Evaluation of an adenosine deaminase (ADA) assay in serum, pleural, pericardial, peritoneal, and cerebrospinal fluids on the Roche cobas c501 analyzer.

OBJECTIVES: Adenosine deaminase (ADA) can be increased in various body fluids during infectious and inflammatory states. The objective of this study was to evaluate the performance characteristics of the Diazyme ADA assay for serum, pleural, pericardial, peritoneal, and cerebrospinal fluids using the Roche cobas c501 analyzer. METHODS: Accuracy, linearity, recovery, precision, sensitivity, specificity, reference interval, and stability studies were conducted. Potential interference of hyaluronidase and ultracentrifugation pre-treatment for viscosity on ADA concentrations were further evaluated. RESULTS: Assay method comparison to two separate external laboratories showed the following results (slope, intercept, %bias): serum (1.053, -0.478, 4.4 %); pleural (1.046, -1.41, 2.6 %). Accuracy (109.6 % recovery) was further demonstrated using a commercially available ADA reference material (BCR647). Linearity and spiked recovery studies showed percent recoveries ranging 94.3-109.3 %. Precision across all specimen types was ≤4.7 %CV. Interference was observed with increasing concentrations of various sources of conjugated and unconjugated bilirubin. Reference intervals were established for serum and pleural fluids, and previously published reference intervals were verified for pericardial, peritoneal, and cerebrospinal fluids. All specimen types were stable for 24 h ambient (8-25 °C), 1 week refrigerated (2-8 °C), and 1 month frozen (-20 °C). Of the two types of hyaluronidase evaluated, one showed positive interference for ADA (Sigma-Aldrich, H3506; 4.59 to 17.90 average % difference from baseline). Ultracentrifugation did not interfere with results (-2.32 to 0.87 average % difference from baseline). CONCLUSIONS: The Diazyme ADA assay was validated for use in our laboratory for all fluid types evaluated. Interference was observed with increasing concentrations of bilirubin and one source of hyaluronidase.

Tobacco and Cannabis Use During Pregnancy.

OBJECTIVES: Nicotine (NIC) use during pregnancy can influence markers used in biochemical maternal serum screening. This study was designed to determine prevalence of disclosed tobacco smokers in our patient population and to compare disclosed tobacco smoking status with the presence of serum nicotine and a common tetrahydrocannabinol (THC) metabolite. METHODS: A deidentified dataset of disclosed smoking status for quadruple (Quad) screens was obtained. Residual serum submitted for Quad screens was obtained from frozen storage and analyzed for NIC and THC metabolites. RESULTS: Of specimens that had corresponding responses to the smoking history question on the patient history form, 7.2% (n = 1,783 of 24,611) specified that the patient was a tobacco smoker. Of the 271 specimens biochemically analyzed for NIC and THC metabolites, disclosed tobacco smokers had the highest prevalence of detectable NIC and THC metabolites. THC product use was most prevalent in patients categorized as probable tobacco smokers based on cotinine concentrations, as well as in younger patients. CONCLUSIONS: Prevalence and concentration of NIC and THC metabolites vary based on disclosed tobacco smoker status. Biochemical testing may increase sensitivity for the identification of NIC and THC status over self-reporting.

Excessively low cholesterol and triglyceride levels in an apparently healthy patient.

Lipid panels are a commonly performed test in clinical laboratories. Due to the high prevalence of cardiovascular diseases around the world, it is common to see serum or plasma specimens with high results for one or more components of the lipid panel. Exceedingly low results, however, are rare and may be attributed to certain genetic, infectious, or autoimmune conditions in addition to analytical interference. Here we report a serum specimen from a 58-year-old female with cholesterol and triglyceride values below the detection limit of the assay, which was investigated to identify the cause of the anomaly. Using vitamin C test strips and high-performance liquid chromatography, the presence of high levels of antioxidant vitamin C in the patient specimen was confirmed. Subsequent treatment of the sample with the enzyme ascorbate oxidase inactivated vitamin C, leading to lipid analyte values falling within the expected range upon repeat analysis. Thus, analytical interference by vitamin C should be considered when suspiciously low lipid panel results are encountered.

Evaluation of L-index interference limits on Roche cobas c502 and c702 immunoturbidimetric assays using endogenously lipemic specimens and intralipid spiking.

OBJECTIVES: Specimen lipemia is a primary concern with turbidimetric and nephelometric assays due the potential interference caused by light scattering or absorption. The purpose of this study was to evaluate lipemic interference thresholds across seven FDA-cleared assays using patient specimens with varying degrees of endogenous lipemia pre- and post-ultracentrifugation (UC) and with Intralipid spiking. METHODS: Using an IRB-approved protocol, residual human serum specimens (n=42; L-indices, 1-1769; H-index ≤85; I-index ≤2) were obtained. Baseline and post-UC testing was conducted across assays on cobas c502 and c702 instruments (Roche Diagnostics; Indianapolis, IN). Serum indices and triglyceride (TRIG) concentrations were also measured pre- and post-UC. Intralipid spiking studies with human AB serum were also conducted. Lipoprotein subfraction analysis (Lipoprint; Quantimetrix; Redondo Beach, CA) was performed on three additional patient specimens with elevated TRIG post-UC to determine which TRIG-containing lipoprotein fraction(s) remain. RESULTS: Several assays showed L-index thresholds derived from endogenously lipemic specimens that were below previously defined limits from the package inserts (PIs) [new (prior)]: AAT 400 (500); CERU 100 (200); HAPTO 450 (600); TRSF 250 (500). L-index limits derived from Intralipid spiking were generally higher than those listed in PIs. UC did not adversely impact results in non-lipemic or lipemic specimens. UC was effective at clearing lipemic interference, although persistence of residual VLDL was often observed. CONCLUSIONS: This study provides an analysis of L-index thresholds for seven immunoturbidimetric assays. Due to the variety of human lipoproteins, limits defined using endogenously lipemic patient specimens may be different from those derived from spiking studies using Intralipid.

Alpha-fetoprotein in pericardial, peritoneal, and pleural fluids: A body fluid matrix evaluation.

OBJECTIVES: Alpha-fetoprotein (AFP) measurement in pericardial, peritoneal (ascites), and pleural fluids is sometimes requested by clinicians as supportive evidence in the evaluation of suspected malignancy. As commercially available, Food and Drug Administration (FDA)-cleared AFP assays are not validated for these fluid types, laboratories must complete additional validation studies to comply with regulatory requirements for body fluid testing. The objective of this study was therefore to conduct a matrix evaluation for these body fluid types using the Beckman Access AFP assay on the UniCel DxI 800 immunoassay system. DESIGN AND METHODS: Using an Institutional Review Board (IRB) approved protocol, previously collected pericardial fluid, peritoneal fluid, pleural fluid, and serum specimens were de-identified and frozen at -20 °C prior to matrix evaluation experiments. Spiked recovery, mixed recovery/linearity, and precision studies were conducted. RESULTS: In spiked and mixed recovery studies, the average percent (%) recovery was within predefined acceptable limits (±15%) for all three body fluids. Linearity was observed over the analytical measurement range (AMR) for all three body fluids (slope, intercept, systematic error): pericardial 0.988, -0.1, 6.1%; peritoneal 0.986, 0.0, 4.1%; and pleural 1.016, 0.0, 1.6%. Imprecision was ≤6.0% CV for all three body fluids at both high and low AFP concentrations. CONCLUSIONS: Matrix interference with AFP testing was not observed for pericardial, peritoneal, or pleural fluids on the Beckman UniCel DxI 800 system.

Lipemic interference of ceruloplasmin assays - An evaluation of lipid removal methods.

BACKGROUND: The present studies were conducted to characterize lipemic interference across three FDA-cleared ceruloplasmin (CERU) assays and to evaluate procedures designed to remove lipemic interference. METHODS: CERU assays on the Abbott ARCHITECT ci8200, Beckman AU5800, and Roche cobas Integra 400 Plus were evaluated. Precision, linearity with dilution, lipemic interference, and three methods for removing lipemia were assessed on each platform: ultracentrifugation (UC), lipemia-clearing reagent LipoClear (LC), and 1:5 dilution (DIL). Lipemia-index (L-index) thresholds were established using endogenously lipemic specimens and sera spiked with human-derived triglyceride-rich lipoproteins. RESULTS: The ci8200 showed greater susceptibility to endogenous lipemic interference than would be expected based on vendor-derived limits established with Intralipid. Endogenous lipemia causes a negative interference on the ci8200 and a positive interference on the Integra. UC was generally the most reliable method of removing lipemic interference without impacting baseline CERU results. CONCLUSIONS: CERU assays on different platforms have varying susceptibility to lipemic interference. L-index thresholds derived using Intralipid may not accurately represent interference caused by endogenous lipemia.

Evaluation and analytical validation of a handheld digital refractometer for urine specific gravity measurement.

OBJECTIVES: Refractometers are commonly used to determine urine specific gravity (SG) in the assessment of hydration status and urine specimen validity testing. Few comprehensive performance evaluations are available demonstrating refractometer capability from a clinical laboratory perspective. The objective of this study was therefore to conduct an analytical validation of a handheld digital refractometer used for human urine SG testing. DESIGN AND METHODS: A MISCO Palm Abbe™ refractometer was used for all experiments, including device familiarization, carryover, precision, accuracy, linearity, analytical sensitivity, evaluation of potential substances which contribute to SG (i.e. "interference"), and reference interval evaluation. A manual refractometer, urine osmometer, and a solute score (sum of urine chloride, creatinine, glucose, potassium, sodium, total protein, and urea nitrogen; all in mg/dL) were used as comparative methods for accuracy assessment. RESULTS: Significant carryover was not observed. A wash step was still included as good laboratory practice. Low imprecision (%CV, <0.01) was demonstrated using low and high QC material. Accuracy studies showed strong correlation to manual refractometry. Linear correlation was also demonstrated between SG, osmolality, and solute score. Linearity of Palm Abbe performance was verified with observed error of ≤0.1%. Increases in SG were observed with increasing concentrations of albumin, creatinine, glucose, hemoglobin, sodium chloride, and urea. Transference of a previously published urine SG reference interval of 1.0020-1.0300 was validated. CONCLUSIONS: The Palm Abbe digital refractometer was a fast, simple, and accurate way to measure urine SG. Analytical validity was confirmed by the present experiments.

Clinical performance evaluation of total protein measurement by digital refractometry and characterization of non-protein solute interferences.

OBJECTIVES: Refractometric methods to measure total protein (TP) in serum and plasma specimens have been replaced by automated biuret methods in virtually all routine clinical testing. A subset of laboratories, however, still report using refractometry to measure TP in conjunction with serum protein electrophoresis. The objective of this study was therefore to conduct a modern performance evaluation of a digital refractometer for TP measurement. DESIGN AND METHODS: Performance evaluation of a MISCO Palm Abbe™ digital refractometer was conducted through device familiarization, carryover, precision, accuracy, linearity, analytical sensitivity, analytical specificity, and reference interval verification. Comparison assays included a manual refractometer and an automated biuret assay. RESULTS: Carryover risk was eliminated using a demineralized distilled water (ddH2O) wash step. Precision studies demonstrated overall imprecision of 2.2% CV (low TP pool) and 0.5% CV (high TP pool). Accuracy studies demonstrated correlation to both manual refractometry and the biuret method. An overall positive bias (+5.0%) was observed versus the biuret method. On average, outlier specimens had an increased triglyceride concentration. Linearity was verified using mixed dilutions of: a) low and high concentration patient pools, or b) albumin-spiked ddH2O and high concentration patient pool. Decreased recovery was observed using ddH2O dilutions at low TP concentrations. Significant interference was detected at high concentrations of glucose (>267 mg/dL) and triglycerides (>580 mg/dL). Current laboratory reference intervals for TP were verified. CONCLUSIONS: Performance characteristics of this digital refractometer were validated in a clinical laboratory setting. Biuret method remains the preferred assay for TP measurement in routine clinical analyses.

Detection of macro-creatine kinase and macroamylase by polyethylene glycol precipitation and ultrafiltration methods.

BACKGROUND: Macroenzymes may cause elevations in serum enzyme activity. Macroenzymes are not common; however their detection is important because they cause diagnostic confusion and therapeutic errors. METHODS: We analyzed 2 of the most prevalent macroenzymes in the literature, macro-creatine kinase (macro-CK) and macroamylase, using 2 methods for detection, polyethylene glycol (PEG) precipitation and ultrafiltration (UF). Enzyme measurements were made using a Roche Modular Analytics P analyzer. Imprecision was assessed using quality control material. We evaluated 125 samples from apparently healthy subjects to establish reference intervals. For macro-CK comparison, 94 samples with activities >200 U/l were analyzed with both PEG precipitation and UF and compared to electrophoresis. PEG precipitation and UF were compared for macroamylase detection using 130 samples with amylase activities >110 U/l. RESULTS: UF was more precise and demonstrated narrower reference intervals for both analytes. PEG precipitation and UF were able to detect true cases of macro-CK with overall agreement with electrophoresis of 79.8% and 80.9%, respectively. Both methods detected the same number of 'positive' macroamylase samples; however PEG precipitation resulted in a greater number of 'indeterminate' cases. CONCLUSION: This is the first report where UF has been shown useful for the detection of both macro-CK and macroamylase.

Performance characteristics of three random access cyclic citrullinated peptide antibody assays.

BACKGROUND: Cyclic citrullinated peptide antibodies (CCP Ab) are useful biomarkers for the early detection and diagnosis of rheumatoid arthritis (RA). METHODS: We evaluated the performance of 3 random access 2nd generation CCP Ab assays, the Abbott AxSYM and Architect analyzers and the Roche Modular Analytics E170 analyzer, for limit of detection (LOD), imprecision, results for samples from healthy subjects, analytic concordance, and interferences. Method comparison testing was performed using the AxSYM analyzer as the comparison method and a 3rd generation INOVA Quanta Lite ELISA assay was included. RESULTS: LOD determinations met the manufacturers' claims. Total CVs ranged from 1.6% to 8.2%. Results from healthy subjects were generally much lower than the manufacturers' decision cutoffs. Comparison to the AxSYM assay resulted in overall concordance ranging from 82.1% to 98.3%. These assays were resistant to interference from hemolysis, icterus, lipemia and rheumatoid factor. CONCLUSION: All 3 random access CCP Ab assays performed according to the manufacturers' claims and have the potential to improve workflow in clinical laboratories.