bluuebunny/crossref_metadata_2025_split huggingface dataset:
A-070 Evaluation of Plain and Serum-Separator Blood Collection Tubes for the Measurement of Steroid Hormones by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)
Authors: Garg U , Ramoo B , Frazee C , Dasgupta A 
Published: October 2024

Abstract

Background
Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) is the preferred method for quantifying steroids. Commonly measured steroids 11-deoxycortisol, androstenedione, testosterone, 17-hydroxyprogesterone and dehydroepiandrosterone (DHEA). Although serum separator blood collection tubes may not be recommended but their frequent use is attributed to the ease of serum separation. Moreover, the serum separator tube may be the only available option for analysis, without the need for additional sample collection. Under such conditions, from certain serum separator tubes, we observed occasional interference in LC-MS/MS steroid method. We pursued to evaluate the performance of blood collection tubes without and those with serum separators, for the quantification of specified steroids by LC-MS/MS.

Methods
Blood samples were collected from 10 volunteers using both plain and serum separator blood collection tubes procured from two major suppliers (Greiner and BD). The specified steroids were measured by LC-MS/MS method. The procedure involved addition of labeled internal standards to the samples and extraction of steroids in methyl tert-butyl ether. Subsequently, the extract dried under a stream of nitrogen, and the residue was reconstituted in the mobile phase. LC-MS/MS analysis involved multiple reaction monitoring (MRM) for the specified steroids and their internal standards.

Results
The figure shows a sample chromatogram from a subject with blood samples collected in a plain (A) and serum separator tubes from Greiner (B) and BD (C1 and C2, a zoomed of C1). In this sample, from the plain tube, the concentrations of 11-deoxycortisol, androstenedione, testosterone, 17-hydroxyprogesterone and DHEA were 27, 55, 690, 99 and 283 ng/mL respectively. No significant interference was observed in Greiner serum separator tube. Whereas BD serum separator tube exhibited several erroneous peaks making the quantitation of several steroids challenging or inaccurate.

Conclusions
In the described LC-MS/MS method, BD serum-separator tubes exhibited erroneous peaks. These peaks were not seen in Greiner serum-separator tubes.
A-077 Evaluation of Diagnostic Thresholds for Adrenal Insufficiency Post-Cosyntropin Stimulation: Assessment Using Siemens Atellica Cortisol Immunoassay
Authors: Li S , Chen L , Calanche A T , Lee J K , Ghalib L , Meng Q H 
Published: October 2024

Abstract

Background
Accurate interpretation of the cosyntropin (adrenocorticotropic hormone [ACTH]) stimulation test relies on method- and assay-specific cortisol cutoffs. While the Endocrine Society recommends an 18 ug/dL cutoff, lower thresholds (14.6 ug/dL) were proposed for the Roche Elecsys II assay in 2021, which employs a monoclonal antibody. However, cutoff values for the new Siemens Atellica platform, utilizing polyclonal antibody-based cortisol assays, remain undetermined. This study aimed to evaluate cortisol thresholds specific to the Siemens Atellica immunoassay by comparing cortisol measurements with the Roche Elecsys II assay.

Methods
The ACTH stimulation test was conducted in patients with suspected adrenal insufficiency (n=28). Serum cortisol levels were measured using the Siemens Atellica and Roche Elecsys II assays. Atellica assay results were also compared with those obtained via liquid chromatography tandem mass spectrometry (LC-MS/MS).

Results
Cortisol concentrations measured with the Atellica assay exhibited approximately 25-30% higher bias compared to LC-MS/MS and the Roche Elecsys II assay. Passing Bablok regression analysis revealed a strong correlation between the Atellica and LC-MS/MS assays (R2 = 0.9312, slope = 1.26, intercept = 0.34). However, the correlation between Atellica and the Roche Elecsys II assay was weaker compared to LC-MS/MS (R2 = 0.7049 at 0 min, slope = 1.28, intercept = 0.19; R2 = 0.8066 at 30 min, slope = 1.28, intercept = 0.59; R2 = 0.8584 at 60 min, slope = 1.17, intercept = 2.03). The optimized cortisol threshold using the Atellica assay was determined to be 18 ug/dL at 30 minutes after stimulation (sensitivity, 100%; specificity, 90%), using the proposed 14.6 ug/dL cutoff at 30 minutes on Roche Elecsys II as the standard.

Conclusions
Employing assay-specific cutoffs is crucial for reducing misclassification and overtreatment in patients with suspected adrenal insufficiency. We recommend a cortisol threshold of 18 ug/dL at 30 minutes after ACTH stimulation for the Atellica assay, consistent with the Endocrine Society's recommendation.
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

Abstract

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

Abstract

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 &lt; 0.01), increasing amyloid PET centiloid results (p &lt; 0.05), as well as worsening MMSE scores (p &lt; 0.05). In addition, NfL levels become elevated with worsening MMSE scores (p &lt; 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

Abstract

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 &lt; -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

Abstract

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

Abstract

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 &gt;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

Abstract

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

Abstract

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

Abstract

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 &gt;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).