Current Issues in Blood Gas Analysis.

BACKGROUND: Blood gas analysis constitutes one of the most widely used tests, especially in critical care settings such as intensive care units, emergency departments, and operating rooms. Blood gas results are key for assessing acid-base balance and ventilatory control in critically ill patients. Because blood gas analysis plays a vital role in management of critically ill patients, this testing is frequently conducted at the point-of-care by users with various educational backgrounds across different hospital departments. CONTENT: When performing blood gas analysis, it is important to be aware of the analytical issues that may affect the different components of this testing. With blood gas analysis, differences in test names and method changes over time have led to several controversies that might affect test result interpretations. Hence, being aware of these controversies is important in ensuring appropriateness of result interpretations. Many blood gas testing programs face challenges with maintaining quality assurance. Having practical approaches to method verification, and choosing the right blood gas analyzer type, will go a long way to ensure quality in blood gas analysis. SUMMARY: We review analytical issues and controversies associated with blood gas testing, as well as practical approaches to deciding on a blood gas analyzer and quality assurance.

Use of Cotton Balls in Diapers for Collection of Urine Samples Impacts the Analysis of Routine Chemistry Tests: An Evaluation of Cotton Balls, Diapers, and Chemistry Analyzers.

OBJECTIVE: To evaluate the suitability of urine samples collected with cotton balls placed into diapers for routine laboratory chemistry analyses. STUDY DESIGN: Twenty pools of residual unpreserved urine samples were separated into control and treated aliquots. The treated samples were absorbed into 2 different brands of cotton balls, wrapped in 3 different brands of diapers, and incubated at 37°C for 1 hour. The urine-soaked cotton balls were placed into a syringe and expressed via plunger depression. Urine sodium, potassium, creatinine, urea, calcium, magnesium, inorganic phosphorus, albumin, and total protein were measured on all samples on 5 automated clinical chemistry platforms: Ortho Vitros 4600, Siemens Dimension Vista 500, Beckman Coulter AU5822, Roche Cobas 6000, and Abbott Architect c8000 at 5 separate hospital laboratories. Criteria used to exclude the presence of significant effects of urine from presoaked cotton balls in a diaper on the measurement of chemistry laboratory tests were R2 >0.95, slope of 0.9-1.1, and mean bias within ±10%. RESULTS: Albumin and total protein measurements demonstrated significant negative bias in urine from both brands of presoaked cotton balls with all brands of diapers on all 5 chemistry platforms compared with the control urine. We did not observe a significant effect of presoaking urine in cotton balls in a diaper on the measurement of sodium, inorganic phosphorus, and urea. The remaining tests demonstrated significant effects when measured in urine from presoaked cotton balls and/or diapers that were specific to the chemistry analyzer platform or diaper. CONCLUSIONS: Diaper and cotton ball-based urine collection significantly impacts the measurement of several common chemistry assays.

The 2018 AACC/SYCL PhD Clinical Chemist Compensation Survey.

BACKGROUND: Doctoral level board-certified clinical chemists play an invaluable role in many facets of laboratory medicine and healthcare. However, information concerning their total compensation is sparse. CONTENT: A confidential self-reported compensation survey was conducted by the American Association for Clinical Chemistry's Society for Young Clinical Laboratorians (AACC SYCL) Core Committee from April 1 to April 17, 2018. Respondents provided information on geographic location, employment sector, gender, and years of experience to account for the influence of these variables on compensation. There were 199 respondents in total from the United States and Canada, however, only respondents employed in the United States with an earned doctoral degree and certification by the American Board of Clinical Chemistry (n = 133), were included in the full analysis. In comparison to compensation reported in AACC SYCL salary surveys conducted in 2010 and 2013, early career median salaries are trending upwards after correction for inflation. SUMMARY: This survey is the first to collect the gender of respondents, and identify a pay gap for some geographic groups. However, this gap could be due in part to a difference in the years of experience, since males were highly represented in the group with >20 years of experience (25 out of 35, 71%). Future studies on compensation trends within clinical chemistry that do not rely on self-report are needed to ensure accuracy and completeness of the dataset.

Implementation of point-of-care testing in a pediatric healthcare setting.

Point-of-care testing (POCT) refers to testing performed outside the clinical laboratory near the patient or at the site of patient care. This could be in critical care settings like the intensive care unit (ICU) and emergency department (ED) or primary care settings like physician offices where testing is performed by nonlaboratory personnel. POCT circumvents several steps in central laboratory testing including specimen transportation and processing resulting in faster turnaround times. Provider access to rapid test results at the site of patient care allows for prompt medical decision making which can lead to improved patient outcomes, operational efficiencies, patient satisfaction, and even cost savings in some cases. In addition to providing results rapidly, POCT devices have small specimen volume requirements compared to central laboratory tests making POCT particularly attractive for pediatric healthcare settings. The availability of published reports on the impact of POCT implementation in pediatric care are helpful resources when evaluating the clinical necessity of POCT prior to implementation. Even though several studies have shown advantages to implementing POCT in different pediatric settings, it is important to note that limitations exist that might limit the utilization of certain POCTs in some pediatric populations. So, it is important that these limitations and the analytical performance of a test are considered while keeping the target patient population in mind. Since POCTs are performed by non-laboratory staff who are not trained laboratory personnel, one challenge with POCT is maintaining regulatory compliance and quality assurance. It is therefore important that regulatory and quality assurance programs be put in place prior to implementing POCT in the pediatric hospital. With advances in POCT technology, most POCT devices have the capability to interface to the laboratory information system (LIS) and electronic medical record (EMR). POCT device interfacing allows for improved compliance to regulatory and quality assurance standards. Maintaining a cost efficient POCT program is becoming increasingly important as hospitals and healthcare systems are undergoing consolidation and harmonization. This includes assessing the clinical and operational benefit of POCT before implementation and inventory management to ensure minimal reagent wastage. This review discusses these different considerations when implementing POCT with a focus on the pediatric healthcare setting.

Comparative analysis of neutrophil gelatinase-associated lipocalin and other laboratory markers for lupus nephritis: a cross-sectional investigation.

Background Lupus nephritis is one of the most serious complications of systemic lupus erythematosus. This study evaluates the prevalence and correlation between neutrophil gelatinase-associated lipocalin and other biomarkers associated with renal involvement in systemic lupus erythematosus. Methods Paired serum and urine specimens from 50 suspected systemic lupus erythematosus patients, characterized by antinuclear antibodies detected by indirect immunofluorescence assay and varying positive concentrations of anti-double stranded DNA antibodies by Crithidia luciliae immunofluorescence assay, were investigated. Of these 50 patients, 18 were identified with renal involvement based upon laboratory serology. Patients and healthy control serum samples ( n = 50) were also evaluated for high avidity double stranded DNA IgG antibodies, anti-C1q IgG antibodies, and serum creatinine. The prevalence and relationship between biomarkers were evaluated using statistical methods. Results Serum and urine neutrophil gelatinase-associated lipocalin concentrations were significantly elevated in patients compared to controls, with a prevalence of 24% and 36%, respectively. These concentrations were also more markedly increased in systemic lupus erythematosus patients with renal involvement than those without. Spearman's correlations between neutrophil gelatinase-associated lipocalin and other biomarkers tested ranged from 0.06 to 0.66 in all patients. Combined concordance as determined by Cronbach alpha coefficient between biomarkers was reduced from 0.71 to 0.58 (serum) and 0.62 (urine) when neutrophil gelatinase-associated lipocalin was removed. Conclusions Neutrophil gelatinase-associated lipocalin concentrations are elevated and demonstrate variable associated with other laboratory markers for renal involvement in systemic lupus erythematosus. Prospective longitudinal studies are needed to determine the optimal biomarker combinations for use in routine management of systemic lupus erythematosus patients at-risk for lupus nephritis.

Immunoassays for the detection of IgA antibodies to tissue transglutaminase: significance of multiples of the upper limit of normal and inter-assay correlations.

BACKGROUND: The presence of IgA antibodies to tissue transglutaminase (anti-tTg) is associated with variable risk for celiac disease. The use of common multiples of the upper limit of normal (ULN) has been suggested to optimize diagnostic pathways as well as improve harmonization between assays. METHODS: The characteristics of four anti-tTG IgA assays relative to endomysial IgA (EMA) by indirect immunofluorescence assay (IFA) as reference test were assessed. Commutability between anti-tTG immunoassays and/or EMA based on manufacturer's recommended cut-off values and three common multiples of ULN (3×, 5× and 10×) was also investigated. Sera from 200 patients and 100 healthy individuals were analyzed. RESULTS: At manufacturer's cut-off; the sensitivities for the tTG assays ranged from 72.5% to 98.6% and specificities from 60.3% to 99.2%. The percent positive agreements between any anti-tTG and EMA or any two anti-tTG immunoassays varied from 56.7% to 98.0% and 46.7% to 100.0%, respectively. At 3×, 5× or 10× ULNs, the inter-rater reliability as measured by Cohen κ between any two anti-tTG assays were quite variable and ranged from 0.28 to 0.96, 0.26 to 0.89 or 0.13 to 0.78, respectively. Furthermore, the percent positive agreements between any two anti-tTg IgA immunoassays ranged from 83.1% to 98.2%, 92.0% to 100%, or 100%, at 3×, 5× or 10×, respectively. CONCLUSIONS: Commutability between tTG IgA immunoassays or tTG IgA and EMA is kit-dependent and common multiples of the ULN are not sufficient to correct for inter-assay variations. Many factors influence the performance of anti-tTG IgA assays which limit their commutability.

Reference interval determination for anabasine: a biomarker of active tobacco use.

Laboratory detection of nicotine exposure is important for establishing eligibility for organ transplant and elective surgery. Nicotine testing is also used to verify compliance with nicotine replacement therapies (NRT), smoking cessation programs and for life insurance purposes. Nicotine metabolites, such as cotinine and trans-3'-hydroxycotinine, are used as biomarkers of nicotine exposure. For some clinical applications, it is important to distinguish between active use of tobacco products versus NRT. Anabasine is a tobacco alkaloid that has been used as a biomarker of active tobacco use. However, the use of anabasine as an insecticide, and its presence in consumables other than nicotine products, suggests that anabasine may not be specific to tobacco use/exposure. Here, we determine the reference interval for anabasine in the urine of nonsmokers and compare it to the range of anabasine concentrations observed in the presence or absence of nicotine metabolites.

Challenging identification of a novel PiISF and the rare PiMmaltonZ α1-antitrypsin deficiency variants in two patients.

OBJECTIVES: α1-Antitrypsin (AAT) deficiency is associated with an increased risk for lung and liver disease. Identification of AAT deficiency as the underlying cause of these diseases is important in correct patient management. METHODS: AAT deficiency is commonly diagnosed by demonstrating low concentrations of AAT followed by genotype and/or phenotype testing. However, this algorithm may miss novel AAT phenotypes. RESULTS: We report two cases of AAT deficiency in two patients: a case of the novel phenotype PiISF, misclassified as PiII by phenotyping, and a case of the rare phenotype PiMmaltonZ misclassified as PiM2Z. CONCLUSIONS: These cases highlight the importance of understanding the limitations of a commonly used diagnostic algorithm, use of further gene sequencing in applicable cases, and the potential for underdiagnosis of AAT deficiency in patients with chronic obstructive pulmonary disease.

Anti-NMDA-receptor antibody encephalitis: performance evaluation and laboratory experience with the anti-NMDA-receptor IgG assay.

BACKGROUND: Antibodies targeting the NR1 subunit of the N-methyl-d-aspartate-receptor (NMDAR) are considered diagnostic for a novel form of autoimmune encephalitis. We report the validation of a qualitative indirect immunofluorescence antibody (IFA) test for the detection of anti-NMDAR IgG and describe the attributes of antibody-positive patients. METHODS: The anti-NMDAR IgG assay (Euroimmun Diagnosika, Lübeck, Germany) was validated with serum and cerebrospinal fluid (CSF) specimens from 30 healthy and 50 disease controls as well as 5 anti-NMDAR IgG-positive individuals. Consecutive specimens (n=1671) for anti-NMDAR IgG antibodies were evaluated and positive specimens titrated to end-point [starting dilutions: CSF; 1:1 and serum; 1:10]. In a subset of antibody-positive patients, we sought clinical information for correlation with diagnostic and treatment outcomes. RESULTS: The assay demonstrated excellent performance characteristics in all groups evaluated. Of the 1671 specimens tested, 1389 were unique cases with a positivity rate of 9.0% (n=123). For the antibody-positive samples, the female to male ratio was 2:1 with a prevalence of 46% in the pediatric population (≤17 years). Antibody titers were titrated to end-point for 106/123 specimens [45 CSF, 41 sera, and 20 CSF and serum pairs] with more than 75% having titers greater than 1:10 (CSF) and 1:20 (serum). Overall, high levels of these antibodies showed correlation to disease severity with variable response to treatment in the subset of patients evaluated. CONCLUSION: Our data suggests a high prevalence for anti-NMDAR antibody encephalitis irrespective of age and gender in our unselected disease cohort with support for measuring antibody titers in the evaluation of these patients.

APhL antibody ELISA as an alternative to anticardiolipin test for the diagnosis of antiphospholipid syndrome.

BACKGROUND: Persistent levels of antiphospholipid (aPL) antibodies [lupus anticoagulant (LA), anticardiolipin (aCL), anti-beta 2 glycoprotein I (aß(2)GPI) IgG and/or IgM] in association with clinical features of thrombosis and/or pregnancy associated morbidity are indicative of antiphospholipid syndrome (APS). Of the aPL antibodies, aCL is the most sensitive for APS, however, their lack of specificity constitute a laboratory and clinical challenge. IgG/IgM antibodies directed against APhL (a mixture of phospholipids) has been reported to predict APS more reliably than aCL tests. The main objective of this study was to evaluate the performance characteristics of the APhL IgG/IgM ELISA, relative to the aCL and aß(2)GPI tests. METHODS: Sixteen (16) clinically confirmed APS and 85 previously tested serum (PTS) samples for aCL and aß(2)GPI IgG/IgM antibodies were evaluated with the APhL IgG/IgM ELISA. Clinical specificity was determined in 100 serum samples (50 healthy and 50 infectious disease controls [parvo- and syphilis-IgG/IgM positive]). RESULTS: The IgG antibody prevalence for aCL and APhL in the APS and PST groups was comparable with marginal differences in clinical specificities. In contrast to the aCL IgM ELISA, the APhL test showed improved clinical specificities (72% aCL vs 94% APhL in the healthy controls; 38% aCL vs 78% APhL in the infectious disease controls) with implications for increased reliability in the diagnosis of APS. The overall agreement of the APhL with the aCL or aß(2)GPI for the IgG tests was 89% and 85% respectively, and that of the APhL IgM to the aCL or aß(2)GPI IgM tests was 72% and 86% respectively. CONCLUSION: Routine use of the APhL IgG/IgM ELISA may substantially reduce the high number of false positives associated with the aCL test without loss in sensitivity for APS.

Evaluation of a high avidity anti-dsDNA IgG enzyme-linked immunosorbent assay for the diagnosis of systemic lupus erythematosus.

The high avidity (HA) anti-dsDNA IgG ELISA is considered highly specific for the diagnosis of systemic lupus erythematosus (SLE). The main objective of this study was to determine the performance of this test with existing assays for detecting anti-dsDNA IgG antibodies as well as assess its analytical characteristics. For method comparison studies, we investigated the correlation between the HA ELISA with 8 other assays for the detection of dsDNA IgG antibodies namely; six anti-dsDNA IgG ELISA, the Crithidia luciliae immunofluorescence test (CLIFT) and an in-house developed Farr radioimmunoassay (RIA). Overall, 125 patient (100 ANA-positive, 25 CLIFT-tested) and 100 healthy control samples were tested. The assay was also evaluated for imprecision, lot-to-lot consistency and the effect of interfering substances using commercial quality control materials based on the manufacturer's claims unless otherwise stated. Of the 100 ANA positive samples, 18 were positive in the HA ELISA with significant levels of antibodies in the six ELISAs and CLIFT. The HA ELISA had a specificity of 100% with an overall agreement of 84% with the RIA. Intra - and inter-assay imprecision ranged from 13.9-16.5% and the reproducibility between lots based on qualitative interpretation was 100%. Hemoglobin, bilirubin and lipemia showed variable interference with assay performance based on the manufacturer's claims and our in-house protocol. Our data suggest that the HA ELISA although less sensitive than the other dsDNA IgG assays evaluated, is specific and predicts high levels of anti-dsDNA IgG antibodies.

Significant differences in the analytic concordance between anti-dsDNA IgG antibody assays for the diagnosis of systemic lupus erythematosus--implications for inter-laboratory testing.

BACKGROUND: Anti-double stranded DNA (anti-dsDNA) autoantibodies are considered hallmark of systemic lupus erythematosus (SLE). METHODS: To determine concordance between assays for the detection of this marker, we analyzed 100 antinuclear antibody (ANA) positive sera with a homogeneous pattern and titers≥1:160 by indirect immunofluorescence assay (IFA) on HEp-2 cells, 100 consecutive anti-dsDNA IgG ELISA-negative as well as 100 healthy control samples using six commercial ELISAs and the Crithidia luciliae immunofluorescence test (CLIFT). RESULTS: The positivity rates for the ELISAs in the ANA positive group ranged from 55.0 to 88.0% with specificities from 84.0 to 98.0%. The CLIFT had a positivity rate of 68.0% and specificity of 84%. In the previously screened anti-dsDNA IgG-negative group, the positivity rates ranged from 1 to 19%. The overall correlations between the ELISAs ranged from 73.0 to 89.5% and varied from 70.0 to 80.0% among specific ELISAs and CLIFT. CONCLUSIONS: Our data show variable degree of concordance between anti-dsDNA IgG ELISAs which may significantly impact inter-laboratory testing as well as the diagnosis and management of SLE patients. Although some of the ELISAs show comparable performance to the CLIFT, the degree of concordance between these assays at high antibody levels suggests that CLIFT is still a relevant confirmatory tool.

A fast and efficient method for quantitative measurement of S-adenosyl-L-methionine-dependent methyltransferase activity with protein substrates.

Modification of protein residues by S-adenosyl-L-methionine (AdoMet)-dependent methyltransferases impacts an array of cellular processes. Here we describe a new approach to quantitatively measure the rate of methyl transfer that is compatible with using protein substrates. The method relies on the ability of reverse-phase resin packed at the end of a pipette tip to quickly separate unreacted AdoMet from radiolabeled protein products. Bound radiolabeled protein products are eluted directly into scintillation vials and counted. In addition to decreasing analysis time, the sensitivity of this protocol allows the determination of initial rate data. The utility of this protocol was shown by generating a Michaelis-Menten curve for the methylation of heterogeneous nuclear ribonucleoprotein K (hnRNP K) protein by human protein arginine methyltransferase 1, variant 1 (hPRMT1v1), in just over 1h. An additional advantage of this assay is the more than 3000-fold reduction in radioactive waste over existing protocols.

Efficient cleavage of problematic tobacco etch virus (TEV)-protein arginine methyltransferase constructs.

Protein arginine methyltransferases (PRMTs) are enzymes that are involved in many biological processes. Several studies have shown that the identity of the N-terminal fusion tag affects the substrate selectivity of PRMTs. Therefore, to accurately study substrate recognition, it is imperative that a tagless PRMT be used. However, cleavage of tagged PRMTs has been problematic. We have developed a successful method by which untagged PRMTs can be made using a tobacco etch virus (TEV) cleavage site at the N-terminal domain. This method may be useful for cleaving other challenging target proteins that have the TEV protease recognition site.