Impact of Unconjugated estriol (uE3) assay interference on prenatal screening tests.

BACKGROUND: Unconjugated estriol (uE3) is an important biomarker in second trimester prenatal screening. Previous studies from our laboratory identified rare interference in the Beckman uE3 assay due to anti-ALP antibodies, which could be mitigated with a scavenger or heat-inactivated ALP (hALP). In the current study, 160 de-identified patient samples previously submitted for the Quad screen with low uE3 multiples of the median (MoM ≤0.50) were investigated for potential interference. METHODS: A reagent pack spiking strategy with hALP was employed to understand if the interference could be identified and mitigated in a scalable manner. The 160 samples were measured using uE3 lot #920861 previously known to be subject to interference, lot #920861 spiked with hALP, and the vendor reformulated lot #922579. Samples were suspected to have interference if the percent difference in uE3 measurements was >50%. Pseudo-risks were calculated using a test patient environment to understand the screening impact due to the change in uE3 result. RESULTS: Seventeen of the 160 samples had uE3 results that were >50% different between the hALP spiked and non-spiked reagent pack. Both original lot #920861 with hALP and reformulated lot #922579 identified the same 17 patients as having interference in lot #920861. Analysis of screening risks using a test patient environment showed that assay interference could result in false positives for one trisomy 21 and three trisomy 18 post-test risk calculations. CONCLUSION: Our experiment of reagent pack spiking with hALP produced similar uE3 results to a reformulated reagent designed to address potential interference, demonstrating that this is a feasible strategy to screen for interference in a scalable manner. The vendor-provided reformulation addressed anti-ALP interference and improved the performance of the screen.

Clinical and Analytical Characterization of the DiaSorin and ScheBo Fecal Pancreatic Elastase 1 Assays.

OBJECTIVES: Fecal pancreatic elastase (PE) assays are screening tests for exocrine pancreatic insufficiency (EPI). We analytically evaluated a new PE assay and retrospectively analyzed data from an academic hospital and reference laboratory to understand the clinical utility. METHODS: Forty stool samples with different PE concentrations were tested on the ScheBo enzyme-linked immunosorbent assay (ELISA) versus DiaSorin LIAISON immunoassay; a simple-to-use extraction device was assessed. The cross-reactivity of porcine enzymes was investigated in the immunoassay. Charts of 207 patients with PE results less than 250 µg/g at an academic hospital were reviewed, and data were analyzed for 5136 patients with repeat PE results from a reference laboratory. RESULTS: The LIAISON immunoassay gave comparable results to the ScheBo ELISA, with 87.5% agreement of PE results in classifying as sufficient, mild/moderate insufficiency, or severe insufficiency. The extraction device worked well compared with manual weighing, and no cross reactivity with porcine enzymes was observed. In agreement with prior studies, our clinical data suggested that PE assays were most useful in detecting severe EPI. CONCLUSIONS: The new DiaSorin LIAISON immunoassay preforms similarly to the well-known ScheBo ELISA. Pancreatic elastase assays can help identify patients with severe EPI but are not as useful in classifying mild/moderate EPI.

Comparison of Next-Generation Assays for Fecal Calprotectin vs the PhiCal Assay.

OBJECTIVES: To compare the PhiCal assay (CALPRO), the first US Food and Drug Administration-approved assay for fecal calprotectin, to 4 next-generation assays. METHODS: Stool samples from 50 patients were selected, and relevant clinical information was collected. Comparisons were performed using the PhiCal, fCAL turbo (BÜHLMANN), LIAISON Calprotectin (DiaSorin), QUANTA Lite Calprotectin ELISA (Inova Diagnostics), and Calprotectin Chemiluminescence ELISA (ALPCO) assays. RESULTS: All 4 assays had acceptable agreement with PhiCal when qualitatively categorizing results. Within the PhiCal reportable range of 16 to 1,250 µg/g, the DiaSorin, Inova Diagnostics, and ALPCO assays had Spearman correlation coefficients of 0.98, 0.97, and 0.95 and positive biases of 17%, 20%, and 15%, respectively. The BÜHLMANN assay ran approximately 2-fold higher than the PhiCal assay but had a correlation coefficient of 0.98, with similar result categorization. CONCLUSIONS: Our results demonstrate good comparison between PhiCal and 4 next-generation assays. Laboratories performing fecal calprotectin assays may have compelling reasons to adopt next-generation fecal calprotectin testing, such as greater automation, a decreased number of replicates needed per test, and the use of stool-extraction devices. These benefits could decrease turnaround times and lower costs. Although the results of the assays correlated, they are not standardized. Laboratories adopting the newer assays will need to further investigate their performance through validation studies.

Performance evaluation of five lipoprotein(a) immunoassays on the Roche cobas c501 chemistry analyzer.

OBJECTIVES: Measurement of lipoprotein(a) [Lp(a)] is used in risk assessment of atherosclerotic cardiovascular disease (ASCVD). The aim of the current study was to evaluate performance characteristic of five different Lp(a) assays using the cobas c501 (Roche Diagnostics) analyzer. DESIGN AND METHODS: Lp(a) was measured using five Lp(a) assays (Diazyme, Kamiya, MedTest, Randox, and Roche) configured to mg/dL units. Assays from Diazyme and Kamiya were also configured using nmol/L units in separate experiments. Studies included sensitivity, imprecision, linearity, method comparison, and evaluation of healthy subjects. Imprecision (intra-day, 20 replicates; inter-day, duplicates twice daily for five days) and linearity were evaluated using patient pools. Linearity assessed a minimum of five patient splits spanning the analytical measurement range (AMR). Method comparison used 80 residual serum samples. Specimens from 120 self-reported healthy subjects (61 females / 59 males) were also tested. Method comparison for two assays in nmol/L units was conducted using 96 residual serum samples. RESULTS: Assay sensitivities met all manufacturer claims. Imprecision studies demonstrated %CVs ranging from 2.5 to 5.2% for the low pool (average concentration from 7.3 to 12.4 â€‹mg/dL); high pool %CVs ranged from 0.8 to 3.0% (average concentrations from 31.5-50.2 â€‹mg/dL). Linearity was confirmed for all assays. Variation in accuracy was observed when comparing results to an all method average. Lp(a) results were higher in females versus males in self-reported healthy subjects. CONCLUSIONS: All assays performed according to manufacturer described performance characteristics, although differences were observed across Lp(a) assays tested when compared to an all method average.

Clinical and Analytical Verification of an Automated Fecal Calprotectin Immunoassay with Extraction Device.

BACKGROUND: Fecal calprotectin (FC) is a screening test for intestinal inflammation, and often used by clinicians to help identify and monitor patients with inflammatory bowel disease (IBD). Improvements in FC assays include moving to more automated immunoassays compared to ELISAs and simple-to-use extraction devices compared to manual weighing for the extraction process. METHODS: A method comparison was performed between the PhiCal ELISA and LIAISON immunoassay for 53 stool samples, and the screening results were compared to the gold standard endoscopy with biopsy results. Clinical accuracy was assessed by comparing the FC results from each assay to the presence or absence of inflammation determined from the biopsy report. The performance of the extraction device was compared to manually weighing. Additional studies were completed to verify the manufacturer's claims. RESULTS: The FC results were compared to the biopsy results for detecting inflammation. PhiCal ELISA had a sensitivity of 86% and specificity of 100%, while the LIAISON immunoassay had a sensitivity of 97% with specificity of 94%. Therefore, the LIAISON immunoassay performed better than the PhiCal ELISA. The extraction device performed well compared to manual weighing if stool samples were <800 µg/g, within Bristol stool types 2-6, and did not contain a significant amount of undigested material, fibrous material, or mucus. CONCLUSION: The LIAISON immunoassay with extraction device has acceptable performance for clinical use in measuring fecal calprotectin.

Detection and characterization of estradiol (E2) and unconjugated estriol (uE3) immunoassay interference due to anti-bovine alkaline phosphatase (ALP) antibodies.

OBJECTIVES: Competitive immunoenyzmatic assays for estradiol (E2) and unconjugated estriol (uE3) on UniCel DxI 800 Access immunoassay systems (Beckman Coulter) utilize bovine alkaline phosphatase (ALP) for amplification. In these assays, rare 'IND' error flags indicate that a relative light unit (RLU) raw result is past the high or low end of the calibration curve but cannot be differentiated from an instrument error or analytical interference. The present studies were conducted to establish a protocol to identify analytical interference and to characterize its mechanism when present. DESIGN AND METHODS: Matrix and recovery studies were conducted to establish a protocol for interference identification. Spiking experiments with inactivated calf intestinal ALP were performed to determine whether interference could be blocked. Commercial anti-ALP antibodies (Abs) were spiked into human serum to model assay interference. Three E2 immunoassays which do not include ALP as a reagent component (cobas e602, Roche; Centaur XP, Siemens; ARCHITECT i2000SR, Abbott) were tested for comparative purposes. RESULTS: 1:2 dilution of specimen into Access Sample Diluent A (Beckman) differentiated IND error flags due to true low results (e.g. less than the analytical measurement range; AMR) from those due to assay interference. Interferences were reduced by pre-incubation with inactivated ALP and could be replicated by spiking with commercial anti-ALP Abs. CONCLUSIONS: Patient anti-bovine ALP Abs can cause interference on DxI 800 E2 and uE3 assays. This model can be used to investigate interference risk with other ALP-dependent assays.

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.

Identifying and Eliminating Laboratory Contamination by Topical Testosterone Therapeutics.

Many prescription and over-the-counter drugs are available as topical formulations. Contamination of clinical laboratory workspaces by topical drugs may increase the risk of potential interference with diagnostic testing. An example of localized workspace contamination attributed to a topical hormonal drug (testosterone, T) is presented to highlight significant challenges in identifying and resolving this potential problem. Investigation included precision studies, instrument service and parts replacement, instrument replacement, airflow analysis, environmental dust sampling, and the development of customized methods for workspace monitoring and cleaning. Laboratory policies and procedures were also revised to minimize future risk.

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.

Performance characteristics of six automated 25-hydroxyvitamin D assays: Mind your 3s and 2s.

BACKGROUND: Measurement of 25(OH)D has evolved rapidly, with an increased number of high-throughput automated immunoassays becoming available in recent years. The aim of this study was to fully evaluate six commercially available automated 25(OH)D immunoassays, including 2 newly available (Beckman Coulter) and one recalibrated (Siemens) assay. Comparisons were made to specifically identify the effect of the absence or presence of 25(OH)D2 on these assays. DESIGN AND METHODS: Access2 and UniCel DxI 800 (Beckman Coulter), ARCHITECT i2000SR (Abbott Diagnostics), ADVIA Centaur XP (Siemens), Liaison XL (DiaSorin) and MODULAR E170 (Roche Diagnostics) assays were assessed for accuracy, imprecision, interference, limit of blank, and linearity. All were compared to an in-house LC-MS/MS method (traceable to NIST SRM 972) using Passing-Bablok regression and Bland-Altman bias plots. Method comparisons used residual serum samples with both endogenous 25(OH)D2 and 25(OH)D3 (n=50) or 25(OH)D3 only (n=86). Comparisons with all 136 samples were intended to simulate real-world laboratory testing. RESULTS: The majority of assays under-recovered 25(OH)D in comparison to LC-MS/MS, with three of six immunoassays affected by the presence of 25(OH)D2. Imprecision was greatest at 25(OH)D concentrations near the decision limits used to assess deficiency. Only two of six immunoassays would meet the recommended bias criteria of <5%. CONCLUSIONS: Although standardization efforts continue, these differences in performance remain a concern. Clinicians should be aware when comparing results among assays and using them to determine adequacy of 25(OH)D stores in various patient populations.

Multiple macroenzymes in a patient with AIDS: diagnosis using ultrafiltration.

OBJECTIVES: Multiple immunoglobulin-bound enzymes (macroenzymes) are reported for the first time in an individual with AIDS. Possible causes and suitable methods of detection are addressed. METHODS: An asymptomatic man with a history of AIDS with hypergammaglobulinemia and elevated creatine kinase, amylase, and liver enzyme concentrations was evaluated before enrollment in a clinical trial. Macroenzymes were considered a possible source of these elevated concentrations. RESULTS: Polyethylene glycol (PEG) precipitation and ultrafiltration (UF) were used to evaluate the presence of seven macroenzymes. PEG results suggested the presence of six of seven macroenzymes tested, while UF revealed three. UF results supported the clinical presentation. CONCLUSIONS: A previous report shows that in cases of excess immunoglobulin, PEG coprecipitates monomeric enzymes along with serum globulins, causing false-positive reporting of macroenzymes. This may explain the discrepancy between PEG and UF results in the presence of hypergammaglobulinemia, making UF a better method of detection in these circumstances.

Human chorionic gonadotropin discriminatory zone in ectopic pregnancy: does assay harmonization matter?

OBJECTIVE: To determine the effect that lack of hCG assay harmonization has on the interpretation of a serum hCG concentration with regards to the hCG discriminatory zone. DESIGN: A multisite method comparison study. SETTING: Clinical laboratories. PATIENT(S): Eighty serum samples containing various concentrations of hCG. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Concentrations of hCG obtained from seven hCG reagent platforms. RESULT(S): The hCG concentrations were significantly different across hCG reagent platforms. Seventy-one percent of assay pairs showed significant differences with samples selected based on hCG concentrations between 1,500 and 3,500 IU/L as determined by a comparative method. Relative to the comparative method, the calculated hCG discriminatory zones for five assays were within 9%, and one assay was within 40% of the target concentrations of 1,500 and 3,500 IU/L. CONCLUSION(S): Despite significant differences in hCG concentrations across hCG immunoassays, an hCG concentration within a discriminatory zone of 1,500-3,500 IU/L can be used for all but one commonly used hCG reagent platform.

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.