Baumann, N

Abstract Background Our institution is facing increasing demand for oncology testing that requires high quality DNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue. The current DNA extraction workflow involves a time and labor-intensive manual process that limits our ability to accommodate increasing downstream clinical testing volumes. We evaluated the KingFisher (KF) Flex utilizing Autolys M Tubes and the MagMAX FFPE DNA/RNA Ultra Kit as a possible high-throughput, automated DNA isolation workflow option that would decrease hands-on time and be conducive to volume growth without the need for additional staff. Methods DNA from 52 FFPE tissue samples (n=24 unique cases previously extracted by the current “manual method”) were extracted using Autolys M tubes, the MagMAX FFPE DNA/RNA Ultra Kit, and the KF Flex (Thermo Fisher Scientific Waltham, MA) over 4 “automated method” runs. Method modifications to the published procedure were introduced over the runs to determine any effects on DNA quality/quantity. Timings were taken during each run to compare hands-on and total run time against that of the manual method. Precision experiments were performed by extracting 4 replicates of sample on a single run (intra-assay precision) and 2 samples from 2 cases over 2 runs (inter-assay precision). Three cases were processed using varying tissue input levels to assess input-yield correlation. DNA yield was measured using HS dsDNA Qubit (Thermo Fisher Scientific, Waltham, MA). A subset of samples was tested by digital droplet PCR (ddPCR) (n=10), chromosomal microsomal microarray (CMA) (n=1), and next-generation sequencing (NGS) (n=3). Variants and QC metrics of the KF DNA were compared to those of the manual method DNA. Results The average total DNA yield was 3% lower from samples extracted following the manufacturer recommended KF procedure compared to matched samples extracted using the manual method (n=28 samples from 23 unique cases). A CV of 2.8% was observed in an intra-assay experiment (n=4 replicates from 1 case) and 3.0% in an average inter-assay experiment (n=2 replicates from 2 cases). There was a direct correlation between the tissue input amount (4 amounts of varied tissue input) and the total DNA yield (n=3 samples). We observed concordant fractional abundance values for ddPCR samples and concordant variant detection for NGS and CMA samples. We did not see a significant difference in the quality metrics tracked for any of the downstream testing assays. Conclusions This evaluation demonstrated that, as compared to our manual method, the automated method resulted in comparable DNA yields and downstream assay results. The incorporation of this automation would significantly increase our FFPE DNA extraction capacity without requiring additional staff while reducing hands-on time per run by about six hours. We would also be able to reduce ergonomic hazards related to tube cap manipulation, eliminate use of hazardous reagents, and leverage the plate format to better integrate extracted DNA into our clinical sample preparation workflows. These operational benefits highlight the value of automating high-throughput workflows.

Abstract Background Clinical laboratories are constantly changing with automated analyzers and total lab automation (TLA) implemented and replaced over time. Some benefits of newer technologies include decreased specimen volume requirements, less need for repeat testing, and greater throughput. Recent implementation of new analyzers and replacement of TLA in the Central Clinical Laboratory (CCL) at Mayo Clinic, Rochester, MN, prompted the laboratory to reassess blood collection volumes for commonly ordered tests. Objectives The aim of this study was to evaluate whether updating the blood collection volumes for clinical chemistry and immunoassay tests performed in the CCL could reduce the volume of blood collected from patients and the number of blood collection tubes utilized. Methods Nearly 100 different serum assays from the CCL test menu were reviewed including the basic and comprehensive metabolic, hepatic function, renal function, electrolyte, and lipid panels as well as thyroid function tests, enzymes, hormones, electrolytes, and iron status tests. Cortisol, ferritin, folate, and Vitamin B12 testing were performed on the Beckman DxI800; all other testing was on Roche Diagnostics Cobas 8000 chemistry (ISE, c701, c502) and immunoassay (e801) analyzers. Updated blood volumes for each assay were calculated utilizing manufacturer’s analytical volumes and “dead volume” requirements. Baseline data retrieved from the laboratory information system (LIS) included blood collection volumes and number of tubes collected for 128 patients with physician-orders for laboratory testing on 5/2/21. The collection logic was then updated in the test LIS environment using the new blood volumes requirements and applied to the same 128 orders. Blood volumes and number of tubes collected per order were compared before and after the volume updates. Results Baseline data from 128 unique orders showed that 414 total tubes were collected with a median(range) of 3 (2–6) tubes per order and 5 (1–18) tests per tube. The total volume of blood requested at baseline was 2065 mL. Post-intervention, 197 tubes were needed with a median(range) of 2 (1–3) tubes per order and 13 (1–30) tests per tube. The total volume of blood requested after updating volumes was 851 mL. Conclusions The practice of reviewing and updating specimen volume requirements when replacing or implementing new analyzers or TLA can greatly reduce the volume of blood collected from patients and the number of blood collection tubes per order. In this study of 128 physician orders for common laboratory tests, updating blood collection information in the LIS reduced the requested blood collection volumes by 59% and decreased the number of blood collection tubes required by 52%. This not only benefits patients because less blood is collected but also conserves blood collection tubes and reduces supply chain demands. Additionally, fewer tubes on the TLA system has the potential to improve throughput and test turnaround time.

Abstract Background Total protein is measured in serum and body fluids as part of routine evaluations of general nutritional status and differentiating exudative and transudative effusions. Spectral interference thresholds for bilirubin (e.g., icterus) is set by assay manufacturers to prevent inaccurate results from being reported. The Roche Cobas serum total protein assay package insert states no significant interference from bilirubin up to an icterus (I) index of 20. Body fluids tend to have lower total protein concentrations; therefore, thresholds were derived during prior validation studies and set to I = 10, a threshold that is exceeded routinely. Laboratories can mitigate some interferences by recollecting (e.g., hemolysis) or ultracentrifuging (e.g., lipemia) specimens. Serial dilutions are another option used; however, it is imperative that the laboratory understands the mechanism of interference and can demonstrate that it is mitigated by sample dilution. The aim of this study was to investigate whether bilirubin interference can be mitigated by sample dilution in pleural fluid, peritoneal fluid, and serum specimens. Methods Residual clinically ordered pleural fluid (n = 3), peritoneal fluid (n = 3), and serum (n = 3) samples submitted for protein testing were split into two equal volume aliquots with concentrations ranging 2.7 to 4.3 g/dL in pleural fluids, 3.9 to 5.4 g/dL in peritoneal fluids, and 3.8 to 11.0 g/dL in serum. One aliquot was spiked (<10% by volume) with a bilirubin conjugate solution (∼1000 mg/dL) to surpass the icterus index threshold (spiked). The second aliquot was spiked with an equal volume of 0.9% saline (control). Total protein was measured on the Roche Cobas c701 instrument in both samples to calculate difference (Spiked-Control). The mean(SD) difference was calculated for each sample type. Each spiked sample was serially diluted (2-fold, 4-fold and 8-fold) with 0.9% saline. Total protein was measured for each diluted sample. Mean(SD) difference was calculated (DilutedX-Control), where X = 2-fold, 4-fold, 8-fold. Linear regression analysis was performed by plotting the measured (x-axis) vs expected (y-axis) total protein concentration. Slope, y-intercept, and R2 were determined from serially diluted samples where the expected concentrations were derived using the spiked concentration to replicate the practice of diluting an icteric sample. Results Bilirubin spiking (average icterus index = 33.5) into pleural fluid, peritoneal fluid, and serum, respectively demonstrated mean(SD) bias (g/dL) = −1.0(0.2), −0.9(0.0), and −0.7(0.0). Subsequent 2-fold dilution demonstrated mean(SD) bias (g/dL) = −1.0(0.2), −1.0(0.0), and −0.8(0.1), 4-fold dilution with mean(SD) bias (g/dL) = −1.1(0.2), −1.0(0.0), and -1.0(0.1), and 8-fold dilution with mean(SD) bias (g/dL) = −1.3(0.2), −1.0(0.0), and −1.4(0.1). Diluting pleural, peritoneal, and serum samples, respectively spiked with bilirubin resulted in slope = 0.99, 1.00, and 0.99; y-intercept = 0.04, 0.01, and 0.07; R2 = 0.99, 0.99, 0.99. Conclusion The dilutions exhibited a linear dilution response, however the dilution results never returned to baseline (non-spiked) concentration. Laboratories should exercise caution when conducting serial dilutions to diminish bilirubin interference in samples when measuring total protein as the mechanism of interference does not appear to be reversible by dilution. This behavior was demonstrated in both body fluids and serum.

Abstract Background Serum cortisol, measured by automated immunoassay, is a rapid test used for the initial assessment of adrenal dysfunction. Physiologic serum cortisol concentrations are influenced by many factors including diurnal variation, stress, and patient age. In the absence of established pediatric reference intervals (RIs), our laboratory has utilized adult RIs to flag pediatric cortisol results as abnormally high or low in the electronic health record. However, pediatric cortisol RIs recently defined by the Canadian Laboratory Initiative on Pediatric Reference Intervals (CALIPER) are considerably lower than the adult RIs currently in use. This could cause pediatric cortisol results to be incorrectly flagged as “low” when they are within the age-specific RI and lead to unnecessary follow-up testing. The aim of this study was to determine the impact of implementing CALIPER pediatric cortisol RIs on the rate of “low” result flagging for pediatric patients. Methods AM cortisol results for patients age <18 years reported between 10/2012- 09/2022 were obtained from the Mayo Clinic data repository. These results contained values measured by both Beckman Access (n = 3 818) and Roche Cobas (n = 625) immunoassays. Results classified as abnormal “low” by current adult RIs (Beckman: 7–25 mcg/dL, Roche: 4.8–20 mcg/dL) were compared to results which would flag low according to the appropriate method- and age-specific RIs defined by CALIPER (Beckman: 1-<13 yrs 2.19–12.8 mcg/dL, 13-<16 yrs 3.03–17.1 mcg/dL, 16-<19 yrs 3.78–19.4 mcg/dL; Roche: <1 yr 0.58–17.2 mcg/dL, 1-<12 yrs 2.39–14.9 mcg/dL, 12-<19 yrs 3.63–17.4 mcg/dL). The proportion of results flagging low by adult versus pediatric RI were compared by chi-squared test, with P < 0.05 considered significant. Results For Beckman, the percentages of pediatric patient cortisol results flagging low using the adult RI versus (vs) the pediatric age-specific RI were: 35% vs 7% for 1–12 years of age (P < 0.001, n = 1866), 22% vs 5% for 13–15 years (P < 0.001, n = 966), and 16% vs 5% for 16–18 years (P < 0.001, n = 986). For Roche, the same analysis yielded: 52% vs 9% for 0–1 years (P = 0.001, n = 23), 14% vs 8% for 1–11 years (P = 0.03, n = 238), and 9% vs 6% for 12–18 years (P = 0.16, n = 364). Conclusion The implementation of pediatric RIs for serum cortisol will have a significant impact on the number of “low” result flags for patient results on both Beckman and Roche platforms. Statistically significant decreases in the percentage of low result flags were observed on both platforms for all age ranges except for the age bracket of 12–18 years by Roche. Furthermore, pediatric RIs appeared to produce more consistent rates of abnormal results across methods and age ranges, with all groups flagging low at a rate of 5–9%, whereas the same age ranges with adult RIs flagged at rates ranging from 9–52%. Lowering the RIs for pediatric cortisol to more appropriate ranges as identified by CALIPER will decrease the number of “low” flag results. This has the potential to decrease unnecessary follow-up, including repeat testing and further diagnostic procedures such as ACTH stimulation testing. Implementation of pediatric RIs has potential benefits regarding stress and cost for patients and parents, alarm fatigue for physicians, and utilization of laboratory resources.

Abstract Background Measuring the concentration of albumin, amylase, bilirubin, cholesterol, creatinine, glucose, lactate dehydrogenase (LDH), lipase, total protein(TP), triglycerides, and urea nitrogen(UN) in body fluids(BFs) provides important information to aid in clinical decision-making. The range of expected values of these analytes in BFs may not match serum, and the distribution by fluid type is not well described. Quality control (QC) materials are designed to reflect clinically relevant concentrations in blood. Hence, the measuring ranges for assays modified for BF testing vary in suitability. Laboratorians and manufacturers would benefit from understanding the overlap in BF and serum analyte concentrations. The aim of this study was to characterize the range of BF analyte concentrations compared to serum and assess the suitability of QC materials used on these assays. Methods A retrospective analysis was performed for the following analytes in BFs with reportable ranges(including maximum dilution factor): albumin = 0.2–12.0 g/dL(×2), amylase = 3–14 000 U/L(×100), bilirubin = 0.1–72.2 mg/dL(×2), cholesterol = 4–8000 mg/dL(×10), creatinine = 0.1–92.0 mg/dL(×4), glucose = 2–3375 mg/dL(×5), LDH = 10–9000 U/L(×10), lipase = 3–300 000 U/L(×1000), TP = 0.2–36.0 g/dL(×3), triglycerides = 4–8850 mg/dL(×10), and UN = 2–300 mg/dL(×3). Chemistry tests were run on a cobas® 6000 c501 (Roche Diagnostics,Indiana). Patient analyte concentration and fluid type results reported between 9/16/2019 and 1/27/2022 at all Mayo Clinic locations were extracted from an institutional database of electronic medical record data. Serum results were included for comparison. Descriptive statistical analyses (median and range) were performed with R (version 4.2.2)/R Studio(version 2022.12.0). QC materials for BF testing were purchased from Bio-Rad (Multilevel Qual, Levels 1 and 3; Hercules, CA, USA). Target concentrations are reported. Results Conclusion BF median (range) concentrations are similar or higher for bilirubin, creatinine, glucose, lipase, and UN. Serum QC material is likely appropriate for these assays in BFs. BF median(range) concentrations are lower for albumin, cholesterol, TP, and triglycerides suggesting that serum QC material may not be well suited for BFs. The need for extended dilutions may impact BF amylase and lipase.

Abstract Background Hemolysis is a pre-analytic issue that challenges clinical laboratories. When a specimen with significant hemolysis is obtained in our laboratory, tests where the hemolysis threshold is exceeded are not reported and blood is recollected. Unfortunately, in a subset of patients, the recollected specimen is also hemolyzed (termed “recurrent hemolysis”). In these cases, the second recollection utilizes special collection and handling protocols (i.e., hand-carried to lab, plasma collection, etc.) to mitigate preventable causes of hemolysis. Lipemia is also a known contributor to specimen hemolysis. Mayo Clinic Rochester experienced an increased rate of hemolysis as well as recurrent hemolysis during the height of the COVID-19 pandemic (January 2022). The objective of this study was to investigate the increased rate of recurrent hemolysis and compare to a rate prior to the COVID-19 pandemic (January 2019). Methods Retrospective data analysis was performed for January 2019 (negative control) and January 2022. The number of Mayo Clinic Rochester hospital admissions, admitting diagnosis, medications administered, and number of test collections performed were obtained using reporting functionality in our Electronic Health Record (EHR), Epic. The Laboratory Information System, SoftLab, was utilized to obtain recollection rates due to hemolysis. Data were normalized to total patient admissions for the specified period to account for inter-year variability. R/R Studio was utilized to analyze and visualize the data. Pearson’s Chi-squared test for independence was performed on categorical variables (significance defined as P < 0.05). Results A 28% decrease in hospital admissions (n = 3958:Jan-22 vs n = 5517:Jan-19) and a 23% decrease in total specimens collected on hospitalized patients (n = 40 557:Jan-22 vs n = 52 373:Jan-19) was observed between Jan-19 to Jan-22. However, the laboratory observed a 6% increase in hemolyzed specimens (n = 256:Jan-22 vs n = 241:Jan-19). Recurrent hemolysis occurred in 24% (n = 62/256) of Jan-22 and 17% (n = 43/241) of Jan-19 hemolyzed specimens and required special handling protocols. COVID positive patients comprised 48% (n = 30/62) of those requiring special handling protocols in Jan-22. Further examination revealed 34% (n = 21/62) of Jan-22 and 12% (n = 5/43) of Jan-19 cases of recurrent hemolysis were due to lipemia (P < 0.01). Chart review highlighted a potential source of lipemia from the administration of propofol, a lipophilic sedative emulsion. Of those COVID positive cases with recurrent hemolysis due to lipemia (n = 15/30), 93% (n = 14/15) were receiving propofol at the time of collection (P < 0.001). Conclusion We observed increased rates of specimen hemolysis during the COVID-19 pandemic. While most hemolysis resolves upon specimen recollection, patients with recurrently hemolyzed specimens often require special specimen collection and handling protocols to mitigate hemolysis. Approximately half of the recurrent hemolysis cases in Jan 2022 were COVID-19 positive patients. Lipemia-induced hemolysis was also more frequent in this patient population presumably due to the administration of propofol, a lipophilic emulsion sedative commonly used for COVID positive patients requiring sedation and ICU-level care. It is critical to understand the variety of factors which may contribute to recurrent hemolysis, as intervening with specialized collection and handling protocols to reduce observed pre-analytical hemolysis is required to provide accurate and timely laboratory results.

Abstract Background Minimizing blood specimen recollection due to specimen integrity issues is a continuous quality improvement goal in the neonatal intensive care unit (NICU). Specimen integrity issues, such as clotting or hemolysis, are often the result of improper specimen collection processes and lead to blood loss requiring transfusion, additional painful procedures, and delays in patient care. Data collected from Mayo Clinic Hospital - Methodist Campus NICU at Mayo Clinic in Rochester, MN between January – July 2019 showed that clotted complete blood count (CBC) specimens accounted for one-third of rejected neonatal specimens followed by hemolyzed specimens for direct bilirubin (DBIL) testing. The aim of this project was to reduce the percent of CBC and DBIL tests canceled/specimens rejected due to specimen integrity issues in the NICU from 17% to 10% using the Plan-Do-Study-Act methodology. Methods A multidisciplinary quality improvement (QI) team was formed, inclusive of both NICU and laboratory representatives. Bedside direct observations (n=28) were performed by NICU nurses to help identify inconsistencies in the blood collection processes. A total of three Plan-Do-Study-Act (PDSA) cycles were conducted during the 2020-2021 time period. An assessment card was created and revised with each PDSA cycle. The card was completed by the nurse(s) present during the blood collection and initially identified the lab tests ordered, collection tubes, collection type (venipuncture, venous, capillary), number of tube inversions required, staff present during collection, any delays during collection, and whether the draw was deemed successful. Additionally, staff education videos for nursing and laboratory phlebotomy staff were created to demonstrate correct collection tube inversion technique. Results Direct observation identified inconsistent practices in tube inversion during specimen collection. The first PDSA cycle (n=207 collections, April 1 – July 13, 2021) introduced a verbal inversion count and utilization of the assessment card. The following cycle (n=336 collections, July 14 – October 10, 2021) implemented an educational video and modified the assessment card to include start and end time of the draw. The third cycle (n=243 collections, October 11 – November 30, 2021) used a simplified assessment card including patient label, tests ordered, tube type and inversions. The combined rejection rates for CBC and DBIL tests were 4.3%, 3.8% and 2.8% for the 1st, 2nd and 3rd PDSA cycles, respectively. Conclusions Our QI initiative reduced the specimen rejection rate for clotted CBC and hemolyzed DBIL specimens from 17% to 2.8% in the NICU. Interventions focused on improving tube inversion techniques and promoting collaborative relationships between phlebotomists and nursing staff. The use of educational videos and completion of assessment cards during collections were effective at reducing specimen rejection rates.

COVID-19 convalescent plasma (CCP) was one of the first therapeutic options available for the treatment of SARS-CoV-2 infections and continues to be used selectively for immunosuppressed patients. Given the emergence of novel SARS-CoV-2 variants which are resistant to treatment with available monoclonal antibody (MAb) therapy, CCP remains an important therapeutic consideration.

Abstract Background Measurement of cholesterol within lipoprotein subfractions may aid in cardiovascular disease prediction. Simple, homogenous enzymatic assays for the direct measurement of lipoprotein subfractions have been developed to measure small dense low-density lipoprotein cholesterol (sdLDL-C), high-density lipoprotein-3 cholesterol (HDL3-C), and triglyceride-rich lipoprotein (TRL-C) cholesterol. The objective of this study was to determine biological variability for sdLDL-C, HDL3-C, and TRL-C in a healthy reference population to facilitate interpretation of these analytes. Methods Serum samples were collected from 24 healthy subjects (n = 14 female/10 male) daily for 3 days while non-fasting, and daily for 5 days, weekly for 4 weeks, and monthly for 6 months after overnight fasting. sdLDL-C, HDL3-C, and TRL-C cholesterol were measured by homogenous enzymatic assays. Sources of variability (between-subject, within-subject, and analytical) were calculated using random-effects regression models. Reference change value (RCV) and index of individuality (II) for each time period were determined from the variance components. Results Analytic variability (daily, weekly, and monthly CVA) was <3% for each analyte. Monthly within-subject variability (CVI) was 17.1% for sdLDL-C, 7.4% for HDL3-C, and 25.7% for TRL-C. Most of the monthly variation was attributed to between-subject variation for all 3 analytes. Overall RCVs for monthly measurements were 18.1 mg/dL for sdLDL-C, 6.1 mg/dL for HDL3-C, and 16.0 mg/dL for TRL-C. IIs were <0.6 for sdLDL-C and HDL3-C, and 0.81 for TRL-C. Conclusions sdLDL-C, HDL3-C, and TRL-C showed moderate within-subject variability, but high between-subject variability, in a healthy reference population. Given the high individuality of each analyte, population-based reference intervals may be inadequate to detect clinically significant changes.

AbstractBackgroundCeramides are bioactive lipid species that mediate numerous cell-signaling events. Elevated plasma ceramides concentration constitutes a risk factor for several pathologies. Multiple studies have affirmed the plasma concentrations of 4 specific ceramides (Cer16:0, Cer18:0, Cer24:0, and Cer24:1) can predict cardiovascular disease risk. Furthermore, these ceramides can be altered by many lipid-lowering therapies. Understanding the biological variability within an individual, and within a population, will further inform the clinical use of plasma ceramides as a biomarker. In this study, we aimed to define the intra- and interbiological variability of ceramides in a healthy reference population in a weekly and monthly manner.MethodsFasting plasma from 24 healthy adults was collected daily (5 days), weekly (4 weeks), and monthly (7 months). Ceramide concentrations were measured with liquid chromatography–mass spectrometry (LC–MS). For analysis, we used random-effects regression models to estimate variance components.ResultsThe analytical variability was smaller compared to the biological variability overall. The greatest variation reported was between-subject variation for all ceramide species. The critical difference-reference change value (RCV) for within-subject variations monthly were 0.07 mcmol/L (Cer16:0), 0.04 mcmol/L (Cer18:0), 1.09 mcmol/L (Cer24:0), and 0.27 mcmol/L (Cer24:1). The index of individuality (IOI) of ceramides were 0.82 (Cer16:0), 0.96 (Cer18:0), 1.06 (Cer24:0), and 0.89 (Cer24:1). The most consistent ceramide species was Cer18:0 with the lowest within- and between-subject critical differences in weekly and monthly measurements.ConclusionsOverall, this study demonstrates that the variability of ceramide concentrations at different time points is minimal within individuals, allowing a single draw to be sufficient at least in a yearly time frame.