after a chest compression provider change compared to other causes for pauses.MethodsCPR recording/feedback defibrillators were used to evaluate CPR quality for victims ≥8 years who received CPR in the PICU/ED. Audiovisual feedback was supplied in accordance with AHA targets. Etiology of CC pauses identified by post-event debriefing/reviews of stored CPR quality data.ResultsAnalysis yielded 205 pauses during 304.8 min of CPR from 20 consecutive cardiac arrests. Etiologies were: 57.1% for provider switch; 23.9% for pulse/rhythm analysis; 4.4% for defibrillation; and 14.6% “other.” Provider switch accounted for 41.2% of no-flow duration. Compared to other causes, CPR epochs following pauses due to provider switch were more likely to have measurable residual leaning (OR: 5.52; CI⁹⁵: 2.94, 10.32; p < 0.001) and were shallower (43 ± 8 vs. 46 ± 7 mm; mean difference: ?2.42 mm; CI⁹⁵: ?4.71, ?0.13; p = 0.04). Individuals performing continuous CPR ≥ 120 s as compared to those switching earlier performed deeper chest compressions (42 ± 6 vs. 38 ± 7 mm; mean difference: 4.44 mm; CI⁹⁵: 2.39, 6.49; p < 0.001) and were more compliant with guideline depth recommendations (OR: 5.11; CI⁹⁵: 1.67, 15.66; p = 0.004).ConclusionsProvider switches account for a significant portion of no-flow time. Measurable residual leaning is more likely after provider switch. Feedback systems may allow some providers to continue high quality CPR past the recommended switch time of 2 min during in-hospital resuscitation attempts.     

Revaluation of biological variation of glycated hemoglobin (HbA(1c)) using an accurately designed protocol and an assay traceable to the IFCC reference system

BackgroundGlycated hemoglobin (HbA_1c) has a key role for diagnosing diabetes and monitoring glycemic state. As recently reviewed, available data on HbA_1c biological variation show marked heterogeneity. Here we experimentally revaluated these data using a well designed protocol.MethodsWe took five EDTA whole blood specimens from 18 apparently healthy subjects on the same day, every two weeks for two months. Samples were stored at ? 80 °C until analysis and assayed in duplicate in a single run by Roche Tina-quant® Gen.2 immunoassay. Data were analyzed by the ANOVA. To assess the assay traceability to the IFCC reference method, we preliminarily carried out a correlation experiment.ResultsThe bias (mean ± SD) of the Roche immunoassay was 0.3% ± 0.7%, confirming the traceability of the employed assay. No difference was found in HbA_1c values between men and women. Within- and between-subject CV were 2.5% and 7.1%, respectively. Derived desirable analytical goals for imprecision, bias, and total error resulted 1.3%, 1.9%, and 3.9%, respectively. HbA_1c had marked individuality, limiting the use of population-based reference limits for test interpretation. The estimated critical difference was ~ 10%.ConclusionsFor the first time we defined biological variation and derived indices for the clinical application of HbA_1c measurements using an accurately designed protocol and an assay standardized according to the IFCC.     

Carotid intima media thickness is related positively to plasma pre ß-high density lipoproteins in non-diabetic subjects

BackgroundLipid-poor or lipid-free high density lipoprotein (HDL) particles, designated pre ß-HDL, stimulate removal of cell-derived cholesterol to the extracellular compartment, which is an initial step in the reverse cholesterol transport pathway. Pre ß-HDL levels may be elevated in subjects with established cardiovascular disease. We determined the relationship of carotid intima media thickness (IMT), a marker of subclinical atherosclerosis, with pre ß-HDL in subjects without clinically manifest cardiovascular disease.MethodsIMT and plasma pre ß-HDL, assayed by crossed immuno-electrophoresis, were determined in 70 non-diabetic subjects (aged 56 ± 9 years; non-smokers only; 27 women).ResultsIMT was correlated positively with pre ß-HDL, both expressed as plasma apolipoprotein (apo) A-I concentration (r = 0.271, p = 0.023) and as% of apo A-I (r = 0.341, p = 0.004). In contrast, IMT was correlated inversely with HDL cholesterol (r = ? 0.253, p = 0.035). IMT was also related positively to pre ß-HDL after adjustment for age, sex, systolic blood pressure (in apoA-I concentration, ß = 0.203, p = 0.043; in% of plasma apoA-I, ß = 0.235, p = 0.023). IMT remained associated with pre ß-HDL after additional adjustment for either body mass index, plasma glucose, cholesterol, triglycerides, HDL cholesterol, apoA-I and apoB.ConclusionSubclinical atherosclerosis may relate to higher plasma pre ß-HDL independently of apoA-I and HDL cholesterol levels.     

Pre-analytic and analytic sources of variations in thiopurine methyltransferase activity measurement in patients prescribed thiopurine-based drugs: A systematic review

ObjectivesLow thiopurine S-methyltransferase (TPMT) enzyme activity is associated with increased thiopurine drug toxicity, particularly myelotoxicity. Pre-analytic and analytic variables for TPMT genotype and phenotype (enzyme activity) testing were reviewed.Design and methodsA systematic literature review was performed, and diagnostic laboratories were surveyed.ResultsThirty-five studies reported relevant data for pre-analytic variables (patient age, gender, race, hematocrit, co-morbidity, co-administered drugs and specimen stability) and thirty-three for analytic variables (accuracy, reproducibility). TPMT is stable in blood when stored for up to 7 days at room temperature, and 3 months at ? 30 °C. Pre-analytic patient variables do not affect TPMT activity. Fifteen drugs studied to date exerted no clinically significant effects in vivo. Enzymatic assay is the preferred technique. Radiochemical and HPLC techniques had intra- and inter-assay coefficients of variation (CVs) below 10%.ConclusionTPMT is a stable enzyme, and its assay is not affected by age, gender, race or co-morbidity.     

Determination of protein-incorporated methylated arginine reference values in healthy subjects whole blood and evaluation of factors affecting protein methylation

ObjectivesProtein arginine methylation is a post-translational modification involved in the regulation of signal transduction, RNA export, and cell proliferation. Reference values of arginine methylation of whole blood proteome remain to be determined.Design and methodsAsymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA) and monomethylarginine (MMA) incorporated in whole blood protein and methionine, cysteine and homocysteine plasma levels from 134 healthy subjects were measured by capillary electrophoresis.ResultsThe mean protein-incorporated concentration of the selected population was 4.11 ± 0.77 nmol/mg protein for ADMA; 1.66 ± 0.42 nmol/mg protein for SDMA and 4.31 ± 1.17 nmol/mg protein for MMA. Multiple correlation analysis showed that plasma Hcy was positively related to incorporated protein ADMA (P = 0.002) and MMA (P = 0.049).ConclusionsThe study was able to define a reference value for protein-incorporated ADMA, SDMA and MMA levels and found a positive association between protein-incorporated ADMA and plasma homocysteine.