Baumann, N

Abstract Background Capillary self-collection (CSC) devices allow patients to collect blood samples at home, potentially reducing outpatient phlebotomy visits. This study aimed to (a) assess the patient experience with multiple commercially available CSC devices; (b) determine which laboratory tests are commonly ordered together, which could reduce the need for appointments; and (c) evaluate the analytical performance of these tests using CSC samples. Methods User experience for 3 CSC devices was evaluated. Clinical feasibility was determined by comparing test results in paired sera collected by venipuncture (VP) and CSC devices. VP samples were centrifuged within 2 h and tested immediately. CSC sera were centrifuged and tested both immediately and after delayed processing to simulate shipping temperature extremes (−20°C or 40°C). Basic metabolic panel, lipid panel, thyroid function cascade, and prostate-specific antigen were evaluated. Differences between VP and CSC collections were characterized according to their statistical and clinical differences. Results Patients reported CSC devices were easy to use and painless. Clinically significant differences between VP and CSC sera processed immediately were limited to potassium and bicarbonate. Following delayed processing of CSC serum samples (48 h at room temperature), clinically significant differences in potassium, bicarbonate, and glucose were observed. Frozen samples could not be analyzed. A 48 h delay at 40°C caused clinically significant differences in all analytes except creatinine, lipid panel, prostate-specific antigen, and the thyroid cascade. Conclusion Potassium, bicarbonate, and glucose were not stable at room temperature in CSC sera. CSC serum specimens may be a viable option but require analyte-specific evaluation and consideration of transportation conditions.

Abstract Background The use of autonomous mobile robots (AMRs) to move materials/supplies is long-standing in manufacturing/warehouse settings and is expanding into hospitals and clinical laboratories. The recently launched Proxie robot (Collaborative Robotics i.e. Cobot) is an AMR that can autonomously move existing carts between defined locations. However, AMRs pose new challenges compared to traditional lab automation. The aims of this study were to: i) perform functional validation to verify Proxie*s manufacturer performance specifications within the laboratory, ii) assess the risk of using Proxie to deliver laboratory specimens, and iii) pilot the use of the Proxie robot to autonomously deliver loaded carts from the Pathology case intake area to the sorting station at Mayo Clinic Laboratories. Methods Functional validation of Proxie consisted of verifying manufacturer’s performance specifications for the following robot parameters (5 test runs): maximum speed, emergency-stop, and ability to maintain localization, travel autonomously, stop and/or avoid moving/fixed objects, and park/unpark. Risk assessment for the robotic use case was performed by a multi-disciplinary team utilizing an AMR risk assessment tool developed by the institutional robotics group. Hazards were categorized as mechanical, electrical, noise, ergonomic, environmental, material/substance or combination risks. Injury severity, exposure(frequency), and avoidance scores were assigned to each hazard to determine negligible-, low-, medium-, high- or very high-risk levels. Proxie’s workstream was evaluated weekly over four weeks by measuring number of cart deliveries, total time/miles traveled, % autonomous deliveries(goal >80%), % on-time deliveries, average±standard deviation(SD) time (min:sec±min:sec) for delivery/return trips, and dwell times at intake/sorting stations. Results Manufacturer’s performance specifications were verified in 100% of test runs. Proxie traveled at human walking speed, stopped within 1 second of emergency stop being enabled, maintained localization, and traveled autonomously for =5 consecutive workflow cycles. Proxie navigated through spaces =4’ wide along fixed pathways and stopped safely when encountering obstacles. Proxie navigated around people and carts in the main 6’ hallway if they were blocking <2’ hallway width. For objects blocking >2’, Proxie stopped and resumed motion after the object moved. After task completion, Proxie side-shifted into the AMR parking space stopping no closer than 7.8” from any object. Twenty-four potential hazards were identified through the risk assessment tool with all risk levels scored as negligible or low. Proxie completed 304 cart deliveries/returns operating 17:35 hours and traveling 11.5 miles during the pilot. The % autonomous deliveries for week 1-4 was 82%,88%,91% and 88%, respectively with =99% on-time deliveries. Delivery from pathology case intake to sorting station averaged 5:43±0:25 min and return trips averaged 3:32±0:34. Proxie dwell times were 13:10±0:33 and 37:34±0:23 min at the pathology intake and sorting stations, respectively. Conclusions The Proxie AMR was capable of successfully and autonomously moving carts with laboratory specimens between the pathology intake and sorting stations of the laboratory during the pilot study. Adding Proxie to this workstream translated to eliminating 5,800 steps and 4 hours 24 min of human cart-pushing per week. Incorporating Proxie into laboratory operations as a cart-moving AMR would increase efficiency and allow laboratory staff to focus on technical, non-repetitive work.

Abstract Background Hemolysis, a common pre-analytical interferent in clinical laboratory testing, can significantly affect accuracy and reliability of laboratory results. One test particularly susceptible to interference is direct bilirubin (D-bil). For example, during December 2024, 40% (500/1239) of specimens rejected due to hemolysis were orders for D-bil. The recently released Roche (Roche Diagnostics) direct bilirubin reagent (BILD2) has a lower threshold for hemolysis (H-index [HI]=25)) than the previous generation (HI=30). The aims of our study were to (1) establish concentration-specific hemolysis thresholds for D-bil and (2) investigate the instrument’s reduced volume function (dilution) as a possible mitigation strategy for samples with hemolysis indices exceeding the established thresholds. Methods Residual serum specimens from physician-ordered D-bil testing were used and analyzed on Roche cobas c701 and c501 analyzers. The instrument’s decreased volume function, intended for results outside the measuring range, was used as a 1:2 dilution and validated using 9 non-hemolyzed negative control specimens with D-bil results from 9.8-13.4 mg/dL. Recovery within 10% neat value was considered acceptable. EDTA whole blood was used to prepare hemolysate pools by washing red cells and lysing by freezing (-80 °C). Twenty-six serum pools with varying D-bil concentrations (0.2-3.4 mg/dL) were prepared. Each pool was split into 10 aliquots and spiked with increasing amounts of hemolysate, not exceeding 10% serum to interferent ratio. The decreased volume function (dilution) was utilized on a subset of the hemolysate-spiked aliquots (N=55) with D-bil results from 0.2-2.5 mg/dL. HI values were estimated to be 50% of neat HI due to the instrument’s inability to measure indices on specimens tested with the decreased volume function. Acceptance criteria for experiments utilizing hemolyzed specimens were ±0.1 mg/dL or 20% of neat sample. Results All non-hemolyzed specimens tested with the decreased volume function passed within acceptance criteria with average recovery of 109% (range: 106-110%) from undiluted specimens. Serum pools with D-bil <0.3 mg/dL (N=7) spiked with hemolysate (HI range: 7-191) had an average difference of -0.1 mg/dL (range: -0.2-0.0 mg/dL) from non-hemolyzed pools, D-bil pools 0.4-0.9 mg/dL (N=10; HI range: 6-173) had an average difference of -0.2 mg/dL (range: -0.5-0.0 mg/dL), and D-bil pools >1.0 mg/dL (N=9; HI range: 5-182) had an average difference of -20.7% (range –45.5-0.0%). Serum pools with D-bil <0.3 mg/dL (N=23; HI range: 43-191) tested using the decreased volume function had an average difference –0.1 mg/dL (range: -0.2-0.0 mg/dL; HI range: 22-96) from the non-hemolyzed specimens, D-bil pools 0.4-0.9 mg/dL (N=20; HI range: 39-173) had an average difference -0.1 mg/dL (-0.3-0.0 mg/dL; HI range: 19-86), and D-bil pools >1.0 mg/dL (N=12; HI range: 37-182 ) had an average difference of -0.2 mg/dL (-0.5-0.2 mg/dL; HI range: 19-91). Conclusion We established a new hemolysis interference threshold of 75 for samples with D-bil <0.3 mg/dL. Using the decreased volume function, we expanded the hemolysis interference threshold to HI=100 for specimens =0.3 mg/dL. All specimens with HI>100 will result in a recollection. With these changes, we estimate that we can reduce our D-bil recollections by up to 6% over six months.

Abstract Background Lymphocyte function and immune competence can be measured by proliferative response to stimuli and is useful in several clinical contexts including primary (or genetic) and secondary immunodeficiencies (e.g., immunosuppression following transplantation). Proliferation assays have traditionally been prepared manually due to the difficulty in maintaining the sterile environment required to prevent bacterial and fungal impact on cell growth. On most automated liquid handlers, the sample and reagents are placed inside a cabinet whose inner surfaces include rails, pullies, pumps, and circuitry that face the sample and are not amenable to sterilization. The Andrew+ (Waters, MA) is a small surface cleanable liquid handler with all machinery enclosed. We demonstrate here the use of an Andrew+ placed in a biological safety cabinet (BSC) to perform sterile assay setup. Methods Peripheral blood monocytes (PBMCs) from five individuals were manually generated via buffy coat isolation, then brought to standardized concentration. An Andrew+ was placed in a 4’ BSC and all surfaces disinfected with Oxivir Tb (Diversey, SC). The Andrew+ prepared three serial dilutions of two stimulant reagents (phytohemagglutinin (PHA) and pokeweed (PWM)), then dispensed stimulant and control reagents horizontally and PBMCs vertically to a 48 well plate. This process was duplicated manually from the same specimens to create a reference plate. The plates were incubated at 37°C and 5% CO2 for three days, then pulsed with EdU (Thermo Fisher, MA) and incubated for one additional day. Cells were harvested and counted via flow cytometry. Overall time and hands-on time were measured. Results A total of five samples were tested inside a using both manual and automation setup under eight conditions including with isotype control, media without stimulant, three concentrations of PHA, and three concentrations of PWM. The percent proliferation for various cell types as induced by each stimulant was calculated from the difference of the highest stimulant signal from the background (media-only) signal. Comparing the automated and manual method results showed a slope of 1.0217 and R2 of 0.9966. For a simulated run of 6 samples, technologist time for automated steps was under 8 minutes with a total time of 56 minutes. When these steps were performed manually, the time was 30 minutes. Automation increased total time by 26 minutes but saved 22 minutes of technologist hands-on time. Conclusion The Andrew+ was able to perform sterile setup of a cell culture assay. While slower overall than manual processing, technologist time was reduced. Ergonomic, inter-tech variability, and sample transcription error issues were subjectively observed to be less likely. The Andrew+ can work with up to four “dominos” (reagent or tip blocks) in width and up to three dominos in depth. Non-custom, standard depth BSCs from a variety of manufacturers (Labconco, Nuaire, ThermoFisher) have room for one domino in depth so, except for timings performed in this evaluation, all steps were broken down to be performed with a max of four dominos. Efficient sterile use of the system would require an extended depth BSC.

Abstract Background We evaluated the Tecan Fluent Mix and Pierce Workstation as an automated platform to aliquot directly from whole blood collection tubes. The Fluent Mix and Pierce Workstation uses the Tube Rotator™, an on-deck rotisserie, to keep whole blood in suspension, and 8 Piercing Tips™ to cross the pierceable caps of collection tubes to aliquot samples. Methods We performed accuracy and precision testing by aliquoting residual whole blood specimens into an on-deck gravimetric scale. To test carryover, we aliquoted alternating blanks and high calibrators for several analytes and performed mass spectrometry on the blanks. We performed 168 runs over a 21-day period to test the robustness of the platform. We aliquoted and analyzed 2 batches of samples on both our clinical platform and the Mix and Pierce Workstation to check concordance. Results We observed that the Fluent Mix and Pierce Workstation can aliquot 200 µL with 0.5% inaccuracy and 0.9%CV and can aliquot 300 µL with 0.1% inaccuracy and 0.7%CV. We were unable to detect any analyte carryover using mass spectrometry. We observed no hardware failures during the instrument stress test. Clinical comparison runs indicated that the performance of aliquots made on the Fluent Mix and Pierce Workstation was equivalent to that of aliquots made by our current clinical method. Conclusions Overall, our evaluation showed that the Tecan Fluent Mix and Pierce Workstation is a viable platform for aliquoting whole blood for downstream work flows and can eliminate labor-intensive sample decapping and recapping in high-throughput settings.

Abstract Magnesium (Mg) is an essential element involved in cellular metabolism. We demonstrated that in patients with diffuse large B-cell lymphoma (DLBCL) undergoing autologous stem cell transplant (SCT), those with a serum Mg < 2.0 mg/dL at the time of transplant had worse outcomes. In this study, we aimed to learn the prognostic value of low serum Mg in patients with untreated DLBCL. We analyzed serum from 408 patients and tested 2 Mg cutpoints—low (<1.7 mg/dL) and low normal (<2.0 mg/dL), a range we found associated with lower survival in the SCT group. We found 3% of patients with low levels and 23% with low normal levels. Low normal serum Mg levels were associated with a higher stage at diagnosis, more extranodal involvement, higher international prognostic index score, lower overall survival (OS), and event-free survival. These data warrant testing Mg replacement to a target of >2.0 mg/dL to learn if survival can be improved.

Abstract Background Bicarbonate stability in serum depends on time, exposure to air, temperature, tube size and sample volume. With implementation of Cobas Connection Module (CCM) automation, the workflow for bicarbonate measurement needed to be re-evaluated. The aim of this study was to determine bicarbonate stability in filled 3.5 mL and 5 mL serum-separator tubes (SSTs) and capped and uncapped aliquot tubes on the CCM automation. Methods Bicarbonate was measured on a Cobas c701 (Roche Diagnostics) at various time points. SSTs (n=8) were centrifuged 6 min at 1649g ∼30 min post-collection. Serum was aliquoted (1 mL) into capped or uncapped tubes and kept ambient (15-25°C) on the CCM track for 0, 60, 90, 150, 180, 240 and 300 minutes (n=2 per time point). Primary SSTs (5mL n=4, and 3.5mL n=5) were uncapped on the CCM track and bicarbonate measured at baseline and 30 minute intervals through 6.5 hours. An absolute difference of +/- 4 mmol/L from baseline was considered acceptable. Results Bicarbonate concentrations decrease with time at rates dependent on whether the aliquot tube is capped or uncapped or the SST is 3.5 mL or 5 mL (Figure 1).Figure 1.Average decrease in bicarbonate concentration with time for capped aliquot tubes (black square), uncapped aliquot tubes (red circle), 3.5 mL serum separator tube (blue up triangle), 5 mL serum separator tube (green down triangle). Bars represent +/- 1SD. Conclusions Bicarbonate in serum is stable for testing for 2.5 hours in uncapped 1 mL aliquot tubes, 3.5 hours in capped 1 mL aliquot tubes, 3.5 hours in uncapped full 3.5 mL SST tubes and 6 hours in uncapped full 5 mL SST tubes at ambient temperatures (15-25°C). Bicarbonate testing can be accommodated by total laboratory automation but processes such as add-ons and retesting must be done with caution.

Abstract Background Thoroughly investigating single patient result errors may identify systemic, large-scale testing errors. The laboratory observed an event where a revision to a Total Bile Acid (TBA, Diazyme Laboratories, Inc.) patient result following a 22S quality control (QC) failure displayed a larger than expected difference (52 to 5mcmol/L). TBA is primarily used for diagnosis and monitoring of intrahepatic cholestasis of pregnancy with results >10mcmol/L considered elevated. Retrospective review of result revisions following QC failures (3/15/2017-5/30/2023) revealed that 50%(20/40) yielded result differences of >10mcmol/L(>20SD based on assay imprecision). The aim of this study was to apply a systematic approach to i) estimate the rate of TBA errors, ii) ensure accurate result reporting during investigation period, iii) perform root cause analysis (RCA), and iv) determine corrective and preventative action(s). Methods Residual samples from TBA testing (6/24-7/5/2023) were retested(n=158), differences >+/-3.0mcmol/L or 15% were confirmed on an alternate analyzer, and reports were revised. Automated repeat testing of TBA samples >10mcmol/L was operationalized 7/6/2023. Initial and repeat TBA results were compared(n=448) and results differing by >+/-20% were remeasured on an alternate analyzer. RCA was conducted using a fishbone diagram. NaOH reagent probe washes were implemented for TBA and the error rate was re-assessed. Assays run prior to TBA samples with errors (n=15) were identified. To assess reagent carryover a residual serum pool (TBA ∼5mcmol/L) was aliquoted into 5 tubes in a sample rack. The first sample in the rack was programmed to run an assay suspected of causing carryover followed by 4 TBA measurements. Mean±standard deviation(SD) TBA concentrations were calculated. TBA was also measured in the liquid reagent for amylase, lipase, acetylcholinesterase (ACE) and fructosamine. Results Initial TBA retesting yielded no errors when initial TBA was ≤10mcmol/L(n=51). For samples with TBA >10mcmol/L(n=107), 9(8.4%) had differences exceeding criteria with 8/9 being revised to ≤10mcmol/L. Analysis of automated patient repeat data showed a 3.8%(17/448) error rate when initial TBA >10mcmol/L. After NaOH reagent probe washes were implemented, the error rate decreased to 0.7%(3/448). Assays run directly before an erroneously high TBA result included: lactate, fructosamine, soluble transferrin receptor(STFR), lipase, and ACE. A serum TBA pool(mean±SD=5.1±0.4 mcmol/L,n=15) measured 37.6±1.2mcmol/L(n=3) and 6.7±0.7mcmol/L(n=3) after lipase and fructosamine, respectively. No other assays demonstrated carryover. TBA in lipase reagent compartment B and C was 653 and 649mcmol/L, respectively, and 653mcmol/L in fructosamine reagent compartment B. Conclusions A single patient TBA result revision was investigated and led to identification of reagent carryover causing erroneously high TBA results on the Roche c502 analyzer. Automatic, real-time, repeat testing was implemented to prevent reporting incorrect patient results until RCA could identify the cause. Occurrence rate of erroneous high TBA results decreased from 3.8% to 0.7% after implementation of NaOH washes on the instruments. Carryover experiments confirmed that lipase and fructosamine assays cause carryover when run prior to a TBA sample due to measurable TBA in the reagent. The lab is pursuing moving the fructosamine and lipase reagent to the Roche Cobas c701 to prevent the issue.

Abstract Background Serum aspartate aminotransferase (AST) is a commonly ordered test useful for diagnosing and monitoring hepatocellular damage. The hemolysis (H) index interference threshold for the Roche Cobas AST reagent is H=20, which is the lowest of all tests on Roche chemistry platforms (Cobas c701, c502, and e801). Previous optimization studies supported the implementation of an AST concentration-specific tiered hemolysis interference threshold schema: AST<100 U/L (H=50), AST=100-200 U/L (H=100), AST=201-300 U/L (H=200) and AST>301 U/L (H=500). This schema significantly reduced redraws; however, a significant number of rejected samples were noted to have AST results within the reference interval (RI). The aim of this study was to establish an H-index threshold for samples with AST results within the RI. Methods Twelve pools of residual serum samples were created with AST concentrations ranging from 17-66 U/L. Each pool was split into 2 aliquots, receiving either 10% of a lab-produced hemolysate solution generating H-indices from 62-508 or 10% of a 0.9% saline control. Aliquots were mixed in equal portions to create a series of 5 samples with increasing hemolysis according to CLSI guideline EP-07 and analyzed in EP Evaluator. Median(interquartile range (IQR)) H-index thresholds were calculated for error limits beginning at +5 U/L and increasing incrementally (+1 U/L) to +10 U/L. Clinical cases with an H-index slightly above the hemolysis interference threshold (50≤H≤70) and results within the RIs (Male:8-48U/L; Female:8-43U/L) were reviewed by the Chemistry Resident/Fellow on-call (n=68) over a period of 8 months. Results were released with a comment if risk of clinical misinterpretation due to a falsely elevated result was low. Previous, non-hemolyzed AST results were compared within 45 days, if available, by paired-Wilcoxon test (Graphpad Prism). Results The median(range) of initial AST results in the serum pools was 20(17-66) U/L. The calculated H-index thresholds (median(IQR)) using error limits of +5, +6, +7, +8, +9, +10 U/L were 49(43-53), 56(52-63), 66(61-78), 78(72-81), 91(84-91) and 101 (96-107), respectively. All slightly hemolyzed AST results (50≤H≤70) within the RI were released with a comment upon chart review. Previously reported AST values (n=23) were compared to current (hemolyzed) concentrations and found to be statistically significantly increased relative to previous results from the same patient (median(range) difference: 3.7(-3.6-11)U/L, p=0.009). However, the median difference was <5U/L or 10%, the laboratory’s criteria used to define a clinically significant difference. Conclusions A median H-index limit of up to 100 can be applied to samples when AST is within the RI. The risk of misinterpretation for results with mild-moderate hemolysis was low in the cases reviewed. Sample rejection rates and reporting delays can be diminished by implementing this practice. Interpretation can be further enhanced by appending the following comment to hemolyzed samples: “Specimen was received with hemolysis slightly above the accepted threshold. This degree of hemolysis is associated with a false increase of approximately 5-10 U/L. Interpret in conjunction with other laboratory and clinical findings.”