Baumann, N

Abstract The guideline-recommended lipid panel for cardiovascular disease (CVD) risk assessment measures total cholesterol, high-density lipoprotein (HDL) cholesterol, triglycerides, and calculated low-density lipoprotein (LDL) cholesterol. Measured cholesterol in subfractions of HDL and LDL purportedly improve CVD risk prediction. Homogenous enzymatic methods are now available for measurement of the cholesterol within small dense LDL (sLDL), small dense HDL (HDL3), and triglyceride-rich lipoproteins (TRL). For meaningful interpretation of these measurements, an understanding of the potential sources and extent of result variability is needed. The smallest difference between serial measurements within a patient that likely reflects a change in clinical status is called the reference change value (RCV). Biological variability and reference change values (RCV) are well-characterized for basic lipids but there is limited information for sLDL, HDL3 or TRL. The objective of this study was to determine intra- and inter-individual variability for sLDL, HDL3, and TRL in a healthy reference population. Serum samples were collected from 24 healthy subjects (n=14 female/10 male) daily for three days (non-fasting), daily for five days (fasting), weekly for four weeks (fasting), and monthly for 7 months (fasting). sLDL, HDL3, and TRL cholesterol were measured in duplicate by enzymatic colorimetric assays (Denka, Japan) on a Roche Cobas c501. Each source of variability (between subject, within subject, and analytical) was calculated using random-effects regression models to estimate each variance component including the overall variation, standard deviation (SD), coefficient of variation (CV), and proportion of total variance (between-subject, within-subject, and analytical). Using these analytical and biological variances, the reference change value (RCV), index of individuality (IoI), and intraclass correlation coefficient (ICC) were determined. Analytic variability (CVa) from monthly testing was 1.2%, 1.1%, and 1.5% for sLDL, HDL3, and TRL, respectively. Monthly within-subject variability (CVw) was 17.1% for sLDL, 7.4% for HDL3 and 25.7% for TRL. Monthly between-subject variability (CVb) was 32.2%, 13.93%, and 33.4% for sLDL, HDL3, and TRL, respectively. Most of the monthly variation was attributed to between-subject variation for all three tests. Within-subject variation accounted for 37% of TRL variation and 22% for both sLDL and HLD3. Within-subject RCVs for monthly measurements were 16.9mg/dL for sLDL, 5.3mg/dL for HDL3, and 15.1mg/dL for TRL. IoIs for monthly testing were 0.81 for TRL, 0.57 for sLDL, and 0.61 for HDL3. Our data demonstrate that sLDL, HDL3, and TRL show low analytical variability, moderate within-subject variability, but high between-subject variability when measured by homogenous assays in a healthy population. The IoI value (>0.6) for TRL suggests use of a reference interval is appropriate for result interpretation. Conversely, clinical cut-points may be more useful than reference intervals for sLDL and HDL3 which had IoIs ~0.6. These findings may be useful for clinical interpretation, particularly when comparing successive measurements of these analytes.

Longitudinal studies assessing durability of the anti-severe acute respiratory syndrome coronavirus 2 (anti-SARS-CoV-2) humoral immune response have generated conflicting results. This has been proposed to be due to differences in patient populations, the lack of standardized methodologies, and the use of assays that measure distinct aspects of the humoral response.

Abstract Objectives To evaluate the analytical and clinical performance characteristics of the fifth-generation troponin T reagent. Methods Troponin T was measured in 2,332 paired serum and plasma samples from emergency department and hospital patients using the fourth- and fifth-generation reagents. Testing was repeated after recentrifugation to determine the frequency of analytical outliers and percentage of patients with elevated values for each assay. We conducted separate experiments to determine the effects of biotin and hemolysis interference, as well as measure interinstrument variability, for fifth-generation troponin T. Results Analytic outliers occurred more frequently using the fifth-generation reagent (3.4%) compared with the fourth-generation reagent (1.0%). The frequency of elevated troponin T above the 99th percentile upper reference limit was 26% for the fourth-generation reagent and 52% for the fifth-generation reagent. Clinically significant assay interference by biotin was observed at 20 ng/mL, but hemolysis interference was not observed until an H index of 150. Instrument-to-instrument variability between e411 and e601/602 instrument platforms is predicted to confound clinical interpretation of troponin changes. Conclusions Analytical outliers and instrument-to-instrument variability are the two analytical variables most likely to confound interpretation of changes in fifth-generation troponin T results over time.

Abstract Background It is important for clinical laboratories to have protocols for investigating suspected biotin interference in patient samples. VeraPrep Biotin™ is a commercial product used to rapidly deplete biotin from serum/plasma samples. The objectives of this study were to verify that VeraPrep Biotin™: (a) does not impact immunoassay analyte recovery in control samples and (b) can effectively deplete biotin from samples (both biotin-spiked and samples from donors who ingested biotin supplements). Methods De-identified residual waste serum/plasma samples were combined to create 9 pools for each immunoassay. Plasma/serum samples (n = 23) were obtained from 6 healthy donors at varying times following ingestion of biotin (20 mg, 100 mg, or 200 mg). Nine Elecsys immunoassays were evaluated using the e 602 (Roche Diagnostics Inc.). Control, biotin-spiked (n = 10, ∼400 ng/mL), and donor samples were assayed pre- and post-VeraPrep treatment. Percentage analyte recovery [(posttreatment/pretreatment) × 100] was calculated for control samples. A laboratory-developed LC–MS/MS method was used to quantify biotin. Results In control samples (n = 81), 90–110% analyte recovery was observed post-VeraPrep treatment in over 95% of samples (77/81). The pre- and post-VeraPrep treatment biotin concentration [mean ± standard deviation (SD)] for specimens spiked with up to 500 ng/mL biotin was 357 ± 47 ng/mL and 1.0 ± 0.6 ng/mL, respectively. The mean (range) biotin concentration for the donor samples pre- and post-treatment was 166 (15–1029) ng/mL and 0.2 (<0.1–3) ng/mL, respectively (P = 0.004). Conclusions These data demonstrate that treatment with VeraPrep Biotin™ does not affect analyte recovery in biotin-negative samples and effectively depletes both spiked and endogenous biotin in serum/plasma.

Abstract Objectives To determine the influence of pH on recovery of analytes in body fluids (BFs), investigate the mechanism of pH interference, measure the frequency of abnormal-pH BFs received, and compare pH measured by meter and paper. Methods We performed pH titration in residual BFs. A low-pH BF was spiked and neutralized to investigate pH interference. We measured analytes on a Roche cobas c501 analyzer (Roche Diagnostics) and calculated the percent recovery. Measurement of pH using a meter and paper was conducted on 122 BF samples received in the laboratory. Results Enzyme activity in BFs was unaffected when pH = 7.4-8.5 lactate dehydrogenase, pH = 7.3-10.2 amylase, pH = 6.0-9.9 lipase, and pH = 1.3-11.7 all other analytes. BFs had mean (range) pH of 8.0 (5.1-8.9), with a mean (range) difference (paper ‒ meter) of ‒0.4 (‒0.6 to 1.1). Conclusions Irreversible loss of enzyme activity occurs in BFs at low pH. Few clinical BFs have pH < 7.0, but laboratories should incorporate pH measurement in BF workflows.

Abstract Objectives Results can vary between different free thyroxine (FT4) assays; global standardization would improve comparability of results between laboratories, allowing development of common clinical decision limits in evidence-based guidelines. Content We summarize the path to standardization of FT4 assays, and challenges associated with FT4 testing in special populations, including the need for collaborative efforts toward establishing population-specific reference intervals. The International Federation of Clinical Chemistry and Laboratory Medicine Committee for Standardization of Thyroid Function Tests has undertaken FT4 immunoassay method comparison and recalibration studies and developed a reference measurement procedure that is currently being validated. Further studies are needed to establish common reference intervals/clinical decision limits. Standardization of FT4 assays will change test results substantially; therefore, a major education program will be required to ensure stakeholders are aware of the benefits of FT4 standardization, planned transition procedure, and potential clinical impact of the changes. Assay recalibration by manufacturers and approval process simplification by regulatory authorities will help minimize the clinical impact of standardization. Summary Significant progress has been made toward standardization of FT4 testing, but technical and logistical challenges remain. Outlook Collaborative efforts by manufacturers, laboratories, and clinicians are required to achieve successful global standardization of the FT4 assays.

Abstract Abstract 3209 Background: The red blood cell folate (RCF) assay has historically been recommended as a more reliable indicator of tissue folate stores compared to the serum folate (SF) assay, as it is not affected by recent ingestion of food. However, the RCF assay suffers from inherent problems with imprecision and accuracy, which are not encountered with SF measurements. Furthermore, following the advent of required folic acid supplementation of many foods by the Food and Drug Administration (FDA) in 1992, folate deficiency is increasingly rare. Very few studies have looked at the value of the RCF versus the SF. We undertook a 10 year retrospective analysis of RCF and SF results to determine the clinical utility of RCF beyond that of SF. Methods: We retrieved all RCF and SF results from the laboratory information system at Mayo Clinic (Rochester, MN) ordered on inpatients and outpatients between 1999–2009. Data for patients who had concurrent orders for SF and RCF were analyzed and chart reviews were conducted on those patients with normal SF but low RCF. Abnormal values were defined by the National Health and Nutrition Examination Surveys (NHANES)/Center for Disease Control (CDC) criteria for folate deficiency (SF< 3.0 ng/ml, RCF < 140 ng/ml). Results: A total of 152,166 SF and 15,708 RCF were performed over the decade of the study. The prevalence of folate deficiency using only SF values was 0.39% and 0.27% using only RCF values. There were 1082 patients in which SFA and RCFA were ordered concurrently (Table 1). Only 1 patient (0.09%) had both abnormal SF and RCF. Chart reviews of the 4 patients with a normal SF but low RCF were as follows: 1) a 6 year old (yo). male with known folic acid transporter deficiency treated with Leucovorin. 2) a 58 yo male with history of gout, hypertension, psoriasis, and hyperlipidemia with normal hemoglobin (Hb) and MCV. 3) a 65 yo male with chronic diarrhea and suspected alcohol abuse; slight macrocytosis (MCV=100.3 fL) but normal Hb. 4) a 51 yo male with multifactorial gait disorder and alcohol abuse. There was a previous history of vitamin B12 deficiency but B12 levels were normal at this time. The CBC was notable for macrocytosis (MCV=115.1 fL) without anemia. Only in patient 4 did the RCF value result in the institution of folic acid supplementation. Conclusions: The RCF provides no additional information beyond that provided by the SF in virtually all situations. Thus SF alone is sufficient for assessment of folate stores. However, there is no evidence to support routine ordering of either SF or RCF, as true folate deficiency in the current era of FDA mandated folic acid supplementation is exceedingly rare. Disclosures: No relevant conflicts of interest to declare.

Abstract Introduction Measurement of hemoglobin A1c (HbA1C) is used for the diagnosis and management of patients with diabetes. Methods for measuring HbA1C are classified on the basis of charge differences (cation exchange chromatography) or structural differences (boronate affinity chromatography). Some cation exchange high-performance liquid chromatography (HPLC) analyzers may be prone to interferences from hemoglobin variants. Historically, our lab used two methods to report HbA1C results: cation exchange HPLC (VariantII) with reflex to boronate affinity HPLC (Ultra2) methods. A new analyzer (BioRad D-100) with improved interference detection and thresholds for interference was evaluated. The objectives of this study were (1) assess the comparability of HbA1c results between D-100, VariantII, and Ultra2; (2) evaluate the need for maintaining a reflex method; and (3) calculate cancellation rates before and after implementing D-100. Methods HbA1c was measured by cation exchange methods using VariantII Turbolink, D-100 (BioRad), and boronate affinity using Ultra2Affinity (Trinity Biotech) according to the manufacturer’s recommendations with the following analytical measuring ranges (AMRs), 4.0% to 17.6%, 4.0% to 18.0%, and 4.0% to 17.6%, respectively. D-100 was compared to VariantII (n = 26) and Ultra2 (n = 31) using residual patient samples from provider-ordered HbA1c having no chromatography flags and with the following chromatography flags on VariantII (n = 113): variant window (n = 41), HbA1c <4.0% (n = 24), HbA1a + HbA1b >5% (n = 19), HbF >5% (n = 9), P3 >10% (n = 6), HbA1c >17.6% (n = 4), unknown peak (n = 3), labile 5% to 15% (n = 3), labile >15% (n = 3), and HbS >60% (n = 1). The cancelation rates were calculated at baseline (January 1-31, 2018) and postimplementation of D-100 (September 20, 2018, to January 22, 2019). Results HbA1c results from D-100 were within ±0.3 or 5% with the following frequencies: Ultra2 in 96.2% (25/26) with VariantII in 96.8% (30/31) using samples with no chromatography flags. A total of 113 samples with abnormal VariantII flags were tested using D-100 with 82% (n = 92) not exceeding interference limits for result reporting. These D-100 results were within ±0.3 or 5% of Ultra2 results with the following frequencies: 38.5% (10/26) of samples below (n = 23) or above (n = 3) the AMR, 85.3% (29/34) of results with Hb variant flags, 68.0% (17/25) with unknown/minor peaks, and 71.4% (5/7) with HbF <15%. The clinical concordance was also assessed according to the following decision limits: normal <5.6%, prediabetic = 5.7% to 6.4%, and diabetic >6.5%. Results were concordant in 88% with variant peaks (12/13 w/HbA1c <5.6%, 8/11 w/HbA1c = 5.7%-6.4%, 10/10 w/HbA1c >6.5%), 84% with minor peaks (5/6 w/HbA1c <5.6%, 0/2 w/HbA1c = 5.7%-6.4%, 16/17 w/HbA1c >6.5%), and 86% with HbF (3/3 w/HbA1c <5.6%, 2/3 w/HbA1c = 5.7%-6.4%, 1/1 w/HbA1c >6.5%). Results above/below AMR had 100% concordance (>16.0% or <4.4%). The frequency of VariantII rule violation was 2.1% in January 2018, with 53 of 117 having results confirmed as <4.0% or >16.0% by Ultra2, 2 of 117 reported by Ultra2, and 62 being cancelled due to HbF >15% (n = 23) or variant Hb >60% (n = 39). The frequency of rule violation using only D-100 since implementation was 2.2%. Conclusion Implementation of D-100 has removed the need for boronate affinity reflex testing and maintained similar cancellation rates.

Abstract Background Body fluid samples (BFs) with low pH have demonstrated underrecovery of enzyme activity. The aims of this study were (1) measure the frequency of abnormal pH in BFs received for clinical chemistry testing, (2) calculate recovery of analytes in BFs undergoing pH titration, and (3) investigate the mechanism of pH interference. Methods Residual BFs submitted for physician-ordered testing to the Central Clinical Laboratory (Mayo Clinic) were utilized for this study. BFs were centrifuged 10 minutes (3,200 rpm) prior to pH measurement (pH meter, Accumet X15; Fisher Scientific) and analysis. A Roche Cobas c501 (Roche Diagnostics) analyzer was used to measure amylase, lactate dehydrogenase (LDH), blood urea nitrogen (BUN), creatinine, glucose, albumin, total protein, bilirubin (all Roche Diagnostics reagents), and lipase (Sekisui Diagnostics). pH was measured in 122 BFs received between 10/23/2018 and 10/26/2018 within 24 hours of receipt. pH titration was performed using pooled BFs of similar source/type. Each pool was titrated over a range of pH (≤2 to ≥10) in 1-unit increments by addition of a fixed volume of acid (6M HCL) or base (6M NaOH). Analytes were measured before and after acid/base addition. Average percent recovery was calculated (measured/expected × 100%) from n = 3 to 9 pH measurements. A low pH BF pool was spiked (<5% by volume) with a high-concentration BF to investigate mechanism of enzyme underrecovery. Additionally, low pH BF pool was neutralized by addition of base and % recovery calculated. For all titration/spiking experiments, a control sample having the same volume of diluent (7% bovine serum albumin or saline) was used to account for the effects of dilution. Results BFs received (n = 122) had mean (SD) pH = 8.0 (0.6) with 6.6% (n = 8) having pH <7.0 and 2.5% (n = 3) having pH <6.0. All recovery (%) and pH data are expressed as mean (range) values. Amylase recovery (initial pH = 8.4-8.9) was 1.3 (0.9-2.2)% at pH = 1.6 (1.0-2.2) (n = 6), 55.6 (37.4-67.0)% at pH = 4.4 (3.5-5.4) (n = 6), 81.4 (74.0-85.2)% at pH = 6.2 (6.1-6.5) (n = 3), 90.1 (84.4-95.4)% at pH = 7.5 (7.3-7.7) (n = 3), 98.8 (96.2-101.7)% at pH = 10.0 (9.8-10.2) (n = 3), and 14.5 (1.4-38.9)% at pH = 11.9 (11.5-12.1) (n = 3). LDH recovery (initial pH = 8.2-8.5) was 1.4 (0.1-3.7)% at pH = 1.7 (1.0-2.4) (n = 6), 18.3 (0.5-39.7)% at pH = 4.5 (3.5-5.4) (n = 6), 63.6 (54.9-68.4)% at pH = 6.2 (6.1-6.5) (n = 3), 85.9 (80.2-90.7)% at pH = 7.2 (6.8-7.4) (n = 3), 46.1 (36.4-61.0)% at pH = 10.0 (9.8-10.2) (n = 3), and 9.8 (0.0-28.9)% at pH = 11.3 (10.4-12.1) (n = 3). Lipase recovery (initial pH = 8.2-8.9) was 10.4 (<1-18)% at pH = 1.8 (1.0-2.4) (n = 6), 73.9 (62.6-85.5)% at pH = 4.2 (3.4-5.2) (n = 3), 92.4 (90.8-93.5)% at pH = 6.2 (6.0-6.5) (n = 3), 96.3 (95.6-96.8)% at pH = 7.3 (6.8-7.7) (n = 3), and 92.9 (89.3-96.8)% at pH = 9.9 (9.8-10.1) (n = 3) and 80.9 (0.0-78.6)% at pH = 11.3 (10.4-12.1) (n = 3). Creatinine, BUN, albumin, glucose, total protein, and bilirubin recovery were 97.9 (92.2-102.9)%, 100 (96.6-103.1)%, 99.9 (97.6-102.4)%, 100.2 (97.8-103)%, 100.2 (98.7-101.9)%, and 100.1 (92.-105.9)%, respectively, between pH 1.3-11.7 (n = 9). Recovery of amylase, LDH, or lipase was <1% after spiking enzymes into BF pool with pH <3. pH adjustment to normal (pH = 8.6-9.0) also resulted in recovery of <1%. Conclusion Enzyme activity in BFs was not affected (90%-110% recovery) when pH = 7.4-8.5 for LDH, pH = 7.3-10.2 for amylase, pH = 6.0-9.9 for lipase, and pH = 1.3-11.7 for all other analytes. An irreversible loss of enzyme activity occurs at low pH. Few clinical BFs have pH <7.0, but laboratories should have awareness.