Faster HbA1c Turnaround Times Using a Dedicated Analyzer Based on Turbidimetric Inhibition Immunoassay Technology

Introduction The Roche Cobas c513 (c513) is a dedicated stand-alone high throughput HbA1c analyzer. We evaluated the performance and the difference in turnaround times (TAT) of the c513 against our Cobas 8000 c502 (c502). Methods We assessed the linearity and precision of the c513, and its agreement (Deming regression and Bland–Altman analysis) with the c502 assay. We compared TAT for these analyzers for a single run of 50 samples and for all samples run over 2 comparable time periods. Results The c513 assay was linear from 4.4–18.3% HbA1c. Interassay precision (CV%) was 1.2 and 0.8 at HbA1c levels of 5.7 and 10.5%, respectively. The c513 assay showed excellent concordance with the c502 assay (r = 0.997) with no significant difference between methods by Bland–Altman analysis (mean difference = 0.021% HbA1c, P = 0.1422). The c513 took 17 min to analyze 50 samples, compared to 40 min on the c502. Over comparable 2-month periods, 90% of samples requiring HbA1c tests only were completed under 25 min (c513) vs 30–35 min (c502). For tubes sharing complete blood count (CBC) testing with HbA1c, the 90th percentile TAT was 35–40 min (c513) compared to 45–50 min (c502). Conclusion The c513 assay performs well with excellent correlation to the c502 assay. The improved TAT of the c513 is suitable when there are demands for rapid HbA1c results and it may forestall requests for point-of-care testing. It is also attractive to sites with heavy workloads with a claimed throughput of 400 tests / h.

Feline plasma adrenocorticotropic hormone: validation of a chemiluminescent assay and concentrations in cats with hypercortisolism, primary hypoadrenocorticism and other diseases

Objectives The aims of this study were to validate a commercially available chemiluminescent assay for measurement of feline plasma adrenocorticotropic hormone concentration (ACTH), to determine the normal reference interval (RI) of plasma ACTH in healthy cats, to assess plasma ACTH in cats with naturally occurring hypercortisolism (HC), primary hypoadrenocorticism (PH) and other diseases (OD), and to evaluate the effect of aprotinin on plasma ACTH degradation. Methods Forty healthy cats, 10 with HC, 11 with PH and 30 with OD, were included. The chemiluminescent enzyme immunometric assay was evaluated by measurement of intra-assay precision, interassay precision and linearity. The RI for plasma ACTH in healthy cats was established using robust methods. Plasma ACTH of samples collected with and without aprotinin, stored at 4°C and assayed over a 6-day period, was measured. Results The intra-assay coefficients of variance (CVs) ranged from 2.7% to 4.3% and interassay CVs from 3.3% to 10.7%. Dilution studies showed excellent accuracy (R2 >0.99). The RI for plasma ACTH in healthy cats was 32–370 pg/ml. Plasma ACTH was not significantly different between healthy cats and the OD group. Cats with pituitary-dependent hypercortisolism (PDH) and PH had significantly higher plasma ACTH than the other groups. Plasma ACTH did not show significant differences when samples collected with and without aprotinin were compared. Conclusions and relevance The Immulite chemiluminescent assay is a valid technique for measuring plasma ACTH in cats and the RI of plasma ACTH is quite wide. Owing to the low overlap between healthy or OD cats and cats with HC or PH, the measurement of plasma ACTH appears to be useful and should be included in the diagnostic work-up when HC or PH are suspected. Furthermore, the measurement of plasma ACTH may be an accurate test for differentiating PDH from adrenal-dependent hypercortisolism.

Determination of apatinib and its three active metabolites by UPLC–MS/MS in a Phase IV clinical trial in NSCLC patients

Aim: To develop and validate a simple method using UPLC–MS/MS for determination of apatinib and its three active metabolites in a Phase IV clinical trial. Materials & methods: All compounds were separated on a Hypersil GOLD™ aQ C18 Polar Endcapped LC column (50 × 2.1 mm, 1.9 μm, Thermo) using 5 mmol/l ammonium acetate with 0.1% formic acid:acetonitrile (20:80, v/v) as the mobile phase after a rapid liquid–liquid extraction. This method was validated over the linear concentration range of 1.00–1000 ng/ml for each compound. Results: The interassay precision and accuracy were less than ±15%. The validated method was successfully applied to determine concentrations of clinical samples in non-small-cell lung cancer patients.

Implementation of a Fully Automated Latex Immunoturbidimetric Assay for the Diagnosis of Heparin-Induced Thrombocytopenia in a High-Volume University Hospital Laboratory

Background Heparin-induced thrombocytopenia (HIT) is an immune-mediated, adverse reaction to heparin in which heparin binds platelet factor 4 (PF4), triggering the development of heparin-PF4 antibodies (HITAb). HITAbs bind and activate platelets, causing thrombosis, platelet consumption, and thrombocytopenia. Heparin is replaced with relatively costly nonheparin anticoagulants until HIT can be ruled out. HIT diagnosis consists of a HITAb immunoassay with reflex to a serotonin release assay (SRA) for confirmation of positive results. Recently, a fully automated latex immunoturbidimetric assay (LIA) for detection of HITAbs received FDA clearance. We sought to verify the performance characteristics of the LIA with the aim of implementing the test in a high-volume university hospital laboratory. Methods The in-house HITAb LIA was performed on the Instrumentation Laboratory TOP700 analyzer using HemosIL HIT-Ab(PF4-H) reagent. The comparator method, a HITAb ELISA, was performed at a reference laboratory with positive results reflexed to SRA. All samples (36 total) sent to the reference laboratory for HIT testing from December 2017 to November 2018 were aliquoted and run in parallel by LIA. Intra-assay precision was assessed by running manufacturer-provided low and high control samples 10 times in succession, while interassay precision was assessed by running low and high samples every day for 10 days. Turnaround time to HITAb result was retrieved from the electronic medical records for HIT testing performed 60 days before and after in-house test implementation. Results The agreement between the LIA and ELISA was 92% (33/36). One discordant sample tested negative by ELISA and was not assessed by SRA. Another tested positive by ELISA and negative by LIA and was confirmed negative by SRA. The final discordant sample tested negative by LIA but positive by ELISA and was confirmed positive by SRA. Thirty-three percent (12/36) of samples tested positive for HITAb by ELISA and were reflexed to SRA. Both the ELISA and LIA showed 83% agreement (10/12) with the SRA. The coefficient of variance (CV) for the intra-assay precision studies was 18% and 5% for the low and high controls, respectively. The CV for the interassay precision studies was 28% and 5% for the low and high controls, respectively. Postimplementation quality control data revealed 61% and 20% imprecision on the low and high level controls, respectively, which declined significantly when reagents were removed from the instrument and refrigerated within 2 hours. The turnaround time for HITAb results was reduced by 74% (10.5 vs 41 hours) after in-house test implementation, significantly reducing the need for administration of nonheparin anticoagulants. Conclusion The LIA and ELISA methods compared favorably, allowing for clinical implementation of the LIA. The shortened turnaround time of the LIA significantly reduced the time to rule out HIT, enhancing patient care and reducing drug costs. The assay imprecision warrants further investigation regarding reagent stability.

Comparison of the Abbott Architect BRAHMS and the Biomérieux Vidas BRAHMS Procalcitonin Assays

Background Procalcitonin (PCT) is a well-established marker for bacterial infection. Recently the US Food and Drug Administration approved the expanded use of this biomarker to guide clinical decisions for antibiotic treatment in patients with lower respiratory tract infections. Both the Architect BRAHMS PCT (PCT-A) and Vidas BRAHMS PCT (PCT-V) are approved for this indication. The aim of this study is to evaluate analytical performance of PCT-A in comparison to PCT-V. Methods PCT-A and PCT-V were evaluated for intra- and interassay precision and functional sensitivity. To assess the accuracy of PCT-A, 108 residual plasma specimens were randomly selected from routine hospital orders, and PCT was measured concurrently with PCT-A and PCT-V. Results Both assays demonstrated excellent precision, with intraassay precision ranging from 2.2% to 4.0% CV and interassay precision ranging from 2.5% to 3.6% CV. The functional sensitivity was verified at 0.01 ng/mL for PCT-A and at 0.05 ng/mL for PCT-V. The Passing–Bablok regression revealed approximately 20% negative bias of PCT-A compared to PCT-V (PCT-A = 0.042 + 0.79 × PCT-V, r = 0.995). The concordance of the 2 methods at diagnostically important cutoffs (0.10, 0.25, 0.50, and 2.0 ng/mL) was excellent, with overall agreement >93% at each threshold. Conclusion The results of our study show improved sensitivity and equivalent clinical performance of PCT-A compared to PCT-V. The availability of this test on common clinical immunoassay analyzers may help accelerate its adoption into antimicrobial stewardship programs and thereby improve antibiotic use and patient outcomes.

Development of a HPLC-DAD Method for the Simultaneous Quantification of 5-O-β-D-galactopyranosyl-7-methoxy-3’,4’-dihydroxy-4-phenylcoumarin and its Aglycone in Feces

A sensitive and specific HPLC-DAD method was devel-oped and validated for the simultaneous quantification of 5-O-β-D-ga-lactopyranosyl-7-methoxy-3’,4’-dihydroxy-4-phenylcoumarin (4-PC) and its aglycone in rat feces. The 4-phenylcoumarins are important antidiabetic and gastroprotective bioactive metabolites of a highly commercialized medicinal plant complex in Mexico, the Copalchi complex. Both the sample preparation and the quantification method were developed; the methodology allows, for the first time ever, the simultaneous determination of the 4-PC (Rt= 2.4 min) and its metabo-lized aglycone (Rt= 7.5 min), aimed to their pharmacokinetic analysis, specially their elimination. Linearity was determined in the range, 0.09–4.5 µg/mL for both compounds (R2= 0.9999). Accuracy was <15.0% for 4-PC and aglycone with an interassay precision of maxi-mum %RSD of 6.1% for 4-PC and 2.7% for the aglycone. The intraassay precision was %RSD < 7.5 for 4-PC and < 3.5% for the aglycone. Analyte recovery from spiked samples was always > 98.02% for 4-PC and > 96.61% for its aglycone. The method was successfully applied to a single-dose preclinical pharmacokinetics preliminary study in rats.

Analytical validation of an automated assay for ferric-reducing ability of plasma in dog serum

We performed analytical validation of an automated ferric-reducing ability of plasma (FRAP) assay in the serum of dogs. Intra- and interassay precision, accuracy, detection limit, and effects of hemolysis and lipemia were evaluated. Intra- and interassay coefficients of variation were <1% and <13%, respectively. The assay showed a high correlation with a FRAP assay described previously, and results were linear when serial sample dilutions were tested. The detection limit was lower than the values observed in sera from healthy dogs; decreased serum FRAP was found in dogs with leishmaniosis. Lipemia and hemolysis caused a significant increase in the results of the assay.

Precision and Reliability of 5 Platelet Function Tests in Healthy Volunteers and Donors on Daily Antiplatelet Agent Therapy

BACKGROUND Anticoagulation protocols used during mechanical circulatory support call for titration of antiplatelet agents. We compared the precision and reliability of 5 platelet function tests in healthy volunteers and donors on daily antiplatelet therapy to distinguish their efficacy for titrating antiplatelet therapy. METHODS We assessed arachidonic acid–induced platelet function by light transmission aggregometry (LTA), Multiplate impedance aggregometry, VerifyNow, and platelet mapping by thromboelastography (TEG PM). We assessed ADP-induced platelet function by the same methods and flow cytometry. Forty healthy volunteers and 10–13 volunteers on daily aspirin and/or clopidogrel therapy were evaluated. We compared tests for intraassay precision, interassay precision (samples from 2 separate blood draws), and reliability coefficient. RESULTS For arachidonic acid–induced platelet aggregation in healthy volunteers, intra- and interassay CVs were ≤10% for all methods. Intra- and interassay precision among donors on daily aspirin was ≤30% for all methods except LTA (38% interassay CV) and TEG PM (95% intraassay and 104% interassay CV). For ADP-induced platelet function, intra- and interassay precision was ≤10% and ≤30% for all methods. Only Multiplate demonstrated moderate or greater (R &gt; 0.40) reliability coefficients for arachidonic acid–induced platelet function among all subjects. All methods of ADP-induced platelet function, except TEG PM, demonstrated substantial or greater (R &gt; 0.60) reliability among all subjects. CONCLUSIONS TEG PM is least suited to monitor effects of antiplatelet agents. Multiplate impedance aggregometry was the only method to demonstrate an acceptable reliability coefficient among healthy volunteers and donors on both aspirin and clopidogrel therapy.

Development of a Fourfold Multiplexed Opsonophagocytosis Assay for Pneumococcal Antibodies against Additional Serotypes and Discovery of Serological Subtypes in Streptococcus pneumoniae Serotype 20

ABSTRACTOpsonophagocytic killing assays (OPAs) are importantin vitrosurrogate markers of protection in vaccine studies ofStreptococcus pneumoniae. We have previously reported the development of a 4-fold multiplexed OPA (MOPA) for the 13 serotypes in Prevnar 13. Because new conjugate vaccines with increased valence are being developed, we developed 4-fold MOPAs for an additional 13 serotypes: serotypes 6C and 6D, plus the 11 serotypes contained in Pneumovax but not in Prevnar 13. A high level of nonspecific killing (NSK) was observed for three serotypes (10A, 15B, and 33F) in multiple batches of baby rabbit complement. The NSK could be reduced by preadsorbing the complement with encapsulated, as well as unencapsulated, pneumococcal strains. The MOPA results compared well with the results of single-serotype OPA for all serotypes except for serotype 3. For serotype 3, the results obtained from the MOPA format were ∼40% higher than those of the single-serotype format. Interassay precision of MOPA was determined with 5 serum samples, and the coefficient of variation was generally <30% for all serotypes. MOPA was also specific for all serotypes except for serotype 20; i.e., free homologous polysaccharide (PS), but not unrelated PS, could completely and efficiently inhibit opsonization. However, serotype 20 PS from ATCC could efficiently inhibit opsonization of one serotype 20 target strain but not three other type 20 target strains even at a high (>80 mg/liter) PS concentration. This suggests the presence of serologic heterogeneity among serotype 20 strains.

Determination of Dodine in Fruit Samples by Liquid Chromatography Electrospray Ionization-Tandem Mass Spectrometry

A rapid and sensitive LC/electrospray ionization-MS/MS method has been developed for the determination of dodine in fruit samples. Based on a liquid–liquid extraction of 10 g solid fruit homogenate using an acetone–dichloromethane– hexane mixture and acetate ammonium buffer (pH 4.5), this LC/MS/MS procedure was characterized by recoveries above 50%, with good intra-assay precision (RSD &lt;13%) and interassay precision (RSD &lt;18%) for seven different matrixes (apple, apricot, cherry, peach, pear, plum, and quince). This method was validated from 5 to 500 μg/kg according to standard guidelines. Its LOD (1 μg/kg) and LOQ (5 μg/kg) were in accordance with recommendations of the European legislation defined for infant food [maximum residue level (MRL) = 10 μg/kg]. The whole procedure was finally tested on 1022 fruit samples intended for commercialization, both infant food samples and samples not intended in particular for babies. In this study, dodine was detected in 27 samples; none exhibited a concentration higher than the MRL.