Passing-Bablok Regression Analysis Research Articles

Liquid Chromatography-Tandem Mass Spectrometry Method for Determination of Gentamicin Concentrations and Comparison with Two Immunoassay Methods (Chemiluminiscent Assay a Polarization Fluoroimmunoassay)

Gentamicin belongs to aminoglycoside antibiotics. Therapeutic drug monitoring (TDM) of gentamicin is recommended for all patients to assure both sufficient and safe concentrations in blood. Gentamicin is composed from a mixture of three drugs (C1, C1A and C2A+C2) with the basic share as follows: C1 25-50%, C1A 10-35% and C2A+C2 25-55%. Gentamicin concentrations are preferably measured using immunoanalytical assays. A new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with simple sample extraction and a relatively short time of gentamicin analysis was developed and validated. 50 µL of serum was precipitated using acetonitrile and formic acid. A RP BEH C18, 1.7 µm, 2.1 x 50 mm column maintained at 30oC and tobramycin as internal standard were used. Mass detection was performed in positive electrospray mode. The results were compared with analyses of polarization fluoroimmunoassay (FPIA) method (Abbott AxSYM) and a chemiluminiscent assay (Advia Centauer, Siemens). Passing-Bablok regression analysis and Bland-Altman analysis were used to compare gentamicin concentrations. Calibration curve was linear in the range of 0.1-50 mg/L. Recovery was 91.6-102.0% with a coefficient of variations of 1.4-8.4% for single types of gentamicin. The sum of gentamicin concentrations correlated significantly with both immunoanylatical assays. Presented LC-MS/MS method is fast and precise with simple sample extraction and can be applied for routine TDM of gentamicin.

EVALUATION OF THE I-STAT PORTABLE CLINICAL ANALYZER FOR MEASUREMENT OF IONIZED CALCIUM AND SELECTED BLOOD CHEMISTRY VALUES IN ASIAN ELEPHANTS (ELEPHAS MAXIMUS).

Thei-STAT® portable clinical analyzer (PCA) provides patient-side results for hematologic, biochemical, and blood gas values when immediate results are desired. This analyzer is commonly used in nondomestic animals; however, validation of this method in comparison with traditional benchtop methods should be performed for each species. In this study, the i-STAT PCA was compared with the Radiometer ABL 800 Flex benchtop analyzer using 24 heparinized whole blood samples obtained from healthy E. maximus . In addition, the effect of sample storage was evaluated on the i-STAT PCA. Analytes evaluated were hydrogen ion concentration (pH), glucose, potassium (K+), sodium (Na+), bicarbonate (HCO3-), total carbon dioxide (TCO2), partial pressure of carbon dioxide (PCO2), and ionized calcium (iCa2+). Statistical analysis using correlation coefficients, Passing-Bablok regression analysis, and Bland-Altman plots found good agreement between results from samples run immediately after phlebotomy and 4 hr postsampling on the i-STAT PCA with the exception of K+, which is known to change with sample storage. Comparison of the results from the two analyzers at 4 hr postsampling found very strong or strong correlation in all values except K+, with statistically significant bias in all values except glucose and PCO2. Despite bias, mean differences assessed via Bland-Altman plots were clinically acceptable for all analytes excluding K+. Within the reference range for iCa2+, the iCa2+ values obtained by the i-STAT PCA and Radiometer ABL 800 Flex were close in value, however in light of the constant and proportionate biases detected, overestimation at higher values and underestimation at lower values of iCa2+ by the i-STAT PCA would be of potential concern. This study supports the use of the i-STAT PCA for the evaluation of these analytes, with the exception of K+, in the Asian elephant.

Measuring Ventilatory Activity with Structured Light Plethysmography (SLP) Reduces Instrumental Observer Effect and Preserves Tidal Breathing Variability in Healthy and COPD.

The use of a mouthpiece to measure ventilatory flow with a pneumotachograph (PNT) introduces a major perturbation to breathing (“instrumental/observer effect”) and suffices to modify the respiratory behavior. Structured light plethysmography (SLP) is a non-contact method of assessment of breathing pattern during tidal breathing. Firstly, we validated the SLP measurements by comparing timing components of the ventilatory pattern obtained by SLP vs. PNT under the same condition; secondly, we compared SLP to SLP+PNT measurements of breathing pattern to evaluate the disruption of breathing pattern and breathing variability in healthy and COPD subjects. Measurements were taken during tidal breathing with SLP alone and SLP+PNT recording in 30 COPD and healthy subjects. Measurements included: respiratory frequency (Rf), inspiratory, expiratory, and total breath time/duration (Ti, Te, and Tt). Passing-Bablok regression analysis was used to evaluate the interchangeability of timing components of the ventilatory pattern (Rf, Ti, Te, and Tt) between measurements performed under the following experimental conditions: SLP vs. PNT, SLP+PNT vs. SLP, and SLP+PNT vs. PNT. The variability of different ventilatory variables was assessed through their coefficients of variation (CVs). In healthy: according to Passing-Bablok regression, Rf, TI, TE and TT were interchangeable between measurements obtained under the three experimental conditions (SLP vs. PNT, SLP+PNT vs. SLP, and SLP+PNT vs. PNT). All the CVs describing “traditional” ventilatory variables (Rf, Ti, Te, Ti/Te, and Ti/Tt) were significantly smaller in SLP+PNT condition. This was not the case for more “specific” SLP-derived variables. In COPD: according to Passing-Bablok regression, Rf, TI, TE, and TT were interchangeable between measurements obtained under SLP vs. PNT and SLP+PNT vs. PNT, whereas only Rf, TE, and TT were interchangeable between measurements obtained under SLP+PNT vs. SLP. However, most discrete variables were significantly different between the SLP and SLP+PNT conditions and CVs were significantly lower when COPD patients were assessed in the SLP+PNT condition. Measuring ventilatory activity with SLP preserves resting tidal breathing variability, reduces instrumental observer effect and avoids any disruptions in breathing pattern induced by the use of PNT-mouthpiece-nose-clip combination.

New liquid chromatography-tandem mass spectrometry method for routine TDM of vancomycin in patients with both normal and impaired renal functions and comparison with results of polarization fluoroimmunoassay in light of varying creatinine concentrations

A new LC-MS/MS method with simple sample extraction and a relatively short period of vancomycin analysis for routine therapeutic drug monitoring was developed and validated. 50μL serum was precipitated using 20μL 33% trichloroacetic acid and 0.5mol/L NH4OH was added to increase pH before analysis. A RP BEH C18, 1.7μm, 2.1×50mm column maintained at 30°C and tobramycin as internal standard were used. Mass detection was performed in positive electrospray mode.The results obtained with LC-MS/MS method were correlated with an FPIA assay (Abbott AxSYM) using mouse monoclonal antibody. Subjects were divided into three groups according to creatinine levels (53.5±19.1, 150.2±48.4, 471.7±124.7μmol/L) and Passing-Bablok regression analysis and Bland-Altman analysis were used to compare vancomycin concentrations. The results of subjects with both normal and higher creatinine levels correlated very well and the linear regression model equations were near ideal (LC-MSVAN=0.947×AbbottVAN+0.192 and LC-MSVAN=0.973×AbbottVAN−0.411 respectively). Dialyzed patients with the highest creatinine levels showed about 14% greater vancomycin concentration with the FPIA assay (LC-MSVAN=0.866×AbbottVAN+2.127). This overestimation probably due to the presence of the metabolite CDP ought not to be of clinical relevance owing to the wide range of recommended vancomycin concentration.

Enzyme-Linked Immunoassay-Based Quantitative Measurement of Apolipoprotein B (ApoB) in Dried Blood Spots, a Biomarker of Cardiovascular Disease Risk

ABSTRACTApolipoprotein B (ApoB) is a strong predictor of cardiovascular disease, which remains the leading cause of mortality in both higher and lower income countries. Here, we adapted an enzyme-linked immunosorbent assay (ELISA) development kit for quantitative determination of ApoB levels in serum and plasma for use with dried blood spots (DBS). After confirming the dilution linearity of the assay for DBS, we measured ApoB in 208 venous DBS samples. Then, using Passing-Bablok regression analysis and Spearman rank correlation analysis, we evaluated the correspondence in ApoB values between matched plasma and finger-prick DBS samples from 40 individuals who had ApoB values spanning the range of ApoB values observed in the 208 vDBS samples. We also evaluated assay precision and recovery, the effects of hematocrit, number of freeze-thaw cycles, and different storage temperatures on ApoB levels in DBS. There was a strong, significant correlation between plasma and DBS ApoB levels with little bias. Assay precision and recovery were within the range recommended by the U.S. government’s industry guidelines for bioanalytical assay validation. The assay was not affected by the DBS matrix or physiological hematocrit levels. This DBS-based ELISA assay will facilitate population-scale assessment of cardiovascular risk in previously unexplored populations.

Clinical performance of cardiac Troponin I: A comparison between the POCT AQT90 FLEX and the Dimension Vista analyzer in an emergency setting

BackgroundCardiac troponin [cTn (I or T)] is the preferred biomarker for the diagnosis of myocardial infarction (MI). AimWe studied the analytical performance of the POCT AQT90 FLEX cTnI assay and its diagnostic accuracy, in comparison to the Dimension Vista cTnI method, in patients presenting to the Emergency Department (ED) with suspect of acute coronary syndrome (ACS). Methods786 consecutive patients were enrolled. cTnI was measured at admission to the ED and about 3 and 6 hours later. The imprecision study was carried out using different lots of quality controls (QCs). ROC curve analysis was conducted using discharge diagnoses in order to verify the global diagnostic accuracy. ResultsThe concentrations measured in the QCs ranging from 0.033 to 1.26μg/L show CVs% ranging from 2.81 to 7.56%, comparable to those declared by the manufacturer. Passing-Bablok and linear regression analysis show a high significant correlation (R2=0.90, p<0.0001); Bland-Altman test describes a statistically significant negative bias (Bias=−0.2336; 95%CI=−0.4217/−0.0456, p=0.0150). ROC curves obtained using Dimension Vista and AQT90 FLEX cTnI assays displayed similar clinical performance being not statistically significant the difference of the corresponding AUC. Comparing sensitivity and specificity of cTnI concentrations obtained from the ROC curve analysis using AQT90 FLEX, we found a “best cut-off” (0.014μg/L) lower than that declared from the manufacturer (0.023μg/L). ConclusionsThe comparison of two different assays of cTnI against a diagnosis of acute MI (AMI) shows that both assays behave equally well with a high degree of sensitivity and specificity. The resulting “best cut-off” suggests that this AQT90 FLEX cTnI concentration could be evaluated as the potentially new “clinically usable” cut-off for AMI/myocardial necrosis diagnoses.

An LC-MS Assay with Isocratic Separation and On-line Solid Phase Extraction to Improve the Routine Therapeutic Drug Monitoring of Busulfan in Plasma.

SummaryBackgroundBusulfan (Bu) requires therapeutic drug monitoring (TDM) in subjects undergoing a conditioning regimen for hematopoietic stem cell transplantation (HSCT). To speed up the procedure and increase reproducibility, we improved our routine LC-MS/MS assay using the on-line solid-phase extraction (SPE) of samples.MethodsA protein precipitation (PP) step was performed before the on-line SPE of Bu from 200 µL of plasma spiked with octa-deuterated Bu (D8-Bu) as the internal standard. Bias was assessed with respect to our routine LC-MS/MS Bu assay with off-line extraction using the Passing-Bablok robust regression. Root cause of bias for individual samples was assessed by analyzing the regression residuals.ResultsThe method was linear in the range 37.75–2,416 ng/mL (r2>0.999), with 19.74 ng/mL LLOQ and 10.5% CV at 20 ng/mL. Precision and accuracy were both within ±5%, and neither appreciable matrix nor carryover effects were observed. The Passing-Bablok regression analysis returned a 0.99 slope (95% CI: 0.97 to 1.01) and –6.82 intercept (95% CI: –15.23 to 3.53). Residuals analysis against the 2.5th–97.5th percentiles range showed four samples with significant bias individually.ConclusionsThe method presented can be successfully employed for the routine analysis of Bu in plasmatic samples, and can replace the LC-MS/MS method with off-line extraction without any statistically significant overall bias. In this regard, samples with individual significant bias were reasonably produced by preanalytical issues which had no relation with the conversion to the on-line SPE extraction.

The Roche Total Mycophenolic Acid® assay: An application protocol for the ABX Pentra 400 analyzer and comparison with LC–MS in children with idiopathic nephrotic syndrome

BackgroundFor TDM of mycophenolate acid (MPA), the Roche Total Mycophenolic Acid® assay based on the inhibition of recombinant inosine monophosphate dehydrogenase (IMPDH) has been shown to be a simple and reliable alternative to chromatographic methods. We have adapted this assay on the ABX Pentra 400 analyzer (HORIBA). ObjectiveTo investigate the analytical performances of the Roche Total Mycophenolic Acid® assay on the ABX Pentra 400 and to compare it to an LC-MS method using samples from children with nephrotic syndrome treated with mycophenolate mofetil (MMF). Material and methodsConfiguration of the open-channel on the ABX Pentra 400 was based on the Roche MPA assay package insert. Precision was determined as described in the CLSI protocol EP5-A2. Comparison with the LC-MS method was performed using 356 plasma samples from 42 children with nephrotic syndrome (8h pharmacokinetic profiles). ResultsThe enzymatic assay demonstrated high precision. The %CV for Within Run Imprecision ranged from 5.5% at 1.2mg/L to 1.5% at 14.1mg/L and Total Imprecision ranged from 9.3% to 2.5%. The method comparison with plasma samples from children yielded overall a good correlation and a good agreement between both methods. The Passing Bablok regression analysis showed the following results: [Roche MPA assay]=1.058 [MPA LC-MS] −0.06; rho=0.996. ConclusionThe Roche Total Mycophenolic Acid® assay is adaptable to the ABX Pentra 400 analyzer, and demonstrates accurate and precise measurement of MPA in plasma obtained from children with nephrotic syndrome.

Inter-Method Variability of Ferritin and Transferrin Saturation Measurement Methods in Patients on Hemodialysis.

Serum ferritin level and transferrin saturation (TSAT) are widely used to evaluate iron status in patients with chronic kidney disease, and are also important variables for performing statistical analyses. Many guidelines have set control targets or upper limits for these markers. Inter-method variability is an important consideration in iron control and statistical analysis. We used 10 ferritin assay kits and five iron/unsaturated iron-binding capacity/total iron-binding capacity assay kits to determine ferritin levels and TSAT in 114 patients on maintenance dialysis, and evaluated measurement bias using Passing-Bablok regression analyses. The variance of distributions categorized by differences in assay kits was examined using Fisher's exact test. Slopes ranged from 1.00 to 1.63 (1.00 to 0.61) for ferritin and 1.00 to 1.10 (1.00 to 0.91) for TSAT. The distribution according to the 2015 JSDT Guideline for Renal Anemia in Chronic Kidney Disease significantly changed (P = 0.01). TSAT thus provides more precise control than ferritin in multi-center comparisons where no particular assay is specified. Developers must reduce variability in serum ferritin assay kits. Researchers must analyze measured values by taking into account the propagation of errors, and clinicians must evaluate laboratory data carefully.

Comparability of the effect of storage time and temperature on serum anti-Müllerian hormone measurement between original and modified enzyme-linked immunosorbent assay

ObjectiveTo explore the effect of modified enzyme-linked immunosorbent assay on the AMH results is increased or decreased, and to investigate the effect of storage time and temperature on AMH measurements with and without sample premixing assay buffer using the Kangrun ELISA method. MethodSerum AMH concentration were measured by ELISA, consistency between two kits, and comparability between original and the modified assay under different stored conditions were analyzed by Passing-Bablok regression analysis and Bland-Altman bias evaluation. ResultThere was a strong consistency between AMH concentrations measured in Kangrun ELISA and Ansh Labs ultra-sensitive AMH ELISA. Pre-mixing serum specimens with assay buffer gave consistent results compared with original assay. Modified protocol can reduce the amplitude of increase affected by sample aged and give the most consistent results regardless of storage conditions. ConclusionPre-mixing protocol did not influence the results of fresh serum or frozen serum incubation <3days at 4°C and −80°C, but when specimens detected after collection and stored in other storage conditions, should be pre-mixed with assay buffer to insure its accuracy.

Analysis of amino acids in human blood using UHPLC-MS/MS: Potential interferences of storage time and vacutainer tube in pre-analytical procedure

ObjectivesTo investigate potential interferences of two pre-analytical variables, the storage time and the vacutainer tube, on the quantification of 20 amino acids using a UHPLC-MS/MS method. Design and methodsBlood samples from 25 apparently healthy subjects were collected into duplicate sets of EDTA-2K, EDTA-3K, coagulation, heparin and citrate tubes, and stored in capped vacutainer tubes at 4°C for 6h, 12h and 24h before sample analysis. A UHPLC-MS/MS method was established for simultaneous quantification of 20 amino acids. ANOVA for repeated measurement was conducted based on the model of Mauchly's test of Sphericity. Student's t-test was applied for comparison between amino acid concentrations obtained from different vacutainer tubes, and consistency of the results was checked through Bland-Altman difference plots and Passing-Bablok regression analysis. ResultsMost of the 20 amino acids showed a least concentration fluctuation with storage time in heparin plasma, followed by EDTA-3K and citrate plasma. The amino acid concentrations were significantly lower in citrate plasma and slightly higher in serum, compared with those in heparin plasma. No fixed bias was observed for amino acid concentrations in EDTA and heparin plasma, but the differences were mostly of statistical significance. Amino acid concentrations in EDTA-3K plasma achieved a good consistency with those in heparin plasma by UHPLC-MS/MS analysis. ConclusionsStorage time and vacutainer tube were important variables for amino acid analysis. They should draw researchers' attention and then be controlled in good laboratory practice to reduce pre-analytical errors.

Boronate-Modified Interdigitated Electrode Array for Selective Impedance-Based Sensing of Glycated Hemoglobin

An impedance-based label-free affinity sensor was developed for the recognition of glycated hemoglobin (HbA1c). Interdigitated gold microelectrode arrays (IDAs) were first modified with a self-assembled monolayer of cysteamine followed by cross-linking with glutaraldehyde and subsequent binding of 3-aminophenylboronic acid (APBA), which selectively binds HbA1c via cis-diol interactions. Impedance sensing was demonstrated to be highly responsive to the clinically relevant HbA1c levels (0.1%-8.36%) with a detection and quantitation limit of 0.024% (3σ/slope) and 0.08% (10σ/slope), respectively. The specificity of the assay was evaluated with nonglycated hemoglobin (HbAo), showing that the impedance response remained unchanged over the concentration range of 10 to 20 g dL-1 HbAo. This demonstrated that the sensor system could be used to specifically distinguish HbA1c from HbAo. Moreover, the binding of HbA1c to the APBA-modified electrodes was reversible, providing a reusable sensing interface as well as showing a stable response after 4 weeks (96% of the initial response). When assaying normal (4.10%) and diabetic (8.36%) HbA1c levels (10 assays per day during a three-day period including a regeneration step after each assay), the overall assay reproducibility, expressed as relative standard error of the mean (n = 30), was 1.1%. The performance of the sensor system was also compared with a commercial method (n = 15) using patient-derived blood samples. A good agreement (Bland-Altman bias plot) and correlation (Passing-Bablok regression analysis) was demonstrated between the boronate-based affinity sensor and the standard method.

Analytical performance of the VERIS MDx system HCV assay for detecting and quantifying HCV RNA

BackgroundThe diagnosis of HCV relies on the detection of viral RNA. ObjectiveTo evaluate the performance of the VERIS/MDx System HCV Assay, a new automated system for quantifying HCV RNA, and to compare with the COBAS® Ampliprep/COBAS® Taqman™ (CAPCTM) HCV Test version 2.0. Study designThe limit of detection was determined by Probit analysis with the 3rd International WHO HCV standard and precision by assaying in duplicate control samples with HCV RNA concentrations of 7.9; 5.0; 3.4; 1.6 and 0logIU/ml over 20 days. Analytical specificity was assessed by assaying 180 samples from negative anti-HCV and HCV RNA blood donors and linearity with replicates of serial dilutions of a clinical plasma (6.4–0.6logIU/ml). We compared the VERIS MDx HCV and CAPCTM HCV assays by testing 209 samples. ResultsThe limit of detection was 6.1IU/ml [CI 95%: 5.0–8.3] and the precision, given by the standard deviation, was ≤0.11logIU/ml. Specificity was 100%. The linearity ranged from 1.5 to 6.4logIU/ml. Passing-Bablok regression analysis gave: VERIS logIU/ml=−0.33+[1.04× CAPCTM] logIU/ml, with biases for the 25th, 50th, 75th percentiles of 0.18, −0.10 and −0.06logIU/ml. The two assays were well correlated (ρ=0.92, p<0.001) and Bland-Altman analysis gave biases of 0.12, logIU/ml for genotype 1, −0.19 for genotype 2, −0.26 for genotype 3, and −0.77 for genotype 4. ConclusionThe VERIS MDx HCV assay performed well. But, we observed an under-quantification of the genotype 4 samples.

Prediction of fermentation index of cocoa beans (Theobroma cacao L.) based on color measurement and artificial neural networks

Several procedures are currently used to assess fermentation index (FI) of cocoa beans (Theobroma cacao L.) for quality control. However, all of them present several drawbacks. The aim of the present work was to develop and validate a simple image based quantitative procedure, using color measurement and artificial neural network (ANNs). ANN models based on color measurements were tested to predict fermentation index (FI) of fermented cocoa beans. The RGB values were measured from surface and center region of fermented beans in images obtained by camera and desktop scanner. The FI was defined as the ratio of total free amino acids in fermented versus non-fermented samples. The ANN model that included RGB color measurement of fermented cocoa surface and R/G ratio in cocoa bean of alkaline extracts was able to predict FI with no statistical difference compared with the experimental values. Performance of the ANN model was evaluated by the coefficient of determination, Bland-Altman plot and Passing-Bablok regression analyses. Moreover, in fermented beans, total sugar content and titratable acidity showed a similar pattern to the total free amino acid predicted through the color based ANN model. The results of the present work demonstrate that the proposed ANN model can be adopted as a low-cost and in situ procedure to predict FI in fermented cocoa beans through apps developed for mobile device.

Performance evaluation of an automated electrochemiluminescent calcitonin (CT) immunoassay in diagnosis of medullary thyroid carcinoma

ObjectivesThe aim was to evaluate the Elecsys® hCT electrochemiluminescence immunoassay on the immunoanalyser Cobas e411. Design and methodsWithin-run, between-run imprecision, linearity, recovery after dilution, the high dose hook effect, as well as the stability of calcitonin were examined. A method comparison to an established immunoradiometric assay was executed in 135 routine plasma samples. ResultsWithin- and between-run imprecision CVs were ≤1.4% and ≤2.5%, respectively. The Elecsys® hCT assay was linear over the measuring range of 0.5–2000ng/L. Recoveries after dilution of a high sample in Diluent MultiAssay were within the range of 95% to 97%. The high dose hook point occurred at an hCT concentration of 56,000ng/L. Calcitonin was found to be stable up to 5h at RT, up to 24h at 2–8°C and up to one month at −20° and −80°C. Passing Bablok regression analysis showed no significant deviation from linearity (p=0.10) with a slope of 1.00 (95% CI 0.95–1.06) and an intercept of −0.81 (95%CI −1.01 to −0.46). ConclusionsThe Elecsys® hCT immunoassay provides precision and reliability in combination with reduced turnaround time as compared to the hCT IRMA.

Evaluation of PIMATM CD4 System for Decentralization of Immunological Monitoring of HIV-Infected Patients in Senegal.

BackgroundHIV infection is a concern in the army troupes because of the risk behaviour of the military population. In order to allow regular access to CD4+ T cell enumeration of military personnel as well as their dependents and civilians living with HIV, the Senegalese Army AIDS program is implementing PIMATM Alere technology in urban and semi-urban military medical centres. Validation such device is therefore required prior their wide implementation. The purpose of this study was to compare CD4+ T cell count measurements between the PIMATM Alere to the BD FACSCountTM.MethodologyWe selected a total of 200 subjects including 50 patients with CD4+ T-cells below 200/mm3, 50 between 200 and 350/mm3, 50 between 351 and 500/mm3, and 50 above 500/mm3. CD4+ T-cell count was performed on venous blood using the BD FASCountTM as reference method and the PIMATM Point of Care technology. The mean biases and limits of agreement between the PIMATM Alere and BD FACSCountTM were assessed with the Bland-Altman analysis, the linear regression performed using the Passing-Bablok regression analysis, and the percent similarity calculated using the Scott method.ResultsOur data have shown a mean difference of 22.3 cells/mm3 [95%CI:9.1–35.5] between the BD FACSCountTM and PIMATM Alere CD4 measurements. However, the mean differences of the two methods was not significantly different to zero when CD4+ T-cell count was below 350/mm3 (P = 0.76). The Passing-Bablok regression in categorized CD4 counts has also showed concordance correlation coefficient of 0.89 for CD4+ T cell counts below 350/mm3 whilst it was 0.5 when CD4 was above 350/mm3.ConclusionOverall, our data have shown that for low CD4 counts, the results from the PIMATM Alere provided accurate CD4+ T cell counts with a good agreement compared to the FACSCountTM.

Commutability and interchangeability of commercial quality control materials with feline plasma for common biochemical analytes.

Species-specific plasma or serum pools are considered the ideal standard material for quality control materials (QCM) instead of commercially available human QCM. However, using plasma or serum pools is limited by volume restrictions, degradation over time, and a narrow range of analyte concentrations. Concentrations of QCM analytes should be consistent or commutable with those from species-specific plasma/serum samples, and the precision from plasma pools should be comparable or interchangeable with commercial human QCM. The aims of this study were to determine the commutability and interchangeability of 2 levels of commercial QCM (MAS chemTRAK-H [CT]) with feline plasma pools (PP) from normal and renal disease cats measured using a commercial laboratory analyzer and a veterinary in-house analyzer. Agreement between the 2 analyzers was assessed for 16 analytes by correlation and Passing-Bablok regression analyses of feline plasma samples. The difference between each CT data point and the regression line (residuals) was determined and standardized, and CT were considered 'commutable' with PP if the standardized residual was within a range of -3 to 3. Coefficients of variation (CVs) for CT and PP for 16 analytes on 2 analyzers were compared by bootstrap analysis to determine interchangeability. Most CT analytes were within the range of patient plasma sample analytes, thus commutable. Only 2 analytes had equivalent precision for both levels of CT and both levels of PP, and 5 additional analytes had similar precision for at least one level of CT compared to at least one level of PP. The QCM assessed is commutable to feline PP within the tested ranges for 2 particular analyzers. Commutability does not grant interchangeability.

Validation of commercially available ELISAs for the detection of circulating sclerostin in hemodialysis patients.

Sclerostin is an endocrine regulator in chronic kidney disease - mineral and bone disorder (CKD-MBD). Validation of assay comparability and pre-analytical handling is mandatory for establishment of sclerostin as a biomarker. Blood samples (serum, EDTA, heparin and citrate plasma) were obtained from 12 hemodialysis (HD) patients after the long dialysis interval. Passing-Bablok regression analysis and Bland-Altman difference plots were used to evaluate the agreement between sclerostin levels measured with two commercially available ELISAs from TECOmedical and Biomedica. Independent of the sample type, the agreement of the two assays was poor with a strong proportional but no systematic bias. Compared to the TECOmedical assay, the Biomedica test yielded almost 2-fold higher sclerostin values throughout all sample types. Spike recovery and linear dilution studies revealed a higher accuracy of the TECOmedical assay (97% and 96%) compared to the Biomedica assay (118% and 78%). Sclerostin levels were stable within 4 hours after sample collection, in particular when analyzed in plasma. In contrast to the Biomedica assay, the TECOmedical showed a systematic but no proportional bias between serum and plasma samples with higher values for plasma samples. Among the 3 different plasma samples no systematic error could be documented. Careful consideration of the pre-analytical handling and comparative assay validation are necessary to facilitate a more differentiated interpretation of studies reporting circulating sclerostin levels. The presence of a proportional bias demonstrates that in HD patients the two ELISAs for measuring sclerostin should not be used interchangeably. Furthermore, caution is necessary when comparing sclerostin results obtained from different blood sample types.

Heparin Reversal After Cardiopulmonary Bypass: Are Point-of-Care Coagulation Tests Interchangeable?

Protamine is used to neutralize heparin after patient separation from cardiopulmonary bypass (CPB). Different bedside tests are used to monitor the adequacy of heparin neutralization. For this study, the interchangeability of the activated coagulation time (ACT) and thromboelastometry (ROTEM; Tem Innovations GmbH, Basel, Switzerland) clotting time (CT) ratios in children undergoing cardiac surgery was assessed. Single-center, retrospective, cohort study between September 2010 and January 2012. University children's hospital. The study comprised children 0 to 16 years old undergoing elective cardiac surgery with CPB. Exclusion criteria were preoperative coagulopathy, Jehovah's witnesses, and children in a moribund condition (American Society of Anesthesiologists score 5). None. After heparin neutralization with protamine, the ratio between ACT, with and without heparinase, and the CT measured with INTEM/HEPTEM (intrinsic test activated with ellagic acid was performed without heparinase [INTEM] and with heparinase [HEPTEM]) using tests of ROTEM were calculated. Agreement was evaluated using Cohen's kappa statistics, Passing-Bablok regression, and Bland-Altman analysis. Among the 173 patients included for analysis, agreement between both tests showed a Cohen's kappa statistic of 0.06 (95% CI: -0.02 to 0.14; p = 0.22). Bland-Altman analysis showed a bias of 0.01, with a standard deviation of 0.13, and limits of agreement between -0.24 and 0.26. Passing-Bablok regression showed a systematic difference of 0.40 (95% CI: 0.16-0.59) and a proportional difference of 0.61 (95% CI: 0.42-0.86). The residual standard deviation was 0.11 (95% CI: -0.22 to 0.22), and the test for linearity showed p = 0.10. ACT, with or without heparinase, and the INTEM/HEPTEM CT ratios are not interchangeable to evaluate heparin reversal after pediatric patient separation from CPB. Therefore, the results of these tests should be corroborated with the absence/presence of bleeding and integrated into center-specific treatment algorithms.

COMPARISON OF TOTAL LEUKOCYTE QUANTIFICATION METHODS IN FREE-LIVING GALAPAGOS TORTOISES (CHELONOIDIS SPP.).

Reptile hematologic data provide important health information for conservation efforts of vulnerable wildlife species such as the Galapagos tortoise (Chelonoidis spp.). Given the reported discrepancies between manual leukocyte counts for nonmammalian species, two manual leukocyte quantification methods, the Natt and Herrick's (NH) and the Eopette (EO), were compared to white blood cell (WBC) estimates from blood films of 42 free-living, clinically healthy, adult female Galapagos tortoises. To investigate the effects of delay in sample processing, estimated WBC counts and leukocyte differentials were compared for blood films prepared at time of collection under field conditions (T0) to blood films prepared from samples that were stored for 18-23 hr at 4°C in the laboratory (T1). Passing-Bablok regression analysis revealed no constant or proportional error between the NH and WBC estimates (T0 and T1) with slopes of 1.1 and 0.9, respectively. However both constant and proportional errors were present between EO and WBC estimates (T0 and T1) with slopes of 3.1 and 2.7, respectively. Bland Altman plots also showed agreement between the NH and WBC estimates where the points fell within the confidence-interval limit lines and were evenly distributed about the mean. In contrast, the EO and WBC estimate comparisons showed numerous points above the upper limit line, especially at higher concentrations. WBC estimates obtained from T0 and T1 films were in agreement, whereas heterophil and monocyte percentages based on differentials were not. Cell morphology and preservation were superior in T0 blood films because thrombocytes exhibited swelling after storage, becoming difficult to differentiate from lymphocytes. In this study, the highest quality and most reliable hematologic data in Galapagos tortoises were obtained by combining immediate blood film preparation with the NH leukocyte quantification method and a confirmatory WBC estimate from the blood film.