The importance of verifying manufacturer's claim on specimen stability: An example in serum angiotensin converting enzyme testing.

Appropriate specimen handling is integral to quality and minimizing medical errors. Clinical laboratories often rely on manufacturer's claims for handling specimens, such as sample stability conditions. Serum angiotensin converting enzyme (ACE) is an example in which manufacturer claims and stability in the literature is limited. The purpose of this study was to demonstrate the importance to verify manufacturer's stability using serum ACE as an example. Serum was collected from 39 healthy volunteers and ACE activity levels measured at baseline, after 4 h, 1, 3, 7 days at room temperature, after 3, 7, and 14 days refrigerated at 4 °C, after 1, 2, 4 and 8 weeks frozen at -20 °C, and after three freeze/thaw cycles. An additional 42 discarded patient serum specimens were re-analyzed after 1 or 2 weeks frozen at -20 °C. To evaluate stability performance, percent difference was compared to the clinical acceptance criteria, which was defined as a ½ total allowable error of ±10.9 %. This study found serum ACE to be stable 4 h at room temperature, 14 days refrigerated at 4 °C, up to 1 week frozen at -20 °C, and up to three freeze/thaw cycles. The preferred storage condition for serum ACE is refrigerated at 4 °C as there was minimal change in percent bias over the 14 day period. The false increase observed in samples stored frozen longer than 1 week could impact clinical decision making. The stability findings differed from manufacturer claims, highlighting the importance of verifying stability, especially for esoteric testing such as serum ACE where specimens travel long distances in varying climates to reach centralized testing locations.

Centralization of multisite reagent lot-to-lot validation for Ortho Clinical Vitros chemistry instruments.

OBJECTIVE: Reagent lot-to-lot comparisons are recommended by accreditation bodies to ensure that the performance of each reagent lot meets acceptable standards for quality patient results. The general approach is comprised of performing quality control (QC) and patient comparison between the old and new reagent lots and evaluating against a pre-defined criteria. Reagent lot comparison practices are often variable despite using the same instrument across different laboratories. This is costly, time consuming, and can lead to variability in acceptance criteria. While Clinical & Laboratory Standards Institute (CLSI) has a recommended guideline for reagent lot validation, it is often difficult to execute for small and rural laboratories due to limited resources. Defining the analytes required for detailed validation is important to allocate appropriate resources to ensure quality patient results. The goal of this study was to develop a standardized approach to reagent lot validation and optimize lab resources on Vitros chemistry instruments. DESIGN AND METHOD: This study consists of a retrospective and prospective analysis of reagent lot changes in dry slide chemistry analyzers (Ortho Clinical Diagnostics Vitros). Two years of retrospective reagent lot comparison data was obtained at a single site. A prospective study was conducted by assessing aliquots of 10 patient sample pools at 9 sites with Vitros analyzers. RESULTS: Of the 19 chemistry analytes evaluated, albumin, sodium, and total protein showed significant differences between reagent lots and also exceeded the pre-defined acceptance criteria. CONCLUSION: For these analytes, our recommendations are to perform a comprehensive lot validation with QC and patient samples. A simple lot validation with a reflex approach comprised of initially assaying QC can be adapted for the more stable analytes to allow achieving quality patient result in a resource constraint rural environment.

Multi-platform analytical evaluation of the Beckman Coulter Access high-sensitivity troponin I assay across different laboratory sites using Barricor plasma.

OBJECTIVES: Previous analytical evaluations of the Beckman Coulter Access high sensitivity troponin (hsTn) I assay have focused on single platforms and laboratory sites. The purpose of this study was to determine assay robustness across different platforms at multiple sites, platform-specific characteristics, and equivalence to other hsTn methods in a large laboratory network. METHODS: Barricor plasma was used to assess imprecision, linearity, sensitivity (limit of blank and detection, LOB/LOD), and comparability to the conventional AccuTnI+3 and other hsTn assays. Various studies were conducted across a total of 9 laboratories using Beckman DxI800 and Access2 platforms. RESULTS: Within-laboratory precision was <10% across all target patient pool concentrations, however, DxI800 mean values were 20% higher than Access2 in the range of 3.6-44.9 ng/L. LOBs and LODs were lower on DxI800, 0.27 and 0.90 ng/L, respectively, compared to 2.9 and 3.2 ng/L, on Access2. Both showed excellent linearity across the full range. In method comparison to AccuTnI+3, DxI800 had a higher slope (0.9417 versus 0.8495) and positive bias (+18.1% versus -9.9%) compared to Access2, a trend further pronounced at concentrations <150 ng/L. At values <150 ng/L, there was good agreement with Abbott hsTnI (slope = 1.017, r = 0.932), but poor agreement with the Roche hsTnT assay (slope = 1.687, r = 0.589). Inter-laboratory split sample comparisons across 2 DxI800 and 7 Access2 sites showed close agreement, except at low concentrations <10 ng/L where DxI800 was 2.8 ng/L higher (p<0.001). CONCLUSIONS: The Beckman hsTnI assay showed robust analytical performance across different laboratories and platforms. However, discrepancies between platforms were found at low concentrations where rapid acute myocardial infarction (AMI) rule-out decisions occur. These differences have important implications for AMI risk assessment, suggesting that laboratories should develop platform-specific parameters rather than using them interchangibly.

A high-throughput test for diabetes care: An evaluation of the next generation Roche Cobas c 513 hemoglobin A1C assay.

OBJECTIVES: The level of glycated hemoglobin A (HbA1C) in blood is the preferred marker for diabetes monitoring and treatment. Here, we evaluate the analytical performance of the Roche Diagnostics Cobas c 513, a stand-alone HbA1C immunoassay analyzer. DESIGN AND METHODS: Performance was assessed with regards to imprecision, accuracy, linearity, method comparison against the Roche Cobas Integra 800 CTS, specimen stability, interference from common hemoglobin variants and hemoglobin F, and throughput. RESULTS: Within-run and between-run precisions were 0.5-0.7 and 0.8-1.3%CV, respectively. An average bias of -1.6% to proficiency survey samples was observed. The c 513 correlated well with the Integra (slope = 0.94, y-intercept = 0.50, and correlation coefficient = 0.998). The effect of hemoglobin variants on this assay was negligible while specimens containing ≥10% HbF demonstrated a negative bias. The c 513 instrument can process up to 340 samples per hour. CONCLUSIONS: The c 513 is a precise, accurate, automated high throughput analyzer for measuring HbA1C in large laboratories.

Analytical Concordance of Diverse Point-of-Care and Central Laboratory Troponin I Assays.

BACKGROUND: Cardiac troponin I (cTnI) 99th percentile cutoffs, used in the diagnosis of acute myocardial infarction, are not standardized across cTnI assays. We compared 3 point-of-care (POC) and 1 central laboratory contemporary cTnI assays against the Abbott high-sensitivity (hs) cTnI to evaluate the analytical concordance and the feasibility of using a single cutoff value for all assays. METHODS: Fresh blood samples collected from 102 inpatients in the coronary care unit were measured on central laboratory instruments (Beckman Coulter DxI AccuTnI+3 TnI, Abbott Architect hs-TnI) and cTnI POC analyzers (Alere Triage Troponin I, Radiometer AQT90, Abbott i-STAT). Agreement and correlation between the contemporary cTnI assays and hs-cTnI assay were assessed using regression analysis. Proportional bias was assessed using Bland-Altman plots. Concordance between the contemporary cTnI and hs-cTnI assays was determined by diagnostic contingency tables at specific cutoffs. RESULTS: Most POC cTnI assays had excellent correlation with the Abbott hs-cTnI method (r 2 = 0.955-0.970) except for Alere Triage (r 2 = 0.617), while proportional bias is evident between all cTnI assays. Overall concordance between POC contemporary cTnI assays and hs-cTnI assay was 80% to 90% at their respective 99th percentile cutoffs. The concordance increased to 90% to 95% when a fixed cutoff of 0.03 to 0.05 ng/mL was used across the assays. CONCLUSIONS: This study demonstrates poor analytical concordance between cTnI assays at the 99th percentile and supports the notion of a single clinical decision limit for cTnI and consequently standardization of diagnostic protocols despite the analytical differences among these assays.

Analytical sensitivity and diagnostic performance of serum protein electrophoresis on the HYDRAGEL 30 PROTEIN(E) ß1-ß2 Sebia Hydrasys system.

BACKGROUND: Serum protein electrophoresis (SPE) and immunofixation electrophoresis (IFE) are used in the diagnosis and monitoring of plasma cell dyscrasias. IFE is considered the most sensitive method for the detection of monoclonal proteins (M-proteins), but it is not quantitative. The goal of this study was to establish the analytical sensitivity and diagnostic performance of SPE on the Sebia Hydrasys using HYDRAGEL 30 PROTEIN(E) ß1-ß2. METHODOLOGY: Patient sera with a previously identified M-protein (IgG, IgA or IgM) were serially diluted with a normal serum pool and electrophoresed on the Sebia Hydrasys using HYDRAGEL 30 PROTEIN(E) ß1-ß2. The SPE gels were individually interpreted by five independent observers and IFE was performed on selected samples. Limit of detection was determined as the lowest concentration of M-protein band visible on the gel. SPE diagnostic performance was evaluated against the "gold standard" IFE according CLSI EP12-A2 guidelines. RESULTS: Detection limit was comparable among all M-proteins migrating in the gamma region, IgG-κ (0.18±0.08g/L; n=6), IgG-λ (0.36±0.25g/L; n=8), IgA-κ (0.40±0.13g/L; n=7), IgA-λ (0.37±0.23g/L; n=4), IgM-κ (0.47±0.20g/L; n=13) and IgM-λ (0.29±0.24g/L; n=6). Percentage agreement with IFE for IgG and IgA in the gamma region ranged from 65% to 100%, whereas IgM migrating in the gamma region and immunoglobulins co-migrating with alpha or beta globulins, showed poor (0-38%) agreement. CONCLUSIONS: This study evaluates the analytical sensitivity and diagnostic performance of SPE on the Sebia Hydrasys using HYDRAGEL 30 PROTEIN(E) ß1-ß2. There was acceptable agreement between SPE and IFE for IgG-κ/λ and IgA-κ/λ migrating in the gamma region, suggesting that repeating IFE for samples with these isotypes, when the previous IFE and second SPE are both negative, may not be necessary.

The use of fructosamine in cystic fibrosis-related diabetes (CFRD) screening.

OBJECTIVE: To determine whether serum fructosamine correlates with glycemic control and clinical outcomes in patients being screened for cystic fibrosis-related diabetes (CFRD). METHODS: Fructosamine and percent predicted forced expiratory volume in 1s (FEV1) were measured in patients undergoing a 2h oral glucose tolerance test (OGTT) for CFRD screening. Fractional serum fructosamine (FSF) was calculated as fructosamine/total protein. RESULTS: FSF exhibited a positive correlation with 2h OGTT results (r2=0.3201, p=0.009), and ROC curve analysis suggested that FSF can identify patients with an abnormal OGTT (AUC=0.840, p=0.0002). FSF also exhibited a negative correlation with FEV1 (r2=0.3732, p=0.035). Patients with FSF≥3.70µmol/g had significantly lower FEV1 (median 47%) compared to those with FSF<3.70µmol/g (median 90%; p=0.015). CONCLUSIONS: FSF correlated with both OGTT results and FEV1, and reliably identified patients with abnormal OGTT results. This simple blood test shows potential as an effective tool in CFRD screening.

Evaluation of thyroid test utilization through analysis of population-level data.

BACKGROUND: Inappropriate laboratory test utilization can result in unnecessary patient testing and increased healthcare costs. While several thyroid function tests are available, thyroid-stimulating hormone (TSH) is recommended as the first-line test for investigating and monitoring thyroid dysfunction. We evaluate thyroid test utilization in Northern Alberta in terms of testing patterns, frequencies, and reflex cutpoints. METHODS: This retrospective study analyzed thyroid test requests from January to December 2014. Each request was designated as appropriate or potentially inappropriate as per clinical practice guidelines and Choosing Wisely recommendations, and the frequencies of each testing pattern were calculated. Sub-analysis was performed to categorize testing patterns based on physician specialty. The number of test requests per patient was determined to assess the appropriateness of testing frequency. Receiver operating characteristic (ROC) curves were generated to define optimal TSH cutpoints for automatic reflex to FT4 testing. RESULTS: Of 752,217 test requests, approximately 10% were potentially inappropriate in terms of testing patterns. Free thyroxine (FT4) and free triiodothyronine (FT3) requested with TSH accounted for 59% of all potentially inappropriate test requests, and 49% of requests from endocrinologists (ENDO) were potentially inappropriate, occurring most frequently among those with less experience. Excessive testing frequencies were observed in 869 patients, accounting for 9382 test requests. Adjustment of our TSH reflex cutpoint would significantly increase specificity for identifying a low FT4 without compromising sensitivity. CONCLUSIONS: This study suggests that questionable testing patterns, excessive testing frequencies, and suboptimal reflexive testing cutpoints contribute to inappropriate thyroid test utilization.

Erroneous HbA1c results in a patient with elevated HbC and HbF.

BACKGROUND: HbA1c is used in the diagnosis and monitoring of diabetes mellitus (DM). Interference from hemoglobin variants is a well-described phenomenon, particularly with HPLC-based methods. While immunoassays may generate more reliable HbA1c results in the presence of some variants, these methods are susceptible to negative interference from high concentrations of HbF. We report a case where an accurate HbA1c result could not be obtained by any available method due to the presence of a compound hemoglobinopathy. METHODS: HbA1c was measured by HPLC, immunoassay, and capillary electrophoresis. Hemoglobinopathy investigation consisted of a CBC, hemoglobin fractionation by HPLC and electrophoresis, and molecular analysis. RESULTS: HbA1c analysis by HPLC and capillary electrophoresis gave no result. Analysis by immunoassay yielded HbA1c results of 5.9% (Siemens DCA 2000+) and 5.1% (Roche Integra), which were inconsistent with other markers of glycemic control. Hemoglobinopathy investigation showed HbC with the hereditary persistence of fetal hemoglobin-2 Ghana deletion. CONCLUSION: Reliable HbA1c results may be unobtainable in the presence of some hemoglobinopathies. HPLC and capillary electrophoresis alerted the laboratory to the presence of an unusual hemoglobinopathy. Immunoassays generated falsely low results without warning, which could lead to missed diagnoses and under treatment of patients with DM.

Stability of specific IgE antibodies to common food and inhalant allergens.

OBJECTIVE: To determine the optimum storage temperature for serum allergen specific IgE antibodies (sIgE) to common food and inhalant allergens. METHODS: Patient sera with sIgE concentrations ≥0.7kIUA/l were pooled accordingly: pool 1-peanut and hazelnut, pool 2-egg white, cow's milk and cod fish, pool 3-soy, wheat and shrimp and pool 4-dust mite Dermatophagoides farinae, dog dander, cat dander, Timothy grass pollen, and silver birch pollen. Aliquots stored frozen, refrigerated and at room temperature were tested in duplicate (Phadia ImmunoCAP® 250) over two weeks. The relative difference was calculated for each sIgE as a percentage of the initial value and compared to the analytical reference change value. RESULTS: Minimal effects on specimen stability were noted for all sIgE analyzed under the three storage conditions tested in this study. All changes observed in sIgE concentrations were related to the assay variability and not to sample deterioration. CONCLUSION: Serum allergen specific IgE concentrations are stable at all temperatures studied for up to 17days.

Effects of hemoglobin C, D, E and S traits on measurements of hemoglobin A1c by twelve methods.

BACKGROUND: Hemoglobin C, D Punjab, E or S trait can interfere with hemoglobin A1c (HbA1c) results. We assessed whether they affect results obtained with 12 current assay methods. METHODS: Hemoglobin AA (HbAA), HbAC, HbAD Punjab, HbAE and HbAS samples were analyzed on one enzymatic, nine ion-exchange HPLC and two Capillary Electrophoresis methods. Trinity ultra(2) boronate affinity HPLC was the comparative method. An overall test of coincidence of least-squared linear regression lines was performed to determine if HbA1c results were statistically significantly different from those of HbAA samples. Clinically significant interference was defined as >7% difference from HbAA at 6 or 9% HbA1c compared to ultra(2) using Deming regression. RESULTS: All methods showed statistically significant effects for one or more variants. Clinically significant effects were observed for the Tosoh G8 variant mode and GX (all variants), GX V1.22 (all but HbAE) and G11 variant mode (HbAC). All other methods (Abbott Architect c Enzymatic, Bio-Rad D-100, Variant II NU and Variant II Turbo 2.0, Menarini HA-8180T thalassemia mode and HA-8180V variant mode, Sebia Capillarys 2 and Capillarys 3) showed no clinically significant differences. CONCLUSIONS: Several methods showed clinically significant interference with HbA1c results from one or more variants which could adversely affect patient care.

Hemoglobin Hirose: A rare beta chain variant causing falsely low HbA1c by HPLC.

OBJECTIVES: To investigate the underlying cause of unexpectedly low HbA1c results (3.3-3.5%) obtained by HPLC in three siblings undergoing routine screening for type 2 diabetes mellitus. DESIGN AND METHODS: HbA1c was measured using an alternate method based on a different analytical principle (the Siemens DCA 2000+ immunoassay). Hemoglobin fractionation was performed by HPLC on the BioRad Variant II, gel electrophoresis at acid and alkaline pH on the Sebia Hydrasys 2, and capillary electrophoresis on the Sebia Capillarys 2. Sequencing of the beta globin gene was also conducted. RESULTS: HbA1c analysis by immunoassay gave significantly higher results, ranging from 5.2-5.5%. Hemoglobin fractionation by HPLC showed an abnormal peak comprising approximately 43% of total hemoglobin, suggesting the presence of a beta chain hemoglobin variant. Gel electrophoresis at alkaline pH revealed a very unusual pattern, with 3 abnormal bands migrating with Hb F, between Hb F and Hb S, and slightly cathodal to Hb S. A single band in the Hb A position was seen on gel electrophoresis at acid pH. Capillary electrophoresis revealed two abnormal peaks, comprising 42% and 5% of total hemoglobin. Sequencing of the beta globin gene showed heterozygosity for Hb Hirose (beta 37(C3) Trp>Ser), an extremely rare variant with a substitution at the α1ß2 interface. CONCLUSIONS: We describe the chromatographic and electrophoretic properties of Hb Hirose, and demonstrate that this rare variant causes falsely low HbA1c results on the BioRad variant II Turbo 2.0. Recognition of this interference is crucial in order to prevent reporting erroneous results.