Citations

Abstract Immune thrombotic thrombocytopenic purpura (iTTP) is a chronically relapsing disorder caused by autoantibody-mediated deficiency of ADAMTS13. Rituximab is frequently administered to prevent relapses, but whether the durability of rituximab effect is maintained with subsequent treatment courses has not been studied. Using the United States Thrombotic Microangiopathy Consortium (USTMA) retrospective iTTP registry, we evaluated clinical relapse-free survival (RFS) with subsequent courses of rituximab treatment in multiply relapsing patients. Separately, we evaluated overall RFS (composite of time to clinical relapse, ADAMTS13 relapse, or preemptive rituximab) in a prospective iTTP cohort from the Johns Hopkins University and the University of Minnesota. In the USTMA registry, median clinical RFS was shorter after the second or subsequent rituximab-treated episode than the first (2.1 vs 6.0 years; P = .04). White patients’ clinical relapse risk after the second and subsequent rituximab courses was not significantly different compared with the first (hazard ratio [HR], 1.86; 95% confidence interval [CI], 0.22-15.80; P = .57), whereas for Black patients, clinical relapse risk was significantly higher after the second or subsequent courses (HR, 2.82; 95% CI, 1.52-5.24; P = .001). In the prospective cohort, overall RFS progressively shortened after each episode of rituximab treatment with the first episode having the longest RFS (2.8 years; interquartile range, 2.0-6.0) and this loss of response durability was most pronounced in Black patients. The durability of rituximab’s effect declines with subsequent treatments, which is more pronounced in Black patients, who may benefit from closer monitoring and alternative immunomodulatory approaches such as maintenance rituximab and consideration of other agents.

Abstract Thromboembolic complication is common in severe coronavirus disease 2019 (COVID-19), leading to an investigation into the presence of prothrombotic antibodies akin to those found in heparin-induced thrombocytopenia (HIT). In a study of samples from 130 hospitalized patients, collected 3.6 days after COVID-19 diagnosis, 80% had immunoglobulin G (IgG) antibodies recognizing complexes of heparin and platelet factor 4 (PF4; PF4/H), and 41% had antibodies inducing PF4-dependent P-selectin expression in CpG oligodeoxynucleotide–treated normal platelets. Unlike HIT, both PF4/H-reactive and platelet-activating antibodies were found in patients with COVID-19 regardless of recent heparin exposure. Notably, PF4/H-reactive IgG antibodies correlated with those targeting the receptor-binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 spike protein. Moreover, introducing exogenous RBD to or removing RBD-reactive IgG from COVID-19 plasma or IgG purified from COVID-19 plasma significantly reduced their ability to activate platelets. RBD-specific antibodies capable of platelet activation were cloned from peripheral blood B cells of patients with COVID-19. These antibodies possessed sequence motifs in the heavy-chain complementarity-determining region 3 (HCDR3), resembling those identified in pathogenic HIT antibodies. Furthermore, IgG+ B cells having these HCDR3 signatures were markedly expanded in patients with severe COVID-19. Importantly, platelet-activating antibodies present in patients with COVID-19 were associated with a specific elevation of platelet α-granule proteins in the plasma and showed a positive correlation with markers for inflammation and tissue damage, suggesting a functionality of these antibodies in patients. The demonstration of functional and structural similarities between certain RBD-specific antibodies in patients with COVID-19 and pathogenic antibodies typical of HIT suggests a novel mechanism by which RBD-specific antibodies might contribute to thrombosis in COVID-19.

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.”

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.

Introduction: Use of validated risk stratification tools for work up of suspected pulmonary embolism (PE) is an endorsed recommendation of the American Society of Hematology and the American College of Emergency Physicians. Due to variations in clinical practice, electronic health record (EHR) systems, and medical provider trainings, the use, documentation, and implementation of these tools vary widely. Current processes do not allow for electronic capture and quality reporting at most institutions. Methods: We sought to design and implement clinical pre-test probability (PTP) tools at three large healthcare institutions for use in emergency departments (ED). At each site, a hematologist partnered with ED physician leadership and local information technology experts to design and implement PTP tools in clinical workflows. Overall, 38 EDs (high volume tertiary EDs and smaller regional EDs) located in the United States along the East Coast and Midwest regions were involved. A major goal of implementation was integration into clinical workflow and automatic documentation. After design and implementation, awareness and education of the new EHR tool was distributed electronically to ED providers. After implementation, use of PTP tools was analyzed between September 12, 2022 and January 11, 2023. PTP use was examined as percent of visits for which patients underwent CT pulmonary angiography (CTPA). Results: Each institution chose the 3 tier Wells' Score for implementation while one site designed an integrated Wells'/PERC or YEARS score calculator. Two sites implemented the tool as an optional flowsheet and one site implemented the tool as mandatory parameters within the orders for PE imaging studies. The mandatory tool could be bypassed with prespecified selections or if a D-dimer existed within 48 hours . Complete details of the site-specific implementation are shown in Table 1. Over the 4-month evaluation timeframe, there was a total of 270,214 ED encounters. Uptake and utilization of the PTP tools are shown in Table 2. Use of PTP was highest at the site with forced PTP documentation which ranged from 49-53% of ordered CTPAs, compared to Site 2 where use was 2-3%, and Site 1 where use ranged from 1-3%. At Site 1, use of PTP increased slightly over the study period with signs that PE yield on imaging was increasing as well (3.4% to 5.9%). At Site 2, PE yield on imaging was overall high (9-10%), and remained similar with similar use of PTP tools over the study period. Use of PTP and PE yield (6-8%) on imaging also remained similar throughout the study at Site 3. Conclusion: Forced use of PTP within the orders for CTPA led to the highest use of PTP (Site 3) but this did not result in meaningfully higher PE yield on CTPA compared to the other sites. PE yield at each site was at or above the 5% yield for PE on CTPA which has been previously reported. Given that PTP uptake was relatively stable over the measurement period at Site 2 and 3, this indicates the process had mostly stabilized and that other strategies are needed to improve uptake of PTP, reduce utilization of CTPA, and further increase yield on CTPA. Increasing PTP use and rising PE yield at Site 1 demonstrated ongoing potential for improvements past this study timeframe.

Introduction: ASH Guidelines for diagnosis of pulmonary embolism (PE) start with risk assessment using a pretest probability tool (PTP) followed by D-dimer testing or CT pulmonary angiography (CTPA) depending on risk. We aimed to develop an electronic clinical quality measure (eCQM) to encourage broader use of a validated PTP scoring tool in emergency departments (EDs). The Centers for Medicare & Medicaid Services require reporting of eCQMs which consist of value sets or lists of standardized codes linked to patient data within the electronic health record. We sought to identify a standardized way to identify CTPA and abnormal D-dimer tests using administrative codes (i.e. CPT, LOINC, SNOMEDCT codes) across three institutions. Methods: Value sets identifying CTPA and D-dimers were reviewed from existing eCQMs. Three institutions representing 38 EDs from across the country submitted data from all ED encounters between September 12, 2022 and January 11, 2023. Imaging types were reviewed from each of the CPT codes and LOINC (Logical Observation Identifiers Names and Codes). We determined the number of ED encounters, pre-test probability tool use, and diagnosis of PE using different codes. We calculated sensitivity, specificity, positive and negative predictive value (PPV and NPV, respectively). Results: Over the 4-month study period, 270,214 encounters were identified across 38 EDs. Each institution identified CTPA with site-specific codes (Table 1). The Full CTPA Value Set identified 55% more encounters than the site-specific codes (Full set 15,054/9679 site-specific codes). LOINC code 88322-3 was only used at one site. CPT code 71275 had the best sensitivity and specificity, but still had false positives (PPV 82%) (Table 2). D-dimer values were identified as LOINC code 48065-7 and 91556-1 at Site 1 and 48067-3 at Site 2. SNOMEDCT codes were not used at any site despite being the way other eCQMs identify elevated D-dimer results. Different D-dimer tests with different normal ranges were used at each site. All sites did not have an electronically extractable positive indicator for D-dimer. Conclusion: Due to persistent false positives using CPT Code 71275 for identification of CTPA, using this code would lead to inclusion of patients for whom PTP was not required or recommended. Additionally, coding for an abnormal D-dimer test result are not standardized across institutions. Therefore, administrative codes cannot be used to develop eCQMs whose aim is to evaluate whether CTPA is ordered appropriately based on the PTP risk level and laboratory testing.