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OBJECTIVES: The pre-analytical stability of various biochemical analytes requires careful consideration, as it can lead to the release of erroneous laboratory results. There is currently significant variability in the literature regarding the pre-analytical stability of various analytes. The aim of this study was to determine the pre-analytical stability of 65 analytes in whole blood, serum and plasma using a standardized approach. METHODS: Blood samples were collected from 30 healthy volunteers (10 volunteers per analyte) into five vacutainers; either SST, Li-heparin, K2-EDTA, or Na-fluoride/K-oxalate. Several conditions were tested, including delayed centrifugation with storage of whole blood at room temperature (RT) for 8â¯h, delayed centrifugation with storage of whole blood at RT or 4⯰C for 24â¯h, and immediate centrifugation with storage of plasma or serum at RT for 24â¯h. Percent deviation (% PD) from baseline was calculated for each analyte and compared to the maximum permissible instability (MPI) derived from intra- and inter-individual biological variation. RESULTS: The majority of the analytes evaluated remained stable across all vacutainer types, temperatures, and timepoints tested. Glucose, potassium, and aspartate aminotransferase, among others, were significantly impacted by delayed centrifugation, having been found to be unstable in whole blood specimens stored at room temperature for 8â¯h. CONCLUSIONS: The data presented provides insight into the pre-analytical variables that impact the stability of routine biochemical analytes. This study may help to reduce the frequency of erroneous laboratory results released due to exceeded stability and reduce unnecessary repeat phlebotomy for analytes that remain stable despite delayed processing.
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Recolección de Muestras de Sangre , Plasma , Suero , Humanos , Recolección de Muestras de Sangre/métodos , Plasma/química , Suero/química , Análisis Químico de la Sangre/métodos , Análisis Químico de la Sangre/normas , Adulto , Masculino , Temperatura , Femenino , Voluntarios Sanos , CentrifugaciónRESUMEN
OBJECTIVES: Monitoring quality indicators (QIs) is an important part of laboratory quality assurance (QA). Here, the Canadian Society of Clinical Chemists (CSCC) Point of Care Testing (POCT) and QI Special Interest Groups describe a process for establishing and monitoring QIs for POCT glucose testing. METHODS: Key, error prone steps in the POCT glucose testing process were collaboratively mapped out, followed by risk assessment for each step. Steps with the highest risk and ability to detect a non-conformance were chosen for follow-up. These were positive patient identification (PPID) and repeat of critically high glucose measurements. Participating sites were asked to submit aggregate data for these indicators from their site(s) for a one-month period. The PPID QI was also included as part of a national QI monitoring program for which fifty-seven sites submitted data. RESULTS: The percentage of POCT glucose tests performed without valid PPID ranged from 0-87%. Sites without Admission-Discharge-Transfer (ADT) connectivity to POCT meters were among those with the highest percentage of POCT glucose tests performed without valid PPID. The percentage repeated critically high glucose measurements ranged from 0-50%, indicating low compliance with this recommendation. A high rate of discordance was also noted when critically high POCT glucose measurements were repeated, demonstrating the importance of repeat testing prior to insulin administration. CONCLUSIONS: Here, a process for establishing these QIs is described, with preliminary data for two QIs chosen from this process. The findings demonstrate the importance of QIs for identification and comparative performance monitoring of non-conformances to improve POCT quality.
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Glucosa , Sistemas de Atención de Punto , Indicadores de Calidad de la Atención de Salud , Canadá , Opinión Pública , Glucosa/química , Pruebas en el Punto de Atención , HumanosRESUMEN
BACKGROUND: We evaluated analytical and Sigma performance for 6 next generation chemistry assays on the Abbott Architect c8000 system. METHODS: Albumin with bromocresol purple or green, amylase, cholesterol, total protein, and urea nitrogen were analyzed using photometric technology. Analytical performance goals were defined based on Accreditation Canada Diagnostics (ACD) and Clinical Laboratory Improvement Amendments (CLIA). Precision study consisted of testing 2 quality control concentrations and 3 patient serum sample pools, twice a day in quintuplicate over 5 days. Linearity testing consisted of 5-6 concentrations of commercial linearity materials. We tested a minimum of 120 serum/plasma specimens on the new and current Architect methods for comparison. We assessed accuracy with reference materials for 5 assays, and a calibration standard for cholesterol. Bias from the reference standard target value was used for Sigma metric analysis. RESULTS: Observed total imprecision of the assays ranged from 0.5 to 4%, meeting pre-defined goals. Linearity was acceptable over the tested range. Measurements on the new and current Architect methods were comparable. Accuracy ranged from 0 to 2.0% absolute mean difference from target value. All 6 next generation clinical chemistry assays demonstrated Six Sigma quality, using CLIA standards. CONCLUSION: Applying ACD recommendations, 5 assays showed Six Sigma, while cholesterol showed Five Sigma performance.
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Química Clínica , Gestión de la Calidad Total , Humanos , Control de Calidad , Estándares de Referencia , CalibraciónRESUMEN
Serological assays detect the presence of specific antibodies in blood. There are urgent and important applications for serological point-of-care (POC) assays. However, available detection methods are either insufficiently sensitive or too complex for POC settings. Here, we demonstrate that lateral flow immunoassay (LFIA), which is arguably the simplest universal molecular detection approach, can serve as a methodological platform for highly sensitive serological POC assays if combined with a simple, fast, and inexpensive electrophoretic step. In this work, we compared such electrophoretically driven LFIA (eLFIA) with conventional LFIA for the detection of immunoglobulins G against hepatitis B and C in serum. The limit of detection of eLFIA was proven to be 1000 times lower than that of conventional LFIA and sufficiently low to support clinical serological tests. eLFIA takes less than 10 min, requires only a minor accessory powered by a small 9 V battery, and can be performed by an untrained person in the POC environment using a 3 µL specimen of finger-prick capillary blood.
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Inmunoglobulina G , Sistemas de Atención de Punto , Humanos , Límite de Detección , Inmunoensayo/métodos , Pruebas SerológicasRESUMEN
OBJECTIVES: Hemolysis, icterus, and lipemia (HIL) are common sources of endogenous interference in clinical laboratory testing. Defining the threshold of interference for immunoassays enables appropriate reporting of their results when they are affected by HIL. METHODS: Pools of residual patient serum samples were spiked with a known amount of interferent to create samples with varying concentrations of hemolysate, bilirubin, and Intralipid that mimicked the effects of endogenous HIL. Samples were analysed on the Alinity i analyser (Abbott Diagnostics) for more than 25 immunoassays. The average recovery relative to the non-spiked sample was calculated for each interference level and was compared to a predefined allowable bias. RESULTS: C-peptide, estradiol, serum folate, free T4, homocysteine, insulin, and vitamin B12 were found to be affected by hemolysis, at hemoglobin concentrations between 0.3 to 20 g/L. Immunoassays for BNP, estradiol, free T3, and homocysteine were affected by icterus at conjugated bilirubin concentrations between 50 to 1,044 µmol/L. BNP, serum folate, and homocysteine were affected by Intralipid with measured triglyceride concentrations between 0.8 to 10 mmol/L. Lastly, serological immunoassays for HIV and hepatitis A, B and C were also affected by interferences. CONCLUSIONS: Immunoassays are impacted by varying degrees of HIL interference. Some measurands, in the presence of interference, are affected in a manner not previously indicated. The data presented herein provide an independent evaluation of HIL thresholds and will be of aid to resource-limited clinical laboratories that are unable to internally verify endogenous interferences when implementing the Alinity i analyser.
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Hiperlipidemias , Ictericia , Humanos , Hemólisis , Hiperlipidemias/diagnóstico , Ictericia/diagnóstico , Inmunoensayo/métodos , Bilirrubina , Estradiol , Ácido FólicoRESUMEN
OBJECTIVES: Immune checkpoint inhibitors (ICIs) cause a variety of toxicities, including immune-related adverse events (irAEs), but there are no biomarkers to predict their development. Guidelines recommend measuring circulating cardiac troponin I (cTnI) during ICI therapy to detect related cardiotoxicities. Moreover, elevated cTnI has also been associated with worse outcomes in non-cardiac patients, including cancer. Thus here, we investigated whether cTnI levels were higher in patients with irAEs. METHODS: The study consisted of three groups; 21 cancer patients undergoing ICI immunotherapies who presented with irAEs, four patients without irAEs, and 20 healthy controls. Patient samples were assessed at baseline (n=25), during ICI treatment (n=25, median=6 weeks of treatment) and at toxicity (n=6, median=13 weeks of treatment). In addition to blood high sensitivity cardiac troponin I (hs-cTnI), anti-thyroglobulin (TG) and anti-thyroid peroxidase (TPO) antibodies were also quantitated to detect thyroid dysfunction, constituting the second leading toxicity (23.8%) after pneumonitis (28.6%). RESULTS: Four patients with irAEs (n=4/21; 19%) and one without irAEs (n=1/4; 25%) showed higher hs-cTnI levels at any time-point; the remaining had physiological levels. None of these patients developed cardiotoxicity. Concurrent elevated levels of anti-thyroid antibodies and hs-cTnI were detected in one patient with thyroid dysfunction (n=1/5, 20%). However, these antibodies were also elevated in three patients (n=3/16, 19%) with non-thyroid irAEs and in up to 40% of healthy controls. CONCLUSIONS: hs-cTnI was not elevated in patients with irAEs, but larger studies are needed to confirm these observations.
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Antineoplásicos Inmunológicos , Inhibidores de Puntos de Control Inmunológico , Neoplasias , Humanos , Antineoplásicos Inmunológicos/efectos adversos , Cardiotoxicidad , Estudios de Casos y Controles , Inhibidores de Puntos de Control Inmunológico/efectos adversos , Neoplasias/tratamiento farmacológico , Neoplasias/complicaciones , Estudios Retrospectivos , Enfermedades de la Tiroides , Troponina IRESUMEN
Lateral flow immunoassay (LFIA) is a rapid, simple, and inexpensive point-of-need method. A major limitation of LFIA is a high limit of detection (LOD), which impacts its diagnostic sensitivity. To overcome this limitation, we introduce a signal-enhancement procedure that is performed after completing LFIA and involves controllably moving biotin- and streptavidin-functionalized gold nanoparticles by electrophoresis. The nanoparticles link to immunocomplexes forming multilayer aggregates on the test strip, thus, enhancing the signal. Here, we demonstrate lowering the LOD of hepatitis B surface antigen from approximately 8 to 0.12â ng mL-1 , making it clinically acceptable. Testing 118 clinical samples for hepatitis B showed that signal enhancement increased the diagnostic sensitivity of LFIA from 73 % to 98 % while not affecting its 95 % specificity. Electrophoresis-driven enhancement of LFIA is universal (antigen-independent), takes two minutes, and can be performed by an untrained person.
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Oro , Nanopartículas del Metal , Humanos , Límite de Detección , Biotina , Inmunoensayo/métodosRESUMEN
BACKGROUND: Manual extraction of immunosuppressants is required before measurement on the Architect immunoassay analyzer. The individual extraction of samples places clinical laboratory staff at risk for ergonomic injury. We evaluated the analytical performance of a batched extraction method for measuring sirolimus, tacrolimus, and cyclosporine using the Architect i2000SR. METHODS: Residual whole blood samples from patients receiving immunosuppressant therapy were used for evaluation. The analytical evaluation included imprecision, linearity, and method comparison. Technologist-to-technologist variation was also assessed. RESULTS: Total imprecision ranged from 2.58 to 3.13% for sirolimus, 2.70-3.77% for tacrolimus, and 7.82-12.41% for cyclosporine. Linearity was verified from 0.44-19.49 µg/l for sirolimus, 0.05-26.15 µg/l for tacrolimus, and 0.15-991.55 µg/l for cyclosporine. Deming regression analysis showed slope and intercept were not significant for either technologist-to-technologist comparison or for batched vs. individual processing comparison. Bland-Altman analysis of individual vs. batched processing revealed a mean bias of 1.29% (LLOA: -14.63%, ULOA: 17.21%) for sirolimus, 2.07% (LLOA: -10.87%, ULOA: 15%) for tacrolimus, and -1.56% (LLOA: -20.05%, ULOA: 16.94%) for cyclosporine. The values were not significantly different from the bias and LLOAs observed for technologist-to-technologist comparison. CONCLUSIONS: The imprecision and linearity of batched methods met analytical goals. The batched method also correlated well with the individual extraction methods. The ULOA and LLOA for all drugs tested exceeded a TAE or ± 15%. However, similar range of differences was observed between technologists, suggesting that batch processing did not increase or reduce variability due to manual preparation steps.
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Ciclosporina , Tacrolimus , Monitoreo de Drogas/métodos , Everolimus , Humanos , Inmunosupresores , SirolimusRESUMEN
INTRODUCTION: A patient presented to hospital with chest pain and shortness of breath on 2 occasions 4 weeks apart. Clinical examination revealed an elevated jugular venous pressure consistent with heart failure or elevated filling pressures. METHODS: The patient was investigated through various modalities including electrocardiogram (ECG), transthoracic echocardiogram, coronary angiography, MRI, cardiac catheterization, positron emission tomography, and an extensive laboratory workup. RESULTS: Serial hs TnI measurements consistently revealed grossly elevated troponin I (>10 000 ng/L). In-lab investigation of increased high sensitivity troponin I (hsTnI) showed evidence of falsely increased troponin due to the presence of heterophilic antibodies. DISCUSSION: This case demonstrates a complex patient presentation and the value of involving the laboratory medicine team when dealing with potentially discrepant results. This is a rare report of grossly elevated troponin due to heterophilic antibodies for high-sensitivity troponin Abbott assay.
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Dolor en el Pecho , Troponina I , Cateterismo Cardíaco , Dolor en el Pecho/diagnóstico , Dolor en el Pecho/etiología , Disnea/diagnóstico , Disnea/etiología , HumanosRESUMEN
BACKGROUND: In North America, both messenger RNA (mRNA) vaccines, Pfizer-BioNTech BNT162b2, and Moderna mRNA-1273, each utilizing a 2-dose regimen, have started to be administered to individuals. METHODS: We evaluated the quantitative serologic antibody response following administration of either a single dose or both doses of an mRNA severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine in a cohort of 98 participants (88 healthcare workers [HCW] and 10 solid organ transplant [SOT] recipients). Antibody levels were compared across 3 immunoassays: Elecsys Anti-SARS-CoV-2 S (Roche Diagnostics), SARS-CoV-2 TrimericS IgG (DiaSorin), and SARS-CoV-2 IgG II Quant (Abbott). RESULTS: Among HCW, sensitivity ranged from 100% (Roche), 99% (Abbott) and 98% (DiaSorin). The SARS-CoV-2 IgG II Quant and SARS-CoV-2 TrimericS IgG assays showed good agreement with a Pearson correlation coefficient of R = 0.95. Pearson correlation coefficients of R = 0.82 and 0.83 were obtained for Elecsys Anti-SARS-CoV-2 S vs SARS-CoV-2 TrimericS IgG and SARS-CoV-2 IgG II Quant vs Elecsys Anti-SARS-CoV-2 S, respectively. Significant differences in antibody levels between HCW and SOT recipients were observed. A decrease in antibody levels from time of vaccine administration to blood draw was evident. Among those with a second dose, an increase in antibody levels with increased time between administration of the first and second dose was observed. CONCLUSIONS: The absolute values generated from each of the assay platforms are not interchangeable. Antibody levels differed with increased time between vaccine administration and with increased time between administration of the first and second dose. Further, significant differences in antibody levels between HCW and SOT recipients were observed.
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COVID-19 , SARS-CoV-2 , Vacuna nCoV-2019 mRNA-1273 , Vacuna BNT162 , Vacunas contra la COVID-19 , Humanos , InmunoensayoRESUMEN
INTRODUCTION: Sodium-glucose cotransporter-2 (SGLT2) inhibitors improve cardiovascular and kidney outcomes through mechanisms that are incompletely understood. In this exploratory post-hoc analysis of the VERTIS RENAL trial, we report the association between the SGLT2 inhibitor, ertugliflozin, and markers of kidney injury, inflammation, and fibrosis in participants with type 2 diabetes (T2D) and stage 3 chronic kidney disease (CKD). METHODS: Participants were randomized to ertugliflozin (5 or 15 mg/d) or placebo, and plasma samples for biomarker analysis were collected at baseline, 26 weeks, and 52 weeks. RESULTS: Ertugliflozin-treated participants had lower plasma levels of kidney injury molecule-1 (KIM-1) at 26 weeks (P = 0.044) and 52 weeks (P = 0.007) and higher eotaxin-1 at 52 weeks (P = 0.007) postrandomization compared with placebo. The change in KIM-1 was not associated with the baseline urine albumin to creatinine ratio (UACR) or the estimated glomerular filtration rate (eGFR, P interaction > 0.05). Additionally, the change in KIM-1 was positively correlated with the change in UACR in participants treated with ertugliflozin (P = 0.0071). No other significant associations between ertugliflozin and changes in the markers of tubular injury, inflammation, fibrosis, oxidative stress, and endothelial dysfunction were observed. CONCLUSION: In conclusion, in participants with T2D and stage 3 CKD, ertugliflozin was associated with a sustained lowering of the tubular injury marker KIM-1 regardless of baseline kidney function.
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Dried blood spots (DBS) are commonly used for serologic testing for viruses and provide an alternative collection method when phlebotomy and/or conventional laboratory testing are not readily available. DBS collection could be used to facilitate widespread testing for SARS-CoV-2 antibodies to document past infection, vaccination, and potentially immunity. We investigated the characteristics of Roche's Anti-SARS-CoV-2 (S) assay, a quantitative commercial assay for antibodies against the spike glycoprotein. Antibody levels were reduced relative to plasma following elution from DBS. Quantitative results from DBS samples were highly correlated with values from plasma (r2 = 0.98), allowing for extrapolation using DBS results to accurately estimate plasma antibody levels. High concordance between plasma and fingerpick DBS was observed in PCR-confirmed COVID-19 patients tested 90 days or more after the diagnosis (45/46 matched; 1/46 mismatched plasma vs. DBS). The assessment of antibody responses to SARS-CoV-2 using DBS may be feasible using a quantitative anti-S assay, although false negatives may rarely occur in those with very low antibody levels.
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Prueba Serológica para COVID-19 , COVID-19/diagnóstico , Pruebas con Sangre Seca , SARS-CoV-2/aislamiento & purificación , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Humanos , Reproducibilidad de los Resultados , SARS-CoV-2/inmunología , Sensibilidad y Especificidad , Glicoproteína de la Espiga del Coronavirus/inmunologíaRESUMEN
BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD)/hemoglobin (Hb) ratio helps detect G6PD deficiency, an X-linked disorder that can be asymptomatic or cause acute hemolytic anemia and chronic hemolysis. We investigated preanalytical, analytical, and postanalytical aspects to optimize G6PD/Hb measurement and interpretation. METHODS: G6PD was measured with the Pointe Scientific assay and Hb with Drabkin's reagent on Alinity c® (Abbott Diagnostics). Stability of G6PD/Hb was assessed after 7 and 14 days while stored at 2-8 °C. Stability of hemolysate prepared for G6PD analysis was assessed using QC and patient samples up to 4 h at room temperature or 2-8 °C. Analytical performance specifications including precision, method comparison, linearity, LOQ, and carry-over were established for the enzymatic reaction of G6PD and spectrophotometric reading of Hb. G6PD/Hb reference interval and cut-offs were established indirectly using truncated maximum likelihood method (TML) using retrospective data (n = 4715 patient data points). RESULTS: Samples were stable after 7 days at 2-8°C, unless grossly hemolyzed. Hemolysate prepared for G6PD measurement remained stable for up to 4 h for QC at room temperature and 2-8°C, but up to 30 min-1 h at room temperature and 1-2 h at 2-8 °C for patient samples. Precision, linearity, LOQ, and carryover were acceptable. G6PD/Hb cut-offs were <3.3, ≥3.3, 3.3-8.9, and ≥8.9 U/g Hb for deficient males/females, normal males, intermediate females, and normal females, respectively. CONCLUSIONS: In vitro hemolysis and delayed hemolysate analysis significantly reduce G6PD/Hb stability. QC material cannot detect the impact of delayed hemolysate analysis. These findings were foundational for optimizing G6PD/Hb protocols for a new platform and establishing laboratory-specific G6PD/Hb cut-offs.
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Deficiencia de Glucosafosfato Deshidrogenasa , Glucosafosfato Deshidrogenasa , Femenino , Deficiencia de Glucosafosfato Deshidrogenasa/diagnóstico , Hemoglobinas , Hemólisis , Humanos , Masculino , Estudios RetrospectivosRESUMEN
BACKGROUND: When a test result is critically abnormal, laboratories notify the responsible caregivers immediately, usually with a phone call. If the same test was ordered repeatedly, our institution has a policy of not notifying the caregiver if the previous result was also critical and within 24 h. We compared our policy with those of several different laboratories in North America and estimated the impact of changing our current policy to calling for all critical results, regardless of the time interval. METHODS: Several North American laboratories (n = 15) were surveyed regarding their critical result notification policy. For our institution, we performed a retrospective analysis focusing on critical values in a 5-month period for common chemistry tests. We estimated the effect on volume of calls and the impact on workload with regard to changing the critical result notification policy and critical thresholds. RESULTS: A majority of surveyed laboratories had some form of restriction for calling about recurring critical results. In our institution, removing the restrictions would increase the average number of daily calls by 11%-155%, depending on the analyte. The choice of critical thresholds also has an effect on the number of calls, and the effect depends on the analyte and the threshold chosen. CONCLUSIONS: Guidelines do not specify how recurring critical results should be communicated. Depending on the institutional resources, some laboratories call only the first critical result for one or more tests if certain criteria are met. Modification of these policies can lead to significant changes in the volume of calls made by the laboratory and can have numerous impacts related to workload, logistics, and patient care.
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Laboratorios , Humanos , Estudios Retrospectivos , Centros de Atención TerciariaAsunto(s)
Diabetes Mellitus Tipo 2 , Insuficiencia Renal Crónica , Biomarcadores , Compuestos Bicíclicos Heterocíclicos con Puentes , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Glucosa , Humanos , Insuficiencia Renal Crónica/tratamiento farmacológico , Sodio , Transportador 2 de Sodio-GlucosaRESUMEN
INTRODUCTION: We observed discordant sodium results from a patient with severe hypernatremia such that whole-blood analysis produced results up to 9.6 mmol/L higher than plasma sodium obtained on the same collection. We investigated this bias by comparing other patients' sodium results and performing comparisons of 3 blood gas and 2 chemistry analyzers. METHODS: First, the laboratory information system was queried for whole-blood sodium results >160 mmol/L, which were used for comparison against plasma results from the same collection. Second, whole blood was collected from a healthy donor, a portion of which was spiked with sodium chloride to generate 8 samples with target concentrations of 140 to 185 mmol/L. Whole-blood sodium was measured in duplicate on the ABL90, RAPIDPoint 500, and GEM 4000. Plasma sodium was then measured in duplicate on the Architect c8000 and Cobas c702. Finally, plasma was injected on the blood gas analyzers to measure sodium in singleton. RESULTS: Overall, 53 paired results from patients showed a significant positive bias on the ABL90 relative to Vitros when sodium was >160 mmol/L. The magnitude of difference was insignificant within the reference range but increased proportionately with concentration. The magnitude and pattern of positive bias in ABL90 sodium results were consistent with the observation in patient results. CONCLUSION: In severe hypernatremia, sodium results produced by blood gas and plasma analyzers can differ significantly.
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Hipernatremia , Análisis de los Gases de la Sangre , Humanos , Hipernatremia/diagnóstico , Valores de Referencia , SodioRESUMEN
Cerebrospinal fluid (CSF) is a circulatory fluid of the central nervous system and it can reflect the biochemical changes occurring in the brain. Although CSF retrieval through lumbar puncture is invasive, it remains the most commonly used fluid in exploring brain pathology as it is less complex and contains a higher concentration of brain-derived proteins than plasma (Reiber, H. Clin. Chim. Acta 2001, 310, 173-186; Macron et al. J. Proteome Res. 2018, 17, 4315-4319). We hypothesize that proteins produced by the brain will have diagnostic significance for brain pathologies. Hence, we expanded the previously in-house-developed 31-protein panel with more proteins classified as brain-specific by the Human Protein Atlas (HPA). Using the HPA, we selected 76 protein coding genes and screened CSF using liquid chromatography-mass spectrometry (LC-MS) and narrowed the protein list to candidates identified endogenously in CSF. Next, we developed a parallel reaction monitoring (PRM) assay for the 21 new proteins and merged it with the 31-protein assay developed earlier. In the process, we evaluated different screening strategies and optimized MS collision energies and ion isolation windows to achieve the highest possible analyte signal resulting in the PRM assay with an average linear dynamic range of 4.3 × 103. We also assessed the extent of Asn (N)-Gln (Q) deamidation, N-terminal pyro-Glu (E) conversion, and Met (M) oxidation and found that deamidation can be misassigned without high mass accuracy and high-resolution settings. We also assessed how many of these proteins could be reliably measured in 10 individual patient CSF samples. Our approach allows us to measure the relative levels of 52 brain-derived proteins in CSF by a single LC-MS method. This new assay may have important applications in discovering CSF biomarkers for various neurological diseases.