bluuebunny/crossref_metadata_2025 huggingface dataset:
A-078 Harmonization of Immunoassay Reference Intervals with LC-MS/MS for Total and Calculated Free Testosterone Measurement
Authors: Li S , Povse P , Lattanzio F , Patel K , Kerlin R , Jones J
Published: October 2024

Background

Management of male hypogonadism relies on accurate serum testosterone testing and reference values. This study aimed to establish harmonized reference ranges for total and calculated free testosterone in adults using the Siemens Atellica immunoassay, validated against a reference method.

Methods

The Siemens Atellica IM total testosterone assay is a fully automated competitive chemiluminescent immunoassay. We conducted 25 de-identified serum samples. Inter-laboratory correlation studies for total testosterone were performed on in-house Siemens Atellica and liquid chromatography-tandem mass spectrometry (LC-MS/MS) at a reference laboratory. Free testosterone was calculated using the International Society for the Study of the Aging Male (ISSAM) Free Testosterone calculator, which can be accessed from the ISSAM website, http://www.issam.ch/freetesto.htm, using the assayed concentrations of testosterone and Sex Hormone Binding Globulin (SHBG) on the Siemens Atellica IM system. The Siemens Atellica IM SHBG assay is a sandwich monoclonal immunoassay. Calculated free testosterone was compared with LC-MS/MS. Deming and linear regression, and Bland-Altman plot were employed for data analysis. Reference intervals were adopted from the reference laboratory and verified using 20 male and 20 female healthy samples. Total testosterone: Male 240-950 ng/dL, Female 8-60 ng/dL; Free testosterone: Male 2.29-20.7 ng/dL, Female 0-1.08 ng/dL.

Results

Strong correlations were observed between Siemens Atellica and LC-MS/MS for total testosterone (R^2 = 0.9915, slope = 0.988, intercept = 35) and for calculated free testosterone (R^2 = 0.9645, slope = 0.988, intercept = -0.4). Adopted reference intervals were verified using local healthy samples.

Conclusions

Harmonized reference intervals for total and calculated free testosterone, validated on Siemens Atellica, offer consistent results comparable to LC-MS/MS. This study demonstrates the feasibility of establishing standardized reference ranges for testosterone assays across laboratories.
A-086 Clinical Performance of a Plasma ATN Panel for Assessment of Alzheimer’s Disease
Authors: Collier B B , Brandon W C , Chappell M R , Harris A B , Le T , Martone R L , Volpe J M , Boles D , Valcour A , Grant R P
Published: October 2024

Background

In recent years, the progression of Alzheimer’s disease has widely been characterized through the utilization of an ATN classification system consisting of biomarkers for three areas of neuropathologic change: development of Amyloid associated plaques, formation of neurofibrillary tangles associated with Tau proteins, and onset of Neurodegeneration. However, the assessment of biomarkers related to each of these stages has predominantly relied on neuroimaging or CSF measurements which are costly and invasive. With the advent of high sensitivity immunoassays, measurements of ATN biomarkers in venous plasma has become possible and would provide a more accessible and affordable tool to assist physicians with the diagnosis of AD.

Methods

Assessment of amyloid status was performed by determining the ratio of amyloid-beta 1-42 to amyloid-beta 1-40 (Aβ42/40) in plasma where measurements for individual analytes were performed in parallel on a SysmexTM HISCL-5000 platform. Assessments of tau and neurodegeneration were performed using a Roche cobas® e801 platform to measure phosphorylated-tau181 (pTau181) and neurofilament light chain (NfL), respectively. As these assays have not yet received FDA approval, each was validated internally using CLSI guidance. The clinical efficacy of each assay was investigated using 200 clinical specimens with amyloid status defined using positron emission tomography (PET) imaging. Assay results were analyzed using receiver operator characteristic (ROC) analysis with respect to amyloid PET results. In addition, results from the clinical cohort were also assessed with respect: amyloid PET centiloid results, clinically defined cognitive status, and mental state as determined using a Mini-Mental State Exam (MMSE).

Results

ROC analysis of clinical sample results from validated assays produced an area-under-the-curve (AUC) of 0.941 for Aβ42/40, 0.847 for pTau181, and 0.666 for NfL. These results are consistent with amyloid PET status as well as the progression of AD along the ATN continuum. With respect to Aβ+ subjects, Aβ42/40 results did not change due to worsening cognitive status, increasing amyloid PET results, or worsening MMSE scores. Levels of pTau181, however, were found to increase with worsening cognitive status (p < 0.01), increasing amyloid PET centiloid results (p < 0.05), as well as worsening MMSE scores (p < 0.05). In addition, NfL levels become elevated with worsening MMSE scores (p < 0.01).

Conclusions

The clinical utility of the plasma ATN panel was found to be appropriate for assisting with diagnosis of AD and is now available for physician use.
A-087 Stability of fecal calprotectin extracted with BÜHLMANN CALEX® Cap
Authors: Shaw C , Voitl A , Trapani-Vondran D , Ohmann A , Guschin D , Schneider M , Gerhold C , Schuster T
Published: October 2024

Background

Calprotectin, a granulocyte-derived alarmin protein, is an established noninvasive biomarker of intestinal mucosal inflammation. Fecal calprotectin testing has become the gold standard for diagnosis of inflammatory bowel diseases. The aim of this work was to determine the stability of calprotectin in stool extracts prepared with the BÜHLMANN CALEX® Cap stool preparation device.

Methods

The analyte stability study was carried out using CALEX® Cap extracts of human fecal left-over samples. Extraction was performed for 24 hours at room temperature and samples were aliquoted and frozen at < -20°C. The baseline calprotectin concentration was measured from thawed aliquots, which were moved to target storage temperatures (2-8 °C, 18 °C, 28 °C, and 37 °C) for up to 22 days. Five stool samples covering the measuring range of the assay were analyzed with the BÜHLMANN fCAL® turbo in four replicates. Stability evaluations were performed according to CLSI guideline EP25-A1 and EP25Ed2.

Results

CALEX® Cap derived extracts stored refrigerated at 2-8°C for up to 20 days and at room temperature (18-28°C) for up to 6 days demonstrated deviations from baseline of less than 20%. CALEX® Cap derived extracts stored up to 15 days at 2-8°C and up to 2 days at room temperature (18-28°C) demonstrated deviations from baseline of less than 10%.

Conclusions

CALEX® Cap provides a stool collection method that stabilizes fecal calprotectin concentrations for an increased processing window. This allows significant timely separation between stool collection and the actual measurement of calprotectin. This specimen stabilization provides for accurate measurements for days after sample collection and thus allows for flexibility for diagnostic laboratories.
A-114 Protective effect of leukocytes in preventing Cold Induced Hyperkalemia
Authors: Mandal S , Bansal R , Sathish S , Rani N , Bhatia P K
Published: October 2024

Background

Cellular components, especially PRBC units stored in blood banks show progressively increasing extracellular potassium because ATP dependent Na+/ K+ATPase that pumps potassium into cells is slowed at cold but K+ leak continues. The extracellular potassium levels in these units may go well above the range of linearity of some electrolyte analyzers and may need dilution to measure accurately. We wanted to see effect of Leukocytes on this progressive cold-storage hyperkalemia. Buffy-Coat, the leukocyte rich layer of spun whole blood, a discard product of blood banks using buffy-coat-method for components, was used as the source of leukocytes.

Methods

Buffy-Coat-bags (from Terumo Quadruple-bag-set, in CPD) were separated from 32 donations from male donors (with IRB, consent). Eight ABO,Rh compatible pools of four buffy each, were prepared and divided into 2 groups of four pools each:Leukoreduced pools (LR group): Each pool leukoreduced by a clinical-grade commercial Leukofilter (Mitra).Non-leukoreduced pools (NLR group) rich in leukocytes.

Potassium/VBG on Cobas B221(Roche), and CBC were done on days 0, 3, 7 on each pool stored at 4oC.

Results

Potassium was significantly lower in NLR than LR pools on days 3 & 7, and potassium had significant negative correlation with WBC count.

Multiple-linear-regression and analysis of additional control experiments consistently showed that independent of days of storage, pH, and Hct, WBC-Count had significant negative correlation with Potassium.

Conclusions

Preanalytic artefacts like “seasonal-pseudohypokalemia” and pseudohypokalemia in a subset of AMLs (opposite of pseudohyperkalemia in fragile hyperleukocytosis) suggest that leukocytes may be capable of reducing extracellular potassium. Our in-vitro closed-system controlled experiments show that leukocyte-rich/Non-Leuko-Reduced (NLR) pools were relatively protected from hyperkaleimia than filtered leukocyte-poor/Leuko-Reduced (LR) buffy pools, and potassium has significant negative correlation with leukocyte count independent of storage-time, pH & Hct.

Though considered otherwise bad/aggravator for “PRBC-storage-lesion”, leukocytes may help abate progressive cold-storage-hyperkalemia.
A-137 Stability of Analytes in Uncapped Specimens
Authors: Kumar V , Gallegos A F
Published: October 2024

Background

Traditionally, analyte stability is tested on the capped specimens. Routine workflow may require sharing uncapped specimens between multiple analyzers. This study was conducted to determine the impact of storing uncapped specimens on stability of analytes in the Comprehensive Metabolic Panel (CMP) tested on Beckman Coulter AU5800 analyzer.

Methods

Serum from patient specimens (SST® tubes) were mixed to prepare 5 sets of pooled patients’ specimens (PPS), aliquoted (2.5 mL) and capped. The 5 PPS were uncapped, analytes tested in duplicate at baseline (0 hr); 5 hr (reflecting more than the expected duration); 21 hr and 27 hr (reflecting 2 extreme times to identify a failure point). Averaged results were used for time-point comparisons. The absolute and percent differences vs 0 hr were compared to the respective total allowable error (TEA)1, index values calculated and averaged for 5 PPS:

Absolute TEA Index = ([Absolute Difference] / [Absolute TEA]) × 100

%TEA Index = ([% Difference] / [% TEA]) × 100

Storage duration that showed both the absolute and %TEA index values >33.3 was considered to introduce substantial error in test results.

Results

Table 1 highlights the analytes that showed substantial error: At 21 hours: Creatine kinase, Carbon dioxide and Sodium. At 27 hours: Creatine kinase, Carbon dioxide, Chloride, Potassium and Sodium.

Conclusions

The results of the study demonstrate that extreme storage in uncapped tubes up to and greater than a day substantially affected only 5 of 19 test results in the CMP. More importantly, none were substantially affected at 5 hr. It is imperative that the laboratories conduct studies to determine the impact of storage conditions of uncapped samples on test results.

Reference:

1. Clinical Laboratory Improvement Amendments of 1988 Proficiency Testing Regulations Related to Analytes and Acceptable Performance. Federal Register, Vol. 87, 131,2022.
A-141 Ferritin Test Utilization to Differentiate Microcytic Anemia
Authors: Keebaugh M
Published: October 2024

Background

Mean corpuscular volume (MCV) of the Red Blood Cells (RBC) is a component test of the commonly ordered Complete Blood Count (CBC) panel and is an important parameter when evaluating a patient diagnosed with anemia. MCV is used to classify anemia as microcytic (low MCV), normocytic (normal MCV), or macrocytic (high MCV). Microcytic anemia occurs most commonly from iron deficiency or from an inherited blood disorder caused when the body does not produce enough hemoglobin (thalassemia trait), often resulting in iron overload. Clinical management for each of these microcytic anemia contributors varies greatly and requires appropriate differentiation. Laboratory testing of serum ferritin level helps to distinguish iron deficiency from thalassemia and is therefore the first laboratory test recommended in the evaluation of microcytosis. However, initial laboratory findings indicate that ferritin tests do not always accompany low MCV results. The objective of this study was to quantify the frequency of ferritin orders proximate to abnormally low MCV results across two hospitals to assess utilization of ferritin testing for differentiating microcytic anemia.

Methods

A retrospective study was performed on the ordering patterns of MCV and ferritin tests at two different hospitals. Abnormally low MCV values were selected from both adult and pediatric populations during an eight-month period from March through October. All ferritin values from January through December of that same calendar year were selected and joined with the MCV data. Patients with a ferritin test 60 days before or after the MCV test were considered to have a ferritin order associated with the abnormally low MCV. The ratio of the total count of these ferritin orders to the total count of low MCV values was calculated as a percent.

Results

Results showed that 46% (619/1351) of all abnormal low MCV values at Hospital One (Pediatric) had a ferritin ordered within 60 days. An additional 57 cases had a ferritin ordered beyond 60 days, and 675 lacked ferritin orders during the calendar year. During that same period and using the same criteria, Hospital Two (Adult) had ferritin ordered 43% (2301/5320) of the time. An additional 135 had a ferritin ordered beyond 60 days, and 2884 lacked ferritin orders during the calendar year.

Conclusions

Establishing test utilization benchmarks can be a useful stewardship tool for clinical laboratories. The data presented here suggested an underutilization of ferritin testing in the presence of abnormally low MCV values across two healthcare organizations. This study lays the groundwork to further assess MCV and ferritin ordering patterns and a standardized means to quantify these observations across diverse healthcare settings to establish a more robust benchmark.
A-155 Development of drop-of-blood QATT heparinase and functional fibrinogen assays
Authors: Pham H , Cummins E M , Attia S A , Kasireddy N , Khismatullin D B
Published: October 2024

Background

Monitoring the blood coagulation status of patients with coagulopathy or on anticoagulant therapy is important for informed treatment decisions. Quasi-static acoustic tweezing thromboelastometry (QATT) can monitor changes in blood coagulation using a small blood drop. Here, we develop the heparinase and functional fibrinogen QATT assays.

Methods

Citrated whole blood (WB) samples were collected from 20 healthy volunteers using IRB-approved protocols No. 520566 and 944474. Half of the samples were used for fibrinogen, platelet count tests and the others were used for QATT measurements. For developing heparinase assay, WB samples from 4 healthy volunteers were randomly selected. For each volunteer sample, QATT experiments were performed for a WB sample, a WB sample spiked with 0.5 IU/mL heparin, and a WB sample spiked with heparin and then heparinase. For developing functional fibrinogen assay, QATT experiments were performed for a WB sample and a WB sample spiked with Cytochalasin-D (4 μM). Coagulation was triggered via intrinsic pathway. Pearson correlation coefficient (rp) or the Spearman rank correlation coefficient (rs) was used for correlation analysis. We focused on the following QATT parameters: CIT, MCF, and MCE.

Results

The heparinase assay effectively reversed heparinized samples (Fig. 1a). CIT of normal and heparinized samples treated with heparinase were within reference range (Fig. 1b). The effect of Cytochalasin-D was shown in Fig 1c. Strong correlation was found between MCE of Cyto-treated WB and fibrinogen level (rp = 0.75). MCE of the difference between normal and Cyto-treated WB, referred to as MCEplatelet, was also strongly correlated with platelet count test (rs = 0.76). The normal range for MCF of the functional fibrinogen assay is efficient in ruling out abnormal fibrinogen level (Fig. 1d).

Conclusions

QATT accurately predicted the inhibition of heparin by heparinase. QATT functional fibrinogen assay detected the contribution of both fibrinogen and platelets to the clot strength.
A-167 Hematologic setpoints are a deep physiologic phenotype which enable personalized blood count reference intervals
Authors: Foy B H , Petherbridge R , Roth M , Mow C , Patel H , Patel C , Ho S , Lam E , Karczewski K , Tozzo V , Higgins J
Published: October 2024

Background

The complete blood count (CBC) is an important screening tool for healthy adults, commonly ordered at periodic physical exams. However, test results are typically interpreted using one-size-fits-all reference intervals (RIs), which don’t account for the low index-of-individuality of most CBC markers. This undermines the goal of precision medicine - to tailor patient care based on individual characteristics.

Methods

Here we utilize a database of >50,000 healthy outpatients with yearly CBCs, to investigate the value of patient-specific reference intervals (PRIs) for 9 CBC markers. We use Gaussian mixture modelling to calculate each patient’s setpoints, and to construct associated PRIs. Mechanistic drivers of setpoints were investigated through GWAS and via analysis of excess clinical specimens. Clinical outcomes were analyzed by comparing sensitivity of RIs and PRIs for detection of future cytopenia/cythemia and mortality.

Results

We found healthy adults had patient-specific setpoints that were stable over decades, and distinctly identifiable from 98% of other patients. Setpoints reflected a deep physiologic phenotype, enabling improved detection of genetic determinants of hematologic regulation (Fig1A), including discovery of multiple novel loci via GWAS analysis. Deviation from PRIs was associated with greater risk of cytopenia/cythemia and mortality (Fig1B) than deviation from the RI for all setpoints except MCHC. Setpoints were significantly correlated with long-term all-cause mortality, even when limited to those within the RI (Fig1C).

Conclusions

We show that hematologic setpoints reflect a deep, patient-specific phenotype. This phenotype can enhance mechanistic studies, and allow for increased diagnostic sensitivity, through calculation of a personalized reference interval. Setpoints are sufficiently stable and patient-specific to help realize the promise of precision medicine for healthy adults.Fig1A.Significance of GWAS hits using setpoints versus single CBCs. Fig1B. 10yr mortality hazard ratio for tests outside the RI and PRI. Fig1C. Association of WBC setpoint with mortality (limited to WBC setpoints inside the RI).
A-170 Hypereosinophilic Syndrome and t(5;15): Case Report and Literature Review
Authors: de Andrade S S , de Queiroz V C , Vicari P , Sthel V M , Perez B B , Figueira S H , Marcelino G A , do Prado G A , Ramadan-Boscolo D R , Tufik S
Published: October 2024

Background

Hypereosinophilic syndrome (HES) is a myeloproliferative disorder characterized by persistent eosinophilia (absolute eosinophil count >1,500/mm3), classified into primary/clonal, reactive and idiopathic. After ruling out secondary causes of eosinophilia (parasitic infections, allergic conditions, vasculitis and collagenosis, drugs, eosinophilic lung disease, allergic gastroenteritis and metabolic conditions such as adrenal insufficiency), the main hypotheses become oncohematological diseases. Main entities to consider are myeloid/lymphoid neoplasms associated with eosinophilia and rearrangement of PDGFRA, FDGFRB, FGFR1 or PCM1-JAK2 and chronic eosinophilic leukemia. Conventional and molecular cytogenetics are the basis for classification and diagnosis of various hematological malignancies. This report describes a rare case of HES with t(5;15) (q33;q22).

Methods

A 29-year-old male patient was admitted with leukocytosis with the following exams: hemoglobin 6.5g/dL, hematocrit 21.7%, platelets 71,000/mm3, leukocytes 56,190/mm3 (60% neutrophils with a left shift to pro-myelocyte; 6% monocytes and 24% eosinophils), DHL 277 U/L, B12 vitamin 932 pg/mL, folic acid 1.72ng/mL, ferritin 913ng/mL. Non-reactive results were detected for antinuclear antibody, rheumatoid Factor, HIV, hepatitis B and C, syphilis and chagas disease. Bone marrow was hypercellular for age with absolute granulocytic predominance (G:E ratio 64:1) and eosinophilia (38%) in all maturational stages without dysplasia. Bone marrow flow cytometry did not detect neither increases in blasts nor anomalous immunophenotype. Bone marrow biopsy demonstrated 99% cellularity with normal blasts. Cytogenetics with translocation between the long arms of chromosome 5 and 15 in 15 of 20 metaphases analyzed - 46,XY,t(5;15)(q33;q22). No other molecular tests were available for this patient.

Results

In the case described t(5;15)(q33;q22) was found, which may be related to the TP53BP1::PDGFRB gene rearrangement. This breakpoint generates a chimeric oncoprotein similar to other tyrosine kinase fusions responsive to Imatinib. 53BP1 is a cellular DNA damage response component that binds wild type (non-mutant) p53. Oncogenesis occurs since the coiled-coil domains of TP53BP1 can mediate the homodimerization of PDGFRB and the constitutive activation of its tyrosine kinase activity; on the other hand, the DNA damage response of TP53BP1 may be disrupted. Gene rearrangements involving PDGFRA and FDGFRB are associated with response to tyrosine kinase inhibitors. Elevated vitamin B12 is a common finding in myeloproliferations with eosinophilia due to the increased production of haptocorrins, which binds to vitamin B12 in serum and in other tissues.

Conclusions

To our knowledge, in the literature there is only one described case of Myeloproliferative Neoplasia with Eosinophilia related to t(5;15)(q33;q22).
A-203 A Disconnect Between Detectable Patient Error Rates and QC Understanding
Authors: Brooks Z
Published: October 2024

Background

ISO 14971 defines “risk evaluation as the process of comparing the estimated risk against given risk criteria to determine the acceptability of the risk. Effective QC processes would detect if a stable acceptable error rate became unacceptable.

Methods

A survey was circulated through LinkedIn and Labvine. Forty respondents who included their credentials were included in the final analysis. Questions included:

1. What is the best choice for acceptable error rate when an analytical process is stable and in control, 2. If an analytical process “fails,” how many errors are you willing to report before the lab should stop reporting, and 3. “If an analytical process has a 5% stable error, how high would the error rate after failure need to become for a 1-2s rule to detect failure with one QC sample in a single QC run?”

This interactive poster will allow viewers to cast their vote for Question 4 and then reveal an LJ chart depicting a stable error rate of 5% with a shift to show the number of 1-2s failures with their chosen acceptable post-failure error rate.

Results

During stable operation, only 8% wanted zero errors while 38% voted that they would allow up to 5% errors;

After failure, 66% wanted to report zero errors and only 10% would allow 5% failure.

69% believed they would see a 1-2s failure in one run if the error rate rose to less than 50%; 19% believed it required a 100% error rate.

Conclusions

There is a strange disconnect where most believed it was acceptable to report more errors during stable operation than if the method failed.

There is a widespread misunderstanding of the logic that connects management of acceptable patient risk with the capabilities of statistical QC.
A-215 Feasibility of a unique homogenous assay for lipoprotein(a) particle number without bias from apo(a) size isoforms
Authors: Contois J , Albert A L , Nguyen R L , Datta P
Published: October 2024

Background

Lipoprotein(a) [Lp(a)] is an LDL-like particle with apolipoprotein(a) [apo(a)] covalently attached to apolipoprotein B. Elevated Lp(a) is associated with increased risk of atherosclerotic heart disease. The structural homology of Lp(a) and plasminogen and the variable number of apo(a) kringle 4 type 2 subunits (k4t2) between individuals, up to 51 repeats and a variable MW of 300-800 kDa, presents an analytical challenge. `Selection of calibrators of specific isoform size does not fix the problem, despite manufacturer’s claims to the contrary.

Methods

We have developed goat antisera to human apo(a)-specific peptides and a prototype immunoturbidimetric assay for Lp(a). We selected and synthesized 10 peptide sequences (15-30 aa each) distinct from the sequences found in k4t2 regions and plasminogen. Peptides were prepared attached to KLH (GenScript) and combined to immunize goats following a standard protocol including monthly boosters (Nittobo). Next, we assessed various sample volumes, reagent formulations and antisera dilutions to establish parameters on the Randox Imola analyzer. Cross-reactivity with recombinant k4t2 (40 mg/dL) and plasminogen (200 mg/dL, Athens Research) was assessed by comparison to the LOD (using delipidated plasma blank and low concentration samples).

Results

Titers to human Lp(a)) were positive to 1:6400 dilution of antisera, showing exceptional reactivity. Working formulation/parameters were 10 µL sample, 100 µL PBS, pH 7.4/PEG6000/detergent and 95 µL R2 (antisera). Sample/R1 was incubated for 5-minutes before addition of R2; reaction was monitored at 340 nm/37°C. LOD and linearity were also assessed. No cross-reactivity with k4t2 or plasminogen up to the concentrations tested was observed. Linearity to 330 nmol/L and LOD of 18 nmol/L were found.

Conclusions

The use of this antisera/assay eliminates the bias associated with apo(a) size polymorphisms and allows for the accurate measurement of Lp(a) particle number. More work is needed to optimize assay parameters and formulation and comprehensively validate assay performance.
A-219 Method Intercomparison Study between Atellica Solution® and CI® Analyzers for Total Cholesterol and Triglycerides
Authors: Ruiz-Alvarez M , Lapeña-Garcia S , Garcia-Valdecasas S , Costales-Lucia T , Moreno-Romero A , Molina-Blas S , Barrionuevo-Gonzalez M
Published: October 2024

Background

Method intercomparison is an essential requirement that every Laboratory must perform when considering a method or instrument change for a specific analyte.

Objective

verify if the results of two analytical techniques (Total Cholesterol and Triglycerides) measured by Atellica Solution® and CI® analyzers (Siemens Healthineers) are interchangeable.

Methods

40 patient samples were selected for each assay, with results distributed across the entire measurement range and analyzed by both instruments. Estatistical program Method Validator was used. To determine the degree of agreement between both instruments, Bland-Altman difference analysis and Passing-Bablok regression were performed. Statistics are expressed with their 95% confidence intervals.

Results

Total Cholesterol: After analyzing Bland-Altman differences, a systematic error was found as the mean difference between the results obtained by Atellica Solution® and CI 1900® was 13.8 with a 95% CI that does not include the null value. Regarding Passing Bablok analysis, a constant systematic difference was observed as the confidence interval of the intercept does not include the zero value, and also a proportional difference based on the 95% CI of the slope that does not include the value 1.• Triglycerides: After analyzing the Bland-Altman differences, a systematic error was found as the mean difference between the results obtained by Atellica Solution® and CI 1900® was -15.6, with higher results in Atellica Solution®. Regarding the Passing Bablok analysis, constant systematic differences were observed as the confidence interval of the intercept does not include the zero value, and also proportional differences based on the slope, whose 95% CI does not include the value 1

Conclusions

It´s concluded that Atellica Solution® and CI® analyzers are not interchangeable for Total Cholesterol and Triglycerides assays due to the presence of a constant systematic error. To use them on both instruments, it would be advisable to modify the reference values for both methods.
A-232 Characterize and reduce positive interference of hemolysis on Fungitell testing for (1, 3)-β-glucan (BDG) in serum
Authors: Zhang Y , Finkelman M , D’ordine R , Kowald G , Roy J
Published: October 2024

Background

Fungitell is an FDA cleared in vitro diagnostic product with an indication for use as an adjunct test for the diagnosis of invasive fungal infection. This assay has been established using a kinetic photometric method. A key aspect of this (1→3)-β-glucan (BDG) activated cascade reaction is its characteristic kinetic optical density profile. Interference which affects the BDG kinetic profile makes data analysis and interpretation difficult. This interference is roughly classified as being associated with hemolysis, icterus, lipemia (collectively HIL), and other non-specific factors.

Methods

In this study, we found that the use of 5% PEG-8000 supplemented serum (1:1 ratio of the volume of serum to 10% PEG-8000 solution with 0.1 M Tris-Cl, pH 7.4), heated at 80°C for 3.5 min, followed by centrifugation at 15,000 x g for 10 minutes (PEG-X Method) can largely eliminate the hemolysis-associated interference.

Results

The treated serum Fungitell BDG reaction curves are more like the ideal BDG kinetic profile. The recovery of introduced β-glucan has been shown to be acceptable (80-120%) for hemolyzed specimens.

Conclusions

This simple and rapid procedure shows promise as a pre-analytical treatment to reduce serum interference in the Fungitell β-glucan test for a substantial fraction of samples that produce currently unreportable results.
A-248 Assessment of Nanopore Sequencing for HIV Genotyping: A Comparative Study with Sanger Sequencing
Authors: Andrade M , Belmok A , Silva L , Vieira A , Vieira C , Velasco L , Jácomo R , Nery L
Published: October 2024

Background

The HIV drug resistance test, conducted through genotyping of the Human Immunodeficiency Virus (HIV), is a critical tool for assessing drug resistance in virus-infected patients and is commonly performed using Sanger Sequencing methodology (first-generation sequencing). Nanopore sequencing (third-generation sequencing) is a new technology that started to be commercialized a few years ago and has recently undergone significant updates with the new Q20+ chemistry. Therefore, this study aims to evaluate the potential use of nanopore sequencing (MinION) for HIV genotyping.

Methods

The genomic region encoding HIV protease and transcriptase was amplified by nested PCR using primer pairs modified with unique barcodes in the second round of amplification. Three libraries were assembled, including samples from 22 HIV-positive patients, one negative control (negative control patient), and one no-template control (NTC) per library, totaling 66 HIV-positive samples. The libraries were constructed using the SQK-LSK114 kit and sequenced on a MinION platform using the new Q20+ chemistry in flowcells or flongle R10.4.1. The sequences were assembled and analyzed using HIVdb-NGS (Stanford). Validation tests, such as accuracy and precision, were conducted following WHO recommendations for HIV genotyping. The same samples were also sequenced using the Sanger method for comparison.

Results

62 HIV sequences were generated by the MinION and analyzed in the described pipeline. The average sequence coverage per sample was 25,866 x. 41 sequences were classified as type B (63.08%), 12 as type C (18.46%), 8 as type F (12.31%), three as CRF02_AG (4.62%), and 1 as a recombinant B+C (1.54%). All identified subtypes were consistent with those found by the Sanger method. The average similarity between the sequences of each sample (MinION vs Sanger) was 98.68% (minimum 94.96%, maximum 99.95%). Basecalling similarity at relevant positions (associated with resistance) was above 98% in 59 out of 62 samples (95.16%), meeting the acceptance criteria recommended by the WHO.

Conclusions

Our results suggest that nanopore sequencing (utilizing Q20+ chemistry) can replace Sanger sequencing for HIV genotyping, which is used to monitor the effectiveness of anti-HIV drugs. This transition will allow for a reduction in test costs in laboratories with high demand for HIV drug resistance testing. Additionally, the use of modified primers proposed here eliminates the need for commercial barcodes, resulting in reduced processing time and improved throughput.
A-267 How Does Rapid HIV Antibody Testing Fit Into the Current Testing Algorithm?
Authors: Bui-Bullock T , Farnsworth C W , Anderson N W
Published: October 2024

Background

Many emergency departments (ED) currently use rapid HIV antibody tests as screening tools, despite limited sensitivity for detecting acute HIV infections. We performed an analysis of standard of care rapid and in-lab fourth generation HIV testing, to 1) assess the performance of rapid HIV testing in our patient population and 2) identify key symptoms of acute HIV infections.

Methods

A four-year retrospective analysis from 1/1/2018-12/31/2021 was conducted at Barnes Jewish Hospital and St. Louis Children’s Hospital in St. Louis, Missouri. During the study period, both rapid HIV testing (SURE CHECK HIV 1/2, Chembio) and in-lab fourth generation HIV testing (ARCHITECT HIV Ag/Ab Combo Assay, Abbott) were available to providers. Comparative performance of rapid testing and in-lab fourth generation testing was assessed using complete Fourth Generation HIV algorithmic testing as the gold standard. Symptomatology was recorded for a subset of patients that presented to the ED to determine associations with different testing patterns.

Results

A total of 28,240 rapid HIV antibody tests were administered throughout inpatient, outpatient, and emergency departments at both hospitals, revealing an average yearly positivity rate of 0.6%. In a subset of 1192 patients who underwent both rapid HIV antibody and fourth generation in-lab testing, the rapid HIV antibody test exhibited a positive percent agreement of 92.5% (95% CI: 84.6-96.5) and negative percent agreement of 98.1% (95% CI: 97.1-98.8). Rapid testing demonstrated a negative predictive value (NPV) of 99.5% (95% CI: 98.8-99.8) and a positive predictive value of 77.9% (95% CI: 68.6-85.1). Notably, the rapid test failed to detect 3/3 (100%) acute HIV infections defined as rapid HIV antibody test negative, Ag/Ab assay and nucleic acid amplification assay positive. The rapid test demonstrated comparable performance in patients with known HIV infections, regardless of antiretroviral therapy (ART) status. The positivity rate was 23/24 (95.8%) in known HIV patients with ART compared to 25/27 (92.6%) in those without (P>0.9999). Among patients on ART, the mean ± SD log10 viral load was 3.15±1.52, whereas those without ART exhibited a viral load of 4.87±0.99 (P=0.0140). In the ED, symptoms such as fever, nausea/vomiting, malaise, headache, and photophobia were significantly correlated with acute HIV infection. Acute HIV patients were more likely to display fever in conjunction with three other symptoms (P<0.0001).

Conclusions

The rapid HIV antibody test demonstrated high sensitivity, specificity, and NPV in our study population, reaffirming its effectiveness as a valuable screening tool. ART did not seem to affect the rapid test performance. However, the low PPV and 100% failure in detecting acute HIV infections underscore the importance of confirmatory in-lab testing. Insights into symptomatology of patients with acute HIV inform ED providers when confirmatory testing is essential for a timely and accurate HIV diagnosis.
A-281 Comparison of C-reactive Protein, Procalcitonin, and Presepsin in Blood and Synovial Fluid as Diagnostic Biomarkers for Septic Arthritis
Authors: Choi H , Choi J , Jeon S , Kim M , Seon J , Kee S
Published: October 2024

Background

Early detection and prompt treatment are crucial for managing septic arthritis (SA). However, diagnosing SA at an early stage can be challenging since the symptoms are non-specific, and there is no definitive diagnostic test available. This study aimed to investigate the potential of biomarkers as a reliable diagnostic tool for SA.

Methods

A prospective study was conducted to collect residual blood and synovial fluid samples from patients who underwent blood sampling and arthrocentesis for differential diagnosis due to suspected SA. The collected samples were analyzed for C-reactive protein (CRP), procalcitonin, and presepsin. The data obtained from the analysis were subjected to statistical analysis.

Results

From January to November 2023, 58 patients were enrolled. They comprised 24 men and 34 women with a median age of 71.1 (interquartile range, IQR 64.0-79.3) years. Twenty-one patients were diagnosed with SA and 37 with non-SA, including osteoarthritis (n=2), osteonecrosis (n=1), meniscus tear (n=1), abscess (n=1), contusion (n=2), and mechanical complications of implants (n=30). There were no significant differences in age or sex distribution between the two groups. The median blood and synovial fluid CRP levels were 4.24 and 1.85 mg/dL, respectively, in the SA group and 0.24 and 0.11 in the non-SA group. The median blood and synovial fluid procalcitonin levels were 0.0383 and 0.0403 ng/mL in the SA group and 0.0358 and 0.0488 in the non-SA group. The median blood and synovial fluid presepsin levels were 307 and 1389 pg/mL in the SA group and 192 and 959 in the non-SA group. Except for blood procalcitonin, all showed statistically significant differences between the SA and non-SA groups. In the ROC curve analysis, blood CRP displayed the highest area under the curve (AUC) of 0.795 with a 0.47 mg/dL cut-off value, 90.5% sensitivity, and 64.9% specificity. Synovial fluid CRP displayed similar results of 0.786 AUC, 0.30 mg/dL cut-off value, 85.0% sensitivity, and 71.4% specificity. Blood and synovial fluid procalcitonin had low AUCs of 0.532 and 0.659, respectively, and were insufficient to differentiate between SA and NS groups. The AUC (0.696 vs. 0.704), sensitivity (76.2% vs. 76.2%), and specificity (67.6% vs. 62.2%) of blood and synovial fluid presepsin were similar; however, synovial fluid showed a significantly higher cut-off value, 4.5 times that of blood (237 pg/mL vs. 1087 pg/mL).

Conclusions

Procalcitonin was not a reliable marker for distinguishing between SA and non-SA, regardless of whether it was measured in blood or synovial fluids. However, CRP and presepsin proved useful markers when measured in either blood or synovial fluid. In particular, when measuring presepsin in synovial fluid, an optimal cut-off value is needed.
A-290 Prevalence and Antimicrobial Resistance Pattern of Microorganisms Recovered From Bloodstream Infections From Public Hospitals of São Paulo City, Brazil
Authors: de Andrade S S , Nurmberger J M , Inoue F M , Lobo A P , Ramadan-Boscolo D R , Tufik S
Published: October 2024

Background

Nosocomial Bloodstream Infections (nBSI) are an important cause of morbidity and mortality. Brazilian studies concerning bloodstream infections are important tools that can bring specific issues related to antimicrobial resistance. The objective of this study was to describe the frequency and antimicrobial susceptibility profile of bacterial isolates from BSI in patients admitted to public hospitals in 2022 and 2023.

Methods

This study was a retrospective analysis from 12 hospitals (January 2022 to December 2023) based on BSI isolates laboratory data detected by the clinical microbiology department of a medical diagnostic laboratory. The identification and susceptibility tests were performed using MALDI-TOF mass spectrometry (Vitek MS) and Vitek®2 systems, according to the manufacture’s recommendations. Coagulase Negative Staphylococci (CoNS) BSI was only considered in the presence of at least two sets of positive blood cultures with the same organism.

Results

During the period (January 2022 to December 2023) a total of 19,588 microorganisms isolated from BSI were obtained from 12 hospitals. The frequency of organism group was Gram-positive (10,707; 54.7%), Gram-negative (7,874; 40.2%), yeasts (961; 4.9%) and filamentous (46; 0.2%). The order of prevalence (top ten) organisms was CoNS (5,149; 26.3%); Staphylococcus aureus (2,769; 14.1%); Klebsiella spp. (2,355; 12.0%); Escherichia coli (1,301; 6.6%); Acinetobacter spp. (1,293; 6.6%); Streptococcus spp. (1,125; 5.7%); Enterococcus spp. (1,112; 5.7%); Candida spp. (937; 4.8%); Pseudomonas spp. (749; 3.8%) and Proteus spp. (553; 2.8%). Extended spectrum beta-lactamase rates for E. coli and Klebsiella pneumoniae were 31.6% (268/848) and 74.9% (1027/1371), respectively. The resistance to carbapenems was detected mainly in 74.1% (661/892) of Acinetobacter spp. isolates, 60.7% (832/1371) of Klebsiella pneumoniae; 24.5% (114/465) of P. aeruginosa; 9.7% (34/349) of Proteus spp. and 1.4% (12/828) of Escherichia coli. Antimicrobial resistance was observed mainly in 87.2% of CoNS (2831/3247) and 44.5% of S. aureus (743/1669) to methicillin; 22.9% (146/637) of Enterococccus spp. to vancomycin.

Conclusions

The frequency of Klebsiella spp. isolates from nBSI has progressively increased and effective infection control measures should be taken to prevent them from spreading. The rates of antimicrobials resistance reported for Klebsiella pneumoniae, Acinetobacter spp., CoNS, S. aureus and Enterococcus spp. reinforce the high resistance rates to antimicrobials in São Paulo city.
A-326 Testing for Multidrug-Resistant Acinetobacter baumannii with a Microfluidic Device
Authors: Gärtner C , Treffon J , Kreuchauff J , Heppner B , Isserstedt-John N , Klemm R R , Omengo B , Mellmann A
Published: October 2024

Background

Multidrug-resistant (MDR) Acinetobacter baumannii in hospitals contribute to high mortality rates and entail expensive infection control procedures. Conventionally, species and resistance determination of MDR bacteria is performed by mass spectrometry and labor-intensive phenotypical assays in clinical microbiology laboratories. Some institutions also apply time-consuming and expensive whole genome sequencing for in-depth resistance and transmission analysis. In contrast, faster and more cost-efficient MDR typing can be achieved by on-site application of real-time PCR-based point-of-care tests (POCTs) that target species- and resistance-specific genes and enable rapid screening of large sample sizes.

Methods

To construct a real-time PCR-based POCT system for species and resistance determination of MDR A. baumannii, we selected two species-specific genes (rpoB, OXA-51-like) and 18 resistance genes found in MDR A. baumannii (OXA-23-like, OXA-24/40-like, OXA-51-like, OXA-58-like, OXA-143-like, mcr, ADC, NDM, GIM, VIM, IMP, GES, PER, TEM, SHV, VEB, CTX-M, KPC) as targets. For each gene, one to six sets of primers and fluorophore-labeled probes that amplify conserved genetic regions were designed and their functionality was tested in vitro. Parallel, a fully automated system comprising microfluidic chips and a bench-top analyzer able to perform DNA extraction and real-time PCR starting from swab eluate was developed.

Results

All oligonucleotides tested so far reliably detected 100 to 100,000 copies of DNA extracted from MDR bacteria in single- and multiplex experiments in vitro. Tests with the microfluidic system showed that lysis of different bacterial species could be accomplished via sonication on a membrane located on the microfluidic chip. In addition, chip implementation of all PCR reagents like lyophilized master mix as well as primers and probes was successful and can be stored on chip at least one year. So far, the system delivered detectable fluorescence signals within one hour when performing test runs with bacteria eluted from swabs.

Conclusions

A real-time PCR-based Point-of-Care screening platform was developed for the detection of antibiotic-resistant A. baumannii from various patient sample materials starting directly from swabs, which detects colonization/infection and at the same time the relevant resistance genes within about an hour.
A-329 Accuracy of the pH, PO2, and PCO2 Tests in the i-STAT® G3+ Cartridge Using the i-STAT 1 System for Venous, Arterial and Capillary Whole Blood Specimens in Clinical Settings
Authors: Kornfeld S F , Gonzalez Aguirre M , Mohammed A , Kovacevic S , Zhang H , Gupta M
Published: October 2024

Background

Precise and accurate measurements of blood gases are important for the diagnosis, monitoring and treatment of respiratory, metabolic, and acid-base disturbances. This study demonstrates the accuracy of the pH, partial pressure of oxygen (PO2), and partial pressure of carbon dioxide (PCO2) tests in the i-STAT G3+ cartridge with the i-STAT 1 System in arterial, venous, and capillary whole blood specimens in point of care or clinical laboratory settings.

Methods

The accuracy of the PO2, and PCO2 tests in the i-STAT G3+ cartridge was assessed at external clinical sites in a method comparison study using whole blood specimens across the measurement range for each test: pH 6.5-7.8, PO2 5-700 mmHg, PCO2 5.0-130.0 mmHg. Specimens used in this study were venous, arterial, and capillary specimens prospectively collected under Institutional Review Board-approved informed consent or were leftover de-identified venous or arterial specimens. The study included venous and arterial whole blood samples from ∼140 adult subjects (≥ 18 years) at 3 clinical sites, and capillary whole blood samples from ∼180 adult subject at 3 clinicals sites and ∼25 neonate subjects (birth to 28 days) at 2 clinical sites. The RapidPoint System was used as comparator. For venous and arterial whole blood, samples were collected and tested in duplicate on the i-STAT G3+ cartridge and in singlicate on the RapidPoint System. For capillary whole blood samples, adult fingerstick samples and neonate heel-stick samples were collected in capillary tubes and tested in singlicate on the i-STAT G3+ cartridge and on the RapidPoint System. Passing-Bablok regression was performed for venous and arterial whole blood samples combined, using the first replicate from the i-STAT G3+ cartridge versus the singlicate result from the comparator, and for capillary whole blood samples (adult and neonate combined) using the singlicate result from the i-STAT G3+ cartridge versus the singlicate result from the comparator.

Results

Passing-Bablok regression of i-STAT G3+ cartridge results versus the comparator was used to calculate slope and correlation coefficient (r). For adult venous and arterial whole blood results, pH had slope = 0.96 and r = 0.99, PO2 had slope = 1.05 and r = 1.00, and PCO2 had slope = 1.04 and r = 0.98. For adult and neonate capillary whole blood results, pH had slope = 1.02 and r = 0.98, PO2 had slope = 1.09 and r = 0.99, and PCO2 had slope = 1.07 and r = 0.96.

Conclusions

These results demonstrate that the i-STAT 1 System delivers accurate pH, PO2, and PCO2 test results with ∼2 minutes using the i-STAT G3+ cartridge for venous, arterial and capillary whole blood samples for adult and neonate subjects when tested by end-users in a point of care or clinical laboratory setting. This study was funded by Abbott Laboratories.