Sonia L. La’ulu

Free Thyroid Hormones in Serum by Direct Equilibrium Dialysis and Online Solid-Phase Extraction–Liquid Chromatography/Tandem Mass Spectrometry

BACKGROUND Measurements of free thyroxine (FT4) and free triiodothyronine (FT3) are important for the diagnosis and monitoring of thyroid diseases. Considerable differences among methods limit their clinical utility, however, and accurate methods are needed for various clinical specimens. We describe a direct equilibrium dialysis (ED)-liquid chromatography (LC)/tandem mass spectrometry (MS/MS) method for FT4 and FT3. METHODS ED was selected as the separation step. Serum samples were dialyzed 1:1 against a simple protein-free buffer for 20 h at 37 degrees C. Thyroid hormones in dialysates were purified by online solid-phase extraction (SPE), then chromatographically separated and quantified in positive ion and multiple reaction monitoring modes. RESULTS For FT4 and FT3, the lower and upper limits of quantification were 1 ng/L (pg/mL) and 400 ng/L with total imprecision <10%. The method correlated well with an ED-RIA, 2 direct immunoassay methods for FT4, and 1 direct immunoassay and 1 tracer dialysis method for FT3. The adult reference intervals were 12.8-22.2 ng/L for FT4 and 3.62-6.75 ng/L for FT3. Reference intervals for the second trimester of pregnancy (14-20 weeks of gestation) were also established. CONCLUSIONS We developed a simple protein-free buffer and ED procedure. The performance characteristics and high throughput of the LC-MS/MS method with online SPE for FT4 and FT3 (also reverse T3) are sufficient for the intended clinical use.

Performance Characteristics of Six Intact Parathyroid Hormone Assays

The aim of this study was to evaluate the performance characteristics of 6 intact parathyroid hormone assays: Access 2 (Beckman Coulter, Fullerton, CA), ARCHITECT i2000(SR) (Abbott Diagnostics, Abbott Park, IL), ADVIA Centaur (Siemens Healthcare Diagnostics, Deerfield, IL), Modular E170 (Roche Diagnostics, Indianapolis, IN), IMMULITE 2000 (Siemens Healthcare Diagnostics), and LIAISON (DiaSorin, Stillwater, MN). Sample collection tubes and storage conditions were compared. Imprecision studies were performed using commercial quality control materials. Linearity was assessed using pools prepared from samples. For method comparison, serum and EDTA plasma samples were tested by all methods, and the ARCHITECT was used as the comparison method. Reference intervals were determined using various vitamin D cutoffs. The types of collection tubes and storage conditions are more important for some methods than others. Total coefficients of variation were 10.9% or less. The maximum deviation from the target recovery for linearity ranged from 5.0% to 82.2%. Bland-Altman plots demonstrated percentage biases ranging from -36.3% to 24.4%. The lower limit of the reference interval was not influenced by vitamin D status, whereas the upper reference limit was affected.

Intermethod differences in results for total PSA, free PSA, and percentage of free PSA.

Serum prostate-specific antigen (PSA) assays differ in calibration and response to different PSA forms. We examined intermethod differences in total PSA (tPSA) and free PSA (fPSA) measurements. We tested 157 samples with tPSA concentrations of 2 to 10 ng/mL (2-10 microg/L) using 6 PSA/fPSA method pairs and 1 tPSA method: ADVIA Centaur (complexed and total; Siemens Diagnostics, Tarrytown, NY), ARCHITECT i 2000(SR) (Abbott Diagnostics, Abbott Park, IL), AxSYM (Abbott Diagnostics), IMMULITE 2000 (Siemens Diagnostics), Modular E170 (Roche Diagnostics, Indianapolis, IN), UniCel DxI 800 (Beckman Coulter, Brea, CA), and VITROS ECi (tPSA only; Ortho-Clinical Diagnostics, Raritan, NJ). Regression analysis was performed for PSA, fPSA, and percentage of fPSA with the ARCHITECT i 2000(SR) comparison method. Differences between test and comparison methods were estimated at 2.5, 4.0, and 10.0 ng/mL (2.5, 4.0, and 10.0 microg/L) for tPSA and 15%, 20%, and 25% for percentage of fPSA. Relative differences were more than 10% at 4.0 ng/mL (4.0 microg/L) tPSA for the Centaur, IMMULITE, ECi, and DxI methods. At 20% fPSA, the relative difference was more than 10% for all methods except the AxSYM. Additional harmonization is needed for tPSA and fPSA methods.

Performance Characteristics of Six Homocysteine Assays

Elevated concentrations of homocysteine (Hcy) are associated with a range of disorders. Linearity, imprecision, interference, method comparison, and accuracy were evaluated on the ADVIA Centaur (Siemens Healthcare Diagnostics, Deerfield, IL), ARCHITECT i2000SR (Abbott Diagnostics, Abbott Park, IL), AxSYM (Abbott Diagnostics), and IMMULITE 2000 (Siemens Healthcare Diagnostics) methods and analyzers and the Catch (Equal Diagnostics, Exton, PA) and Diazyme (Diazyme Laboratories, San Diego, CA) methods, both on the Modular P analyzer (Roche Diagnostics, Indianapolis, IN). All methods were linear with maximum deviations from target recoveries of less than 10%. Total coefficients of variation ranged from 1.7% to 9.4%. The effects of hemolysis, icterus, and lipemia were assessed. Method comparisons were performed using high-performance liquid chromatography as the comparison method. Correlation coefficients were 0.95 to 0.99. Bland-Altman plots demonstrated percentage bias of -29.3% (IMMULITE) to 7.2% (Centaur). Accuracy using the National Institute of Standards and Technology Standard Reference Material 1955 showed varying results with only 1 method within the certified range for all 3 levels. All methods demonstrated acceptable performance except the IMMULITE, which is less precise and accurate. Standardization of most methods seems acceptable, although continuing efforts are warranted.

Effects of 49 Different Rare Hb Variants on HbA1c Measurement in Eight Methods

Background: Previous studies have shown interference with HbA1c measurement from the 4 most common heterozygous Hb variants (HbAS, HbAE, HbAC, and HbAD) with some assay methods. Here we examine analytical interference from 49 different less common variants with 7 different HbA1c methods using various method principles. Methods: Hb variants were screened using the Bio-Rad Variant or Variant II beta thal short program, confirmed by alkaline and acid electrophoresis, and identified by sequence analysis. The Trinity ultra2 boronate affinity high-performance liquid chromatography (HPLC) method and Roche Tinaquant immunoassay were used as primary and secondary comparative methods, respectively, since these methods are least likely to show interference from Hb variants. Other methods included were the Tosoh G7 and G8, Bio-Rad D-10 and Variant II Turbo, Diazyme Enzymatic, and Sebia Capillarys 2 Flex Piercing. To eliminate any inherent calibration bias, results for each method were adjusted using regression verses the ultra2 with nonvariant samples. Each method’s calibration-adjusted results were compared and judged to be acceptable if within the 99% prediction interval of the regression line for nonvariant samples. Results: Almost all variant samples were recognized as such by the ion-exchange HPLC methods by the presence of abnormal peaks or results outside the reportable range. For most variants, interference was seen with 1 or more of the ion-exchange methods. Following manufacturer instructions for interpretation of chromatograms usually, but not always, prevented reporting of inaccurate results. Results: Laboratories must be cautious about reporting results when the presence of a variant is suspected.

Pediatric Reference Intervals for Free Thyroxine and Free Triiodothyronine by Equilibrium Dialysis-Liquid Chromatography-Tandem Mass Spectrometry

Objective: Thyroid hormone concentrations fluctuate during growth and development. To accurately diagnose thyroid disease in pediatric patients, reference intervals (RIs) should be established with appropriate age groups from an adequate number of healthy subjects using the most exact methods possible. Obtaining statistically useful numbers of healthy patients is particularly challenging for pediatric populations. The objective of this study was to determine non-parametric RIs for free thyroxine (fT4) and free triiodothyronine (fT3) using equilibrium dialysis-high performance liquid chromatography-tandem mass spectrometry with over 2200 healthy children 6 months-17 years of age. Methods: Subjects were negative for both thyroglobulin and thyroid peroxidase autoantibodies and had normal thyrotropin concentrations. The study included 2213 children (1129 boys and 1084 girls), with at least 120 subjects (average of 125) from each year of life, except for the 6 month to 1 year age group (n=96). Results: Non-parametric RIs (95th percentile) for fT4 were: 18.0-34.7 pmol/L (boys and girls, 6 months-6 years) and 14.2-25.7 pmol/L (boys and girls, 7-17 years). RIs for fT3 were: 5.8-13.1 pmol/L (girls, 6 months-6 years); 5.7-11.8 pmol/L (boys, 6 months-6 years); 5.7-10.0 pmol/L (boys and girls, 7-12 years); 4.5-8.6 pmol/L (girls, 13-17 years); and 5.2-9.4 pmol/L (boys, 13-17 years). Conclusion: Numerous significant differences were observed between pediatric age groups and previously established adult ranges. This emphasizes the need for well-characterized RIs for thyroid hormones in the pediatric population.

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.

Evaluation of Test Strips for the Rapid Identification of Ethylenediaminetetraacetic Acid (EDTA) Specimens

BACKGROUND Identification of specimens that contain ethylenediaminetetraacetic acid (EDTA) is frequently necessary when investigating potentially mislabeled or improperly collected specimens. OBJECTIVE To evaluate the performance of rapid EDTA detection test strips in clinical specimens. METHODS We applied specimens to test strips designed to detect EDTA (QUANTOFIX EDTA) using a pipet (drop mode). Reactions were scored visually on a scale from red (no EDTA) to orange (low/indeterminate EDTA) to yellow (contains EDTA). RESULTS Test strips reliably identified specimens from EDTA-containing tube types. Although test strips did not detect strong reactivity in other specimens, tubes containing NaFl/K-oxalate produced an orange (low/indeterminate EDTA) reaction. Bismuth and citrate levels were higher in specimens after we dipped test strips into solution (dip mode). CONCLUSIONS Test strips detected the presence of EDTA in concentrations found in EDTA-containing primary tubes. Test strips were less effective in evaluating low-level EDTA concentrations expected with intravenous line contamination or backflow. Indeterminate reactions required further investigation. Dip mode can produce analytical problems for assays that measure (or are interfered by) the contents of test strips.

Antibodies to small ubiquitin-like modifier activating enzyme are associated with a diagnosis of dermatomyositis: results from an unselected cohort

The aim of this study was to examine the frequency and significance of antibodies targeting the small ubiquitin-like modifier 1 activating enzyme (SAE) in patients under serologic evaluation for idiopathic inflammatory myopathies. Patient sera (n = 17) recognizing bands at approximately 40 (SAE1) and 90 (SAE2) kDa were identified in 6445 consecutive samples for myositis autoantibody evaluation by immunoprecipitation (IP) of S35-labeled K562 cell lysate. All 17 positive samples, 176 disease, and 67 healthy controls were evaluated for SAE1 antibodies using a line immunoblot assay (LIA). Clinical data of SAE antibody-positive patients were obtained by retrospective chart review. Positivity with both methods was associated with a diagnosis dermatomyositis with characteristic skin manifestations of varying severity and muscle involvement. Majority of the patients were female (73.7%), mean age of 55.0 (range 12.0–82.0) years at the time of testing. Using the IP as reference, the SAE1 LIA had a sensitivity of 100% (95% CI 82.4–100%), specificity of 99.6% (95% CI 97.7–100%), positive predictive value of 95.0% (95% CI 75.1–99.9%), and negative predictive value of 100% (95% CI 98.5–100%). This study confirms the association of SAE antibodies in patients with dermatomyositis. A combination of IP and the LIA specific for SAE1 may be useful in antibody detection.

46 Evaluation of Hemolysate Hemoglobin to Estimate Patient Hematocrit in RBC Folate Testing

immunoassays before and after treatment of samples with heterophile blocking reagent (HBR) nonmurine solution (Beckman assay) and heterophile blocking tubes (HBT) (Roche assay; Scantibodies Laboratory). The absolute difference and the percent difference between untreated and treated results were calculated. Serial dilutions were also performed (primary method) and percent recovery calculated. Results: From the 112 physician requests, five cases of HA interference were identified. The HA frequency was 6.7% (n = 3) for the Beckman assay and 3.0% (n = 2) for the Roche assay. The range of hCG concentrations on all the evaluated samples was 0.1-2,797.0 IU/L (mean 71.1 IU/L; median: 10.2 IU/L). The presence of HA was detected using HBR/HBT reagents in three cases with a percent difference from the untreated sample of –80%, –51%, and –64% from the initial value of 13, 5.5, and 9 IU/L, respectively. The two cases not detected by HBR/ HBT reagents were detected due to discrepant results with the secondary method (50.44 vs 1.74 IU/L and 56.6 vs <1.0 IU/L) and nonlinear serial dilution (recovery outside 80%-120%). HA-negative cases showed an absolute difference of ±0.9 IU/L at <10 IU/L and percent difference ≤+/–20% from the untreated at concentrations >10 IU/L for the Beckman assay; and an absolute difference of ±1.2 U/L at <10 IU/L and a ≤+/–10% difference from the untreated at concentrations >10 IU/L for the Roche assay. Conclusions: Frequency of HA interference was similar between the Beckman and the Roche assays (6.7 vs 3.0%, P = .357). The HBR/HBT blocking reagents failed to detect 40% of HA interference cases and should not be solely used for these investigations. Multiple strategies including the use of serial dilutions and measurement using an alternative platform are critical to identify the presence of HA.