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 > 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 > 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.

Local verification and assignment of mean normal prothrombin time and International Sensitivity Index values across various instruments: recent experience and outcome from North America.

Warfarin dosing relies on accurate measurements of international normalized ratio (INR), which is calculated from the prothrombin time (PT), International Sensitivity Index international sensitivity index (ISI) of the thromboplastin, and the geometric mean of normal PT (MNPT). However, ISI assignments of certain reagent/instrument combinations are frequently unavailable, especially when the reagent and instrument are not from the same manufacturer. The effort to be in compliance with widely endorsed Clinical and Laboratory Standards Institute (CLSI) guidelines by locally verifying or assigning an ISI to an unsupported reagent/instrument combination is further hindered by the lack of US Food and Drug Administration (FDA)-approved certified plasmas designated for a particular reagent/instrument combination. The objectives of the study include development of a process to verify/assign ISI and MNPT of a single thromboplastin reagent from one manufacturer across multiple instruments including several from another manufacturer and across several campuses of a single institution, the Mayo Clinic. In this study, RecombiPlasTin 2G (R2G), was evaluated on the ACL TOP 700 (IL), STA-R Evolution, STA Compact, and STA Satellite. Random normal donor samples (n = 25) were used to verify/assign MNPT. A subset of the normal donors (n = 8) and 13 warfarin pools (INR range: 1.3-3.9), created from stable warfarin patient plasma, were used for ISI verification/assignment. The manufacturer's assigned ISI was first verified on the ACL TOP 700 (reference method), then assigned on three unsupported instruments using orthogonal regression analysis. The MNPT and manufacturer assigned ISI (11.0, 0.95) were verified on the ACL TOP 700 and subsequently assigned on the STA-R Evolution (11.6, 1.04); STA Compact (11.5, 1.02); and STA Satellite (10.9, 0.99). Linear correlations of the INR results from all the four instruments demonstrated an r2 > 0.99. This process provides a reproducible approach to assigning ISIs on unsupported reagent/instrument combinations. Our data also confirm that ISIs of the same PT reagent differ significantly on different instruments, thus confirming the requirement for evaluations and validation of ISIs for different reagent/instrument combinations.

Bridging the gap: The critical role of laboratory developed tests in clinical toxicology.

•Toxicology testing provides valuable information for patient management.•Current in vitro diagnostics (IVDs) are unable to meet all clinical needs.•Lab-developed tests (LDTs) in toxicology can be used to close clinical care gaps.•LDTs in clinical toxicology are almost exclusively mass spectrometry-based methods.

Resolving icteric interference by a novel photoisomerization device: Bilibox.

Icterus, a phenomenon caused by bilirubin elevation in the blood, is a common endogenous interference in chemistry testing, occurring either spectrally or through chemical reactivity with assay reagents. Often, laboratories have few options other than to dilute or reject samples exceeding icteric thresholds. However, recent studies have optimized in vitro photoisomerization of bilirubin to a 17-minute bilirubin half-life using 500 nm light at 37 °C. Using an enzymatic creatinine assay as a model, due to its prevalence in routine laboratory testing and susceptibility to icteric interference, our study explores the usage of in vitro photoisomerization by replicating these conditions in a device, the Bilibox, as means of resolving icterus in laboratory testing. Left-over icteric and non-icteric clinical samples, collected by lithium heparin vacutainer (n = 10), were analyzed for baseline creatinine, diluted creatinine (1:4 0.9 % NaCl), total bilirubin, direct bilirubin, and hemolysis, icterus and lipemia (HIL) indices. Samples were then placed in the Bilibox in two intervals of 30 min with repeat measurements of the aforementioned analytes. On average, icteric-index, total bilirubin (TBIL), and direct bilirubin (DBIL) decreased by 33.5, 39.1 and 39.9 % respectively following 30 min of Bilibox treatment; and by 47.6 %, 63.7 % and 59.8 % following 60 min. The average percent difference between the pre-exposure diluted and undiluted creatinine was 5.8 %, demonstrating the icterus interference. Following Bilibox treatment, the difference between undiluted (post-exposure) and diluted (pre-exposure) creatinine decreased to 0.02 % (p = 0.0232) and 2.2 % (p = 0.0021) at 30 and 60 min of treatment respectively, demonstrating resolution of interference. Consequently, photoisomerization can be utilized as an additional and reasonably quick method for resolving icterus when dilutional methods cannot be applied.

Rapid serum clot tubes reduce haemolysis due to pneumatic tube transport.

AIMS: Pneumatic tube systems (PTSs) are critical for modern hospital operations, allowing for rapid sample transport. Despite widespread use, PTSs can compromise specimen integrity and affect laboratory values. Our objective was to prove that rapid serum clot tubes (RST) provide protective benefits over plasma during PTS transport and can be a practical solution for certain PTS routes. METHODS: In this study, we compared the effects of PTS transport on cell lysis indicators: h-index, lactate dehydrogenase (LDH) and potassium (K+), in RST versus lithium heparin gel separator tubes using 10 volunteers. RESULTS: In comparison with plasma, RST showed a median reduction in PTS-induced haemolysis of 80.4% (p=0.0049), with a reduction in post-PTS median LDH concentration (49.7%, p=0.04) and K+ concentration (50.0%, p=0.0273). CONCLUSION: This study demonstrates RST tubes can significantly reduce PTS-induced haemolysis and can be recommended for poor PTS routes.

The ALT upper reference interval debate: Blame it on the alcohol.

BACKGROUND: In 2017, the American College of Gastroenterology (ACG) and the North American Society of Pediatric Gastroenterology, Hepatology and Nutrition published clinical guidelines recommending the use of alanine aminotransferase (ALT) upper reference limits (URL) of 33, 25, 26, and 22 U/l for men, women, boys, and girls, respectively. This was opposed by laboratory experts who advocated for the use of higher URL of 59, 41, 33, and 24 U/l instead. We suspected that the variable inclusion of individuals who consumed alcohol to be a major contributing source of URL variability and debate. METHODS: Outpatient ALT data (n = 7379) were collected from unique individuals ≥13 y with BMIs of ≥19 and ≤25. A total of 222 (3%) were excluded due to suspected liver disease. Patients were split into a pediatric group (age 13-17 y), an alcohol-restricted adult group (age 18-20 y), and adults with access to alcohol by decade (i.e., age 21-29, 30-39, 40-49, 50-59, 60-69, 70-79, and ≥ 80 y). All ALT values were measured on Roche Cobas 8000 with pyridoxal phosphate and traceable to the IFCC-reference measurement procedure. RESULTS: We derived URL similar to CALIPER for our pediatric population, but closer to ACG-proposed URL in our alcohol-restricted adult group. The URL increased significantly in men and women for all other age groups. CONCLUSIONS: The discrepancy in ALT URL in clinical laboratories may be attributable in part due to the variable inclusion of individuals who recently consumed alcohol in local population derivation studies.

Determination of 97.5th and 99th percentile upper reference limits for heart-type fatty acid-binding protein (H-FABP) in a US population.

BACKGROUND: Heart-type fatty acid binding protein (H-FABP), a low molecular weight protein found primarily in myocardial tissue, has been identified as a potential biomarker in the detection of acute coronary syndrome and acute kidney injury. To further investigate clinical utility, we sought to establish an upper reference limit (URL) of H-FABP within a healthy U.S. METHODS: Serum samples of healthy donors were acquired from the AACC Universal Sample Bank. We analyzed 355 samples for H-FABP concentration using the Randox Laboratories immunoturbidimetric assay on the Roche Cobas 8000 series analyzer. RESULTS: The final sample population consisted of individuals aged 18-74 y, with 170 males and 185 females. The distribution of the population exhibited a strong positive skew, affecting outlier analysis and URL determination. The 97.5th-percentile URL was found to be 7.4 ng/ml (95% CI: 6.3-9.2), while the 99th-percentile URL was 12.1 ng/ml (8.6-14.9). CONCLUSION: As the URL for H-FABP is highly affected by population distribution and outlier removal, final determination for an assay cutoff should be made in the context of clinical utility, either as a standalone assay or in conjunction with other biomarkers, and the desired clinical sensitivity and specificity.

Identification of novel ADAMTS1, ADAMTS4 and ADAMTS5 cleavage sites in versican using a label-free quantitative proteomics approach.

The chondroitin sulfate proteoglycan versican is important for embryonic development and several human disorders. The versican V1 splice isoform is widely expressed and cleaved by ADAMTS proteases at a well-characterized site, Glu441-Ala442. Since ADAMTS proteases cleave the homologous proteoglycan aggrecan at multiple sites, we hypothesized that additional cleavage sites existed within versican. We report a quantitative label-free approach that ranks abundance of liquid chromatography-tandem mass spectrometry (LC-MS/MS)-identified semi-tryptic peptides after versican digestion by ADAMTS1, ADAMTS4 and ADAMTS5 to identify site-specific cleavages. Recombinant purified versican V1 constructs were digested with the recombinant full-length proteases, using catalytically inactive mutant proteases in control digests. Semi-tryptic peptide abundance ratios determined by LC-MS/MS in ADAMTS:control digests were compared to the mean of all identified peptides to obtain a z-score by which outlier peptides were ranked, using semi-tryptic peptides identifying Glu441 -Ala442 cleavage as the benchmark. Tryptic peptides with higher abundance in control digests supported cleavage site identification. We identified several novel cleavage sites supporting the ADAMTS1/4/5 cleavage site preference for a P1-Glu residue in proteoglycan substrates. Digestion of proteins in vitro and application of this z-score approach is potentially widely applicable for mapping protease cleavage sites using label-free proteomics. SIGNIFICANCE: Versican abundance and turnover are relevant to the pathogenesis of several human disorders. Versican is cleaved by A Disintegrin-like And Metalloprotease with Thrombospondin type 1 motifs (ADAMTS) family members at Glu441-Ala442, generating a bioactive proteoform called versikine, but additional cleavage sites and the site-specificity of individual ADAMTS proteases is unexplored. Here, we used a label-free proteomics strategy to identify versican cleavage sites for 3 ADAMTS proteases, applying a novel z-score-based statistical approach to compare the protease digests of versican to controls (digests with inactive protease) using the known protease cleavage site as a benchmark. We identified 21 novel cleavage sites that had a comparable z-score to the benchmark. Given the functional significance of versikine, they represent potentially significant cleavages and helped to refine a substrate site preference for each protease.The z-score approach is potentially widely applicable for discovery of site-specific cleavages within an purified protein or small ensemble of proteins using any protease.

Preventing pseudohyponatremia: Intralipid®-based lipemia cutoffs for sodium are inappropriate.

BACKGROUND: Pseudohyponatremia describes an artifactual decrease in plasma sodium result in samples with high proteins and/or lipids when measured by an indirect ion-selective electrode (ISE) method. We suspected that Intralipid®-based lipemia cutoffs are inappropriate for detecting interfering lipids in human samples and a major contributing factor to the existence of pseudohyponatremia. METHODS: We evaluated 2 approaches to derive a lipemia cutoff for sodium, one in which patient plasma samples were pooled and spiked to simulate hyperlipidemia using Intralipid® (commonly used approach by in-vitro diagnostics manufacturers), and another in which endogenous hyperlipidemic samples (n = 31) were measured by methods not affected by hyperlipidemia (i.e., direct ISE and post-ultracentrifugation indirect ISE). Triglycerides, lipemic index (L-index) and indirect ISE sodium concentrations of samples were measured on Roche Cobas® 8000 and direct ISE on Radiometer® ABL835 Flex analyzers. Endogenous hyperlipidemic samples were also ultracentrifuged on Beckman Coulter® Airfuge to clear excess lipids and re-analyzed for sodium by indirect ISE. RESULTS: We discovered that Intralipid® is not an accurate emulation of the lipemic interference seen in pseudohyponatremia because it showed no effect up to the maximum level of lipemia tested (L-index = 2000). By contrast, endogenous hyperlipidemic samples demonstrated significant deviations in sodium concentration (≥4 mmol/l) when L-index approached or exceeded 700, and a strong positive correlation between L-index and the difference between the indirect and direct methods (i.e., extent of pseudohyponatremia). CONCLUSIONS: Clinical laboratories should lower their tolerance for lipemia from the currently recommended L-index cutoff of 2000 on Roche Cobas 8000®. We recommend reflexing to direct ISE when L-index exceeds 700. Manufacturers and laboratories with other indirect ISE methods should evaluate the effect of lipid interference on their method using hyperlipidemic human samples not Intralipid®.

Aggrecan in Cardiovascular Development and Disease.

Aggrecan is a large proteoglycan that forms giant hydrated aggregates with hyaluronan in the extracellular matrix (ECM). The extraordinary resistance of these aggregates to compression explains their abundance in articular cartilage of joints where they ensure adequate load-bearing. In the brain, they provide mechanical buffering and contribute to formation of perineuronal nets, which regulate synaptic plasticity. Aggrecan is also present in cardiac jelly, developing heart valves, and blood vessels during cardiovascular development. Whereas aggrecan is essential for skeletal development, its function in the developing cardiovascular system remains to be fully elucidated. An excess of aggrecan was demonstrated in cardiovascular tissues in aortic aneurysms, atherosclerosis, vascular re-stenosis after injury, and varicose veins. It is a product of vascular smooth muscle and is likely to be an important component of pericellular matrix, where its levels are regulated by proteases. Aggrecan can contribute to specific biophysical and regulatory properties of cardiovascular ECM via the diverse interactions of its domains, and its accumulation is likely to have a significant role in developmental and disease pathways. Here, the established biological functions of aggrecan, its cardiovascular associations, and potential roles in cardiovascular development and disease are discussed.

Urinary cannabinoid mass spectrometry profiles differentiate dronabinol from cannabis use.

BACKGROUND: Dronabinol is used to treat a variety of conditions, including loss of appetite in people with AIDS and severe nausea and vomiting caused by cancer chemotherapy. Its therapeutic potential for pain management is now being explored in specific populations. Monitoring dronabinol compliance is challenging because its active ingredient, Δ-9-tetrahydrocannabinol (THC), is also present in cannabis. We developed a rapid LC-MS/MS assay with minimal specimen preparation to quantitate 11 cannabinoids in urine. Using this assay coupled with urine samples from normal controls, cannabis, and dronabinol users, we show the ability to differentiate cannabis from dronabinol use. METHODS: Residual clinical urine samples from 55 cannabinoid positive subjects and 31 negative controls, as well as prospective samples from 5 patients receiving dronabinol therapy were obtained for analysis. RESULTS: In the dronabinol group, only the THC metabolites 11-nor-9-carboxy-tetrahydrocannabinol (THC-COOH) and 11-hydroxy-Δ-9-tetrahydrocannabinol (THC-OH) were detected. Minor cannabinoids were detected in 91% of cannabis group samples and their detection was more frequent in samples with increased THC metabolite concentrations. Of minor cannabinoids evaluated, cannabigerol (CBG) and cannabidiol (CBD) had the greatest sensitivity in detecting cannabis use. CONCLUSIONS: This method has a high sensitivity for the detection of cannabis use with implications for evaluating dronabinol compliance.

Massive aggrecan and versican accumulation in thoracic aortic aneurysm and dissection.

Proteoglycan accumulation is a hallmark of medial degeneration in thoracic aortic aneurysm and dissection (TAAD). Here, we defined the aortic proteoglycanome using mass spectrometry, and based on the findings, investigated the large aggregating proteoglycans aggrecan and versican in human ascending TAAD and a mouse model of severe Marfan syndrome. The aortic proteoglycanome comprises 20 proteoglycans including aggrecan and versican. Antibodies against these proteoglycans intensely stained medial degeneration lesions in TAAD, contrasting with modest intralamellar staining in controls. Aggrecan, but not versican, was increased in longitudinal analysis of Fbn1mgR/mgR aortas. TAAD and Fbn1mgR/mgR aortas had increased aggrecan and versican mRNAs, and reduced expression of a key proteoglycanase gene, ADAMTS5, was seen in TAAD. Fbn1mgR/mgR mice with ascending aortic dissection and/or rupture had dramatically increased aggrecan staining compared with mice without these complications. Thus, aggrecan and versican accumulation in ascending TAAD occurs via increased synthesis and/or reduced proteolytic turnover, and correlates with aortic dissection/rupture in Fbn1mgR/mgR mice. Tissue swelling imposed by aggrecan and versican is proposed to be profoundly deleterious to aortic wall mechanics and smooth muscle cell homeostasis, predisposing to type-A dissections. These proteoglycans provide potential biomarkers for refined risk stratification and timing of elective aortic aneurysm repair.

Versican Proteolysis by ADAMTS Proteases and Its Influence on Sex Steroid Receptor Expression in Uterine Leiomyoma.

Context: Leiomyomas have abundant extracellular matrix (ECM), with upregulation of versican, a large proteoglycan. Objective: We investigated ADAMTS (a disintegrin-like and metalloprotease with thrombospondin type 1 motifs) protease-mediated versican cleavage in myometrium and leiomyoma and the effect of versican knockdown in leiomyoma cells. Design: We used quantitative reverse transcription polymerase chain reaction (qRT-PCR), western blotting, immunohistochemistry, and RNA in situ hybridization for analysis of myometrium, leiomyoma and immortalized myometrium and leiomyoma cells. Short interfering RNA (siRNA) was used to knockdown versican in leiomyoma cells. Setting: This study was performed in an academic laboratory. Patients: Study subjects were women with symptomatic or asymptomatic leiomyoma. Main Outcome Measures: We quantified messenger RNAs (mRNAs) for versican splice variants. We identified ADAMTS-cleaved versican in myometrium and leiomyoma and ADAMTS messenger RNAs and examined the effect of VCAN siRNA on smooth muscle differentiation and expression of estrogen and progesterone receptors. Results: The women in the symptomatic group (n = 7) had larger leiomyoma (P = 0.01), heavy menstrual bleeding (P < 0.01), and lower hemoglobin levels (P = 0.02) compared with the asymptomatic group (n = 7), but were similar in age and menopausal status. Versican V0 and V1 isoforms were upregulated in the leiomyomas of symptomatic versus asymptomatic women (P = 0.03 and P = 0.04, respectively). Abundant cleaved versican was detected in leiomyoma and myometrium, as well as in myometrial and leiomyoma cell lines. ADAMTS4 (P = 0.03) and ADAMTS15 (P = 0.04) were upregulated in symptomatic leiomyomas. VCAN siRNA did not effect cell proliferation, apoptosis, or smooth muscle markers, but reduced ESR1 and PR-A expression (P = 0.001 and P = 0.002, respectively). Conclusions: Versican in myometrium, leiomyomas and in the corresponding immortalized cells is cleaved by ADAMTS proteases. VCAN siRNA suppresses production of estrogen receptor 1 and progesterone receptor-A. These findings have implications for leiomyoma growth.

Analytical performance of three whole blood point-of-care lactate devices compared to plasma lactate comparison methods and a flow-injection mass spectrometry method.

OBJECTIVES: Point of care (POC) whole blood lactate testing may facilitate rapid detection of sepsis. We evaluated three POC methods against both plasma lactate comparison methods and a flow-injection mass spectrometric (MS) method. DESIGN AND METHODS: Nova StatStrip, Abbott i-STAT CG4+ and Radiometer ABL90 POC lactate methods were evaluated against the mean of Cobas Integra 400 and Vitros 350 plasma lactate. POC methods were also compared to a flow-injection mass spectrometric assay measuring lactate in ZnSO4-precipitated whole blood extracts. Intra- and inter-assay precision was determined using quality control material. Method comparison included specimens from normal donors at rest, after exertion, and after spiking with lactic acid. RESULTS: Intra- and inter-assay coefficient of variation was <5% for i-STAT and ABL90; but ranged from 3.1-8.2% on two StatStrip meters. Mean (±SD) bias between POC and plasma lactate ranged from -0.2±0.9 (i-STAT and ABL90) to -0.4±1.2 (StatStrip) mmol/L. At concentrations >6mmol/L, all POC methods showed proportional negative bias compared to plasma methods; but this bias was not observed when compared to the MS method. Despite proportional negative bias, all POC methods demonstrated acceptable concordance (94-100%) with plasma lactate within the reference interval (<2.3mmol/L) and >4mmol/L, commonly used clinical cut-offs for detection of sepsis. CONCLUSIONS: POC lactate methods demonstrate acceptable concordance with plasma lactate across commonly used clinical cut-offs for detection of sepsis. Due to systematic negative bias at higher lactate concentrations, POC and plasma lactate should not be used interchangeably to monitor patients with elevated lactate concentrations.