Goce Dimeski

Ion Selective Electrodes (ISEs) and interferences--a review.

Ion Selective Electrodes (ISEs) are used to measure some of the most critical analytes on clinical laboratory and point-of-care analysers. These analytes which include Na(+), K(+), Cl(-), Ca(2+), Mg(2+) and Li(+) are used for rapid patient care decisions. Although the electrodes are very selective, they are not free of interferences. It is important for laboratories to have an understanding of the type and extent of interferences in order to avoid incorrect clinical decisions and treatment.

Measurement of salivary cortisol in 2012 – laboratory techniques and clinical indications

The utility of measuring salivary cortisol has become increasingly appreciated since the early 1980s. Salivary cortisol is a measure of active free cortisol and follows the diurnal rhythm of serum or plasma cortisol. The saliva sample may be collected by drooling or through the use of absorbent swabs which are placed into the mouth until saturated. Salivary cortisol is therefore convenient for patients and research participants to collect noninvasively on an outpatient basis. Several assay techniques have been used to measure salivary cortisol, including radioimmunoassay and more recently liquid chromatography–tandem mass spectrometry. The analytical sensitivity varies between these assay methods, as does the potential for cross‐reactivity with other steroids. The interpretation of salivary cortisol levels relies on rigorous standardization of sampling equipment, sampling protocols and assay technology with establishment of a local reference range. Clinically, the commonest use for salivary cortisol is measuring late‐night salivary cortisol as a screening test for Cushings syndrome. Several studies have shown diagnostic sensitivities and specificities of over 90%, which compares very favourably with other screening tests for Cushings syndrome such as the 24‐h urinary‐free cortisol and the 1‐mg overnight dexamethasone suppression test. There are emerging roles for the use of salivary cortisol in diagnosing adrenal insufficiency, particularly in conditions associated with low cortisol–binding globulin levels, and in the monitoring of glucocorticoid replacement. Finally, salivary cortisol has been used extensively as a biomarker of stress in a research setting, especially in studies examining psychological stress with repeated measurements.

One-step homogeneous C-reactive protein assay for saliva.

BACKGROUNDnCardiovascular disease is the leading cause of death in the world. Human C-reactive protein (CRP) has been used in the risk assessment of coronary events. Human saliva mirrors the bodys health and well-being and is non-invasive, easy to collect and ideal for third world countries as well as for large patient screening. The aim was to establish a saliva CRP reference range and to demonstrate the clinical utility of salivary CRP levels in assessing the coronary events in a primary health care setting.nnnMETHODSnWe have used a homogeneous bead based assay to detect CRP levels in human saliva. We have developed a rapid 15 min (vs 90 min), sequential, one-step assay to detect CRP in saliva. Saliva was collected from healthy volunteers (n=55, ages 20-70 years) as well as from cardiac patients (n=28, ages 43-86 years).nnnRESULTSnThe assay incubation time was optimised from 90 min to 15 min and generated a positive correlation (n=29, range 10-2189 pg/mL, r2=0.94; Passing Bablok slope 0.885, Intercept 0, p>0.10), meaning we could decrease the incubation time and produce equivalent results with confidence. The mean CRP level in the saliva of healthy human volunteers was 285 pg/mL and in cardiac patients was 1680 pg/mL (p<0.01). Analysis of CRP concentrations in paired serum and saliva samples from cardiac patients gave a positive correlation (r2=0.84, p<0.001) and the salivary CRP concentration capable of distinguishing healthy from diseased patients.nnnCONCLUSIONSnThe results suggest that this minimally invasive, rapid and sensitive assay will be useful in large patient screening studies for risk assessment of coronary events.

The impact of saliva collection and processing methods on CRP, IgE, and Myoglobin immunoassays

BackgroundOwing to its ease of collection, saliva is potentially the sample of choice in diagnosis. Salivary biomolecules have provided a porthole in surveying a person’s health and well-being. Our study aims were (1) to demonstrate the effects of pre-analytical steps, collection and pre-processing techniques on salivary protein detection and (2) to establish an indication of salivary reference intervals for 3 biomolecules of clinical interest.MethodsSaliva samples were collected from participants (nu2009=u200925, ages 20–35 years) using the following methods: no stimulation (resting/unstimulated), mechanical, and acid stimulation. The saliva was prepared for analysis by: unprocessed, post standard centrifugation in a container without any additives, and centrifugation using Centrifugal Filter Unit (Amicon® Ultra-0.5). AlphaLisa® assays were used to measure the levels of C-Reactive Protein (CRP), Immunoglobin (IgE) and myoglobin in saliva samples.ResultsSaliva flow rates were lowest with the resting/drooling collection method. The lowest total protein concentration was with acid stimulation. Unstimulated and mechanically stimulated collections produced no effect on the CRP and IgE levels while myoglobin levels were highest with the unstimulated collection. Acid stimulation had a negative impact on the measured concentrations of IgE and myoglobin (except for CRP levels).ConclusionMechanical stimulation was the most viable option for collecting saliva without affecting the levels of CRP and myoglobin. The processing methods had an adverse effect on the concentration of total protein as well as on CRP and IgE concentrations.

Correction and reporting of potassium results in haemolysed samples

Background: Potassium is usually the most important analyte affected by in vitro haemolysis and the result obtained may falsely indicate or disguise a life-threatening abnormality and so give rise to inappropriate treatment. The purpose of the study was to provide a solution to the problem of reporting potassium on haemolysed samples, taking into account both clinical needs and analytical concerns (inter-individual and inter-sample variability). Methods: Using a new procedure that mimics the collection process in an actual clinical setting, haemolysed samples were prepared from 41 volunteers with a range of inter-individual factors - haemoglobin 80-173 g/L, red blood cells 2.42-6.77 x 1012/L, leucocytes 3.0-306 x 109 /L and platelets 31-710 x 109/L - in order to develop a more accurate correction equation using a haemolytic index (HI) corresponding to g Hb/L in plasma. Results: The mean (range) potassium increase was 0.0036 mmol/L (0.0029-0.0053 mmol/L) per unit HI. The following equation was developed to estimate potassium increase per HI, in order to compensate approximately for potassium leakage in haemolysed samples: Corrected K+=Measured K+ -(HI x 0.004). Conclusion: The balanced solution is this: instead of reporting the post-haemolysis corrected potassium result a qualitative comment is given, indicating the likely range of the potassium concentration. If the potassium result is in a critically low or high range, it is communicated promptly to the requesting clinician.

Newer cardiac troponin I assays have similar performance to troponin T in patients with end-stage renal disease

Background: Troponin T is present in the blood of a majority of patients with endstage renal disease (ESRD) undergoing regular dialysis and presence of troponin T is a predictor of adverse outcome in these patients. With several new formulations of troponin I assays available, this study was performed to see whether these newer assays were able to detect troponin I in these patients more effectively than the older assays. Methods: One hundred and forty-three patients undergoing regular haemodialysis or peritoneal dialysis had plasma collected and troponin T and troponin I measured by a variety of assays. Results: The newer troponin I assays (Abbott Architect, Bayer Centaur and Beckman Accu-TnI) were able to detect troponin I (>75% of samples) as effectively as the Roche assay was able to detect troponin T, while other troponin I assays had a much lower rate of detection of troponin - DPC Immulite 2000 16% and Abbott AxSYM 35%. However, the troponin T assay had more samples detected at concentrations corresponding to an assay CV of 10% (59% of samples) than did the newer troponin I assays (highest on the Bayer Centaur at 37%). Conclusions: Newer assays demonstrate that troponin I is present in a similar number of samples as is troponin T, in the blood of patients with dialysis-dependent renal failure, and these newer troponin I assays identify patients at risk of experiencing a cardiac event.

SYNbiotics Easing Renal failure by improving Gut microbiologY (SYNERGY): a protocol of placebo-controlled randomised cross-over trial

BackgroundEmerging evidence suggests modulating the microbiota in the large bowel of patients with chronic kidney disease (CKD) through pre- and/probiotic supplementation may inhibit the development of key nephrovascular toxins. To date, quality intervention trials investigating this novel treatment in CKD are lacking. The aim of SYNERGY is to assess the effectiveness of synbiotics (co-administration of pre- and probiotics) as a potential treatment targeting the synthesis of uremic toxins, specifically, indoxyl sulphate (IS) and p-cresyl sulphate (PCS).Methods/designThirty-seven patients with moderate to severe CKD (Stage IV and V, pre-dialysis) will be recruited to a double-blind, placebo-controlled, randomised cross-over trial. Patients will be provided with synbiotic therapy or placebo for 6 weeks, with a 4u2009week washout before cross-over. The primary outcome is serum IS, total and free (unbound) concentrations, measured using ultra-performance liquid chromatography. Secondary outcomes include serum PCS, total and free (unbound) concentrations; cardiovascular risk, measured by serum lipopolysaccharides, serum trimethylamine-N-oxide (TMAO) and inflammation and oxidative stress markers; kidney damage, measured by 24u2009hour proteinuria and albuminuria, estimated glomerular filtration rate and renal tubule damage (urinary kidney injury molecule-1); patients’ self assessed quality of life; and gastrointestinal symptoms. In addition, the effects on the community structure of the stool microbiota will be explored in a subset of patients to validate the mechanistic rationale underpinning the synbiotic therapy.DiscussionIS and PCS are two novel uremic toxins implicated in both cardiovascular disease (CVD) and progression of CKD. Preliminary studies indicate that synbiotic therapy maybe a promising strategy when considering a targeted, tolerable and cost-efficient therapy for lowering serum IS and PCS concentrations. This trial will provide high quality ‘proof-of-concept’ data to elucidate both the efficacy of synbiotic therapy for lowering the toxins and whether reductions in serum IS and PCS translate into clinical benefits. Considering the potential of pre- and probiotics to not only shift toxin levels, but to also impede CVD and CKD progression, SYNERGY will provide vital insight into the effectiveness of this innocuous nutritional therapy.Trial RegistrationUniversal Trial Number: U1111-1142-4363. Australian New Zealand Clinical Trials Registry Number: ACTRN12613000493741, date registered: 2nd May 2013.

Disagreement between ion selective electrode direct and indirect sodium measurements: estimation of the problem in a tertiary referral hospital.

PURPOSEnWe estimated the proportion of indirect ion selective electrode (ISE) plasma sodium analyses in intensive care unit (ICU) and hospital wide, exhibiting important disagreement with direct ISE results in relation to abnormal plasma protein concentrations.nnnMATERIALS AND METHODSnDirect and indirect ISE plasma sodium measurements were performed on 346 clinical specimens selected to reflect low, normal, or high total protein concentrations. Important intermethod disagreement was defined as |4| mmol/L or higher. Results were extrapolated to a 3-month laboratory series of 48,033 indirect ISE assays, including 2877 samples from intensive care.nnnRESULTSnIntermethod sodium disagreement at |4| mmol/L or higher was predicted for 25% of ICU samples. Almost all (97%) occurred in hypoproteinemic samples where indirect tended to exceed direct ISE estimates. Hospital wide, such disagreement was projected to occur in 8% of samples, of which the majority (70%) were also hypoproteinemic.nnnCONCLUSIONSnImportant disagreement between indirect and direct ISE sodium measurements may exist in up to 1 in 4 ICU specimens and 1 in 12 hospital-wide samples. The main problem is indirect ISE overestimation associated with hypoproteinemia, potentially leading to misclassifications of pseudohypernatremia and pseudonormonatremia. We recommend that hospital laboratories consider standardization using direct ISE sodium measurement.

Outliers as a Cause of False Cardiac Troponin Results:Investigating the Robustness of 4 Contemporary Assays

BACKGROUNDnIt is important that cardiac troponin be measured accurately with a robust method to limit false results with potentially adverse clinical outcomes. In this study, we characterized the robustness of 4 analytical platforms by measuring the outlier rate between duplicate results.nnnMETHODSnWe measured cardiac troponin concurrently in duplicate with 4 analyzers on 2391 samples. The outliers were detected from the difference between duplicate results and by calculating a z value: z = (result 1 - result 2) ÷ √(SD1(est)² + SD2(est)²), with z > 3.48 identifying outliers with a probability of 0.0005.nnnRESULTSnThe outlier rates were as follows: Abbott Architect i2000SR STAT Troponin-I, 0.10% (0.01%-0.19%); Beckman Coulter Access2 Enhanced AccuTnI, 0.44% (0.25%-0.63%); Roche Cobas e601 TroponinT hs, 0.06% (0.00%-0.13%); and Siemens ADVIA Centaur XP TnI-Ultra, 0.10% (0.01%-0.19%). The occurrence of outliers was higher than statistically expected on all platforms except the Cobas e601 (χ² = 2.7; P = 0.10). A conservative approach with a constant 10% CV and z > 5.0 identified outliers with clear clinical impact and resulted in outlier rates of 0.11% (0.02%-0.20%) with the Architect i2000SR STAT Troponin-I, 0.36% (0.19%-0.53%) with the Access2 Enhanced AccuTnI, 0.02% (0.00%-0.06%) with the Cobas e601 TroponinT hs, and 0.06% (0.00%-0.13%) with the ADVIA Centaur XP TnI-Ultra.nnnCONCLUSIONSnOutliers occurred on all analytical platforms, at different rates. Clinicians should be made aware by their laboratory colleagues of the existence of outliers and the rate at which they occur.

NT-ProBNP Levels in Saliva and Its Clinical Relevance to Heart Failure

Background Current blood based diagnostic assays to detect heart failure (HF) have large intra-individual and inter-individual variations which have made it difficult to determine whether the changes in the analyte levels reflect an actual change in disease activity. Human saliva mirrors the body’s health and well being and ∼20% of proteins that are present in blood are also found in saliva. Saliva has numerous advantages over blood as a diagnostic fluid which allows for a non-invasive, simple, and safe sample collection. The aim of our study was to develop an immunoassay to detect NT-proBNP in saliva and to determine if there is a correlation with blood levels. Methods Saliva samples were collected from healthy volunteers (nu200a=u200a40) who had no underlying heart conditions and HF patients (nu200a=u200a45) at rest. Samples were stored at −80°C until analysis. A customised homogeneous sandwich AlphaLISA(R) immunoassay was used to quantify NT-proBNP levels in saliva. Results Our NT-proBNP immunoassay was validated against a commercial Roche assay on plasma samples collected from HF patients (nu200a=u200a37) and the correlation was r2u200a=u200a0.78 (p<0.01, yu200a=u200a1.705× +1910.8). The median salivary NT-proBNP levels in the healthy and HF participants were <16 pg/mL and 76.8 pg/mL, respectively. The salivary NT-proBNP immunoassay showed a clinical sensitivity of 82.2% and specificity of 100%, positive predictive value of 100% and negative predictive value of 83.3%, with an overall diagnostic accuracy of 90.6%. Conclusion We have firstly demonstrated that NT-proBNP can be detected in saliva and that the levels were higher in heart failure patients compared with healthy control subjects. Further studies will be needed to demonstrate the clinical relevance of salivary NT-proBNP in unselected, previously undiagnosed populations.