Neurofilament light chain: serum reference intervals in Danish children aged 0-17 years.

Elevated levels of neurofilament light chain (NfL) in the blood is an unspecific biomarker for damage to neuronal axons. The measurement of NfL levels in the blood can provide useful information for monitoring and prognostication of various neurological disorders in children, but a reference interval (RI) is needed before the clinical implementation of the biomarker. We aimed to establish a RI for children aged 0-17 years. Serum samples from 292 healthy reference subjects aged 0.4-17.9 years were analysed by a single-molecule array (Simoa®) established for routine clinical use. Non-parametric quantile regression was used to model a continuous RI, and a traditional age-partitioned non-parametric RI was established according to Clinical and Laboratory Standard Institute (CLSI) guideline C28-A3. Furthermore, we investigated the effect of hemolysis on assay performance. The traditional age-partitioned non-parametric RI for the age group <3 years was 3.5-16.6 ng/L and 2.1-13.9 ng/L in the age group ≥3 years, respectively. The continuous RI showed an age-dependent decrease in median NfL levels in the first three years of life which was also evident in the age-partitioning of the traditional RI. We found no difference between sexes and no impact of hemolysis on the NfL test results. This study establishes a pediatric RI for serum NfL and lays the groundwork for its future use in clinical practice.

Serum GFAP - pediatric reference interval in a cohort of Danish children.

OBJECTIVES: Glial fibrillary acidic protein (GFAP) in blood is an emerging biomarker of brain injury and neurological disease. Its clinical use in children is limited by the lack of a reference interval (RI). Thus, the aim of the present study was to establish an age-dependent continuous RI for serum GFAP in children. METHODS: Excess serum from routine allergy testing of 391 children, 0.4-17.9 years of age, was measured by a single-molecule array (Simoa) assay. A continuous RI was modelled using non-parametric quantile regression and presented both graphically and tabulated as discrete one-year RIs based on point estimates from the model. RESULTS: Serum GFAP showed a strong age-dependency with declining levels and variability from infants to adolescents. The estimated median level decreased 66 % from four months to five years of age and another 65 % from five years to 17.9 years of age. No gender difference was observed. CONCLUSIONS: The study establishes an age-dependent RI for serum GFAP in children showing high levels and variability in the first years of life.

Glial fibrillary acidic protein is a robust biomarker in cerebrospinal fluid and peripheral blood after traumatic spinal cord injury: a prospective pilot study.

PURPOSE: Biochemical biomarkers to determine the injury severity and the potential for functional recovery of traumatic spinal cord injury (TSCI) are highly warranted; however, it remains to be clarified whether cerebrospinal fluid (CSF) or peripheral blood (PB) is the ideal sample media. This study aims to measure and compare biomarker concentrations in CSF and PB and to explore associations between biomarker concentrations and injury severity, i.e., American Spinal Injury Association (ASIA) Impairment Scale (AIS) grade, and biomarker concentrations and clinical outcome, i.e., AIS grade improvement and Spinal Cord Independent Measure version III (SCIM-III) score. METHODS: From 2018 to 2020, we conducted a single-center prospective pilot study of TSCI patients (n=15) and healthy controls (n=15). Sample collection and clinical outcome assessment were performed at median 13 h [IQR: 19], 9 days [IQR: 2], and 148 days [IQR: 49] after TSCI. Concentrations of neuron-specific enolase (NSE); glial fibrillary acid protein (GFAP); neurofilament light chain (NfL); interferon-γ (IFN-γ); interleukin (IL)-1ß, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, and IL-13; and tumor necrosis factor α (TNF-α) were measured and associated to clinical outcomes. RESULTS: The biomarker concentrations were higher in CSF than PB. CSF concentrations of GFAP, NSE, IFN-y, TNF-a, IL-2, IL-12p70, IL-4, IL-10, and IL-13 and PB concentrations of GFAP and IFN-y were significantly associated with AIS grade, but not with AIS grade improvement or SCIM-III score. CONCLUSIONS: Our results support GFAP as a potential diagnostic biomarker that may be measured in CSF as well as PB.

Platelet Function in Acute Kidney Injury: A Systematic Review and a Cohort Study.

Acute kidney injury (AKI) patients have increased bleeding risk, which could be partially due to acquired platelet dysfunction. We conducted a systematic review and a cohort study to investigate platelet function and count in AKI and their association with AKI-related bleeding and mortality. Through a systematic literature search in PubMed and Embase, we identified 9 studies reporting platelet function and 56 studies reporting platelet count or platelet indices in AKI patients. Overall, platelet aggregation was reduced in AKI patients in nonintensive care unit (ICU) settings but not in ICU settings, except that reduced aggregation was associated with renal replacement therapy. Thrombocytopenia in AKI was frequent and often predictive of mortality. In our cohort study, we prospectively included 54 adult ICU patients who developed AKI within 24 hours of ICU admission and 33 non-AKI ICU controls. Platelet function was measured with light transmission aggregometry and flow cytometry. AKI patients bled more frequently than non-AKI patients (p = 0.04), and bleeding was associated with increased 30-day mortality in AKI (p = 0.02). However, platelet function was not different between AKI and non-AKI patients (aggregation: all p > 0.52; flow cytometry: all p > 0.07) and platelet function was not associated with bleeding in AKI. In conclusion, a reduced platelet count is frequent in AKI, but the literature on platelet function in AKI is sparse. In a cohort study, we demonstrated that patients with AKI within 24 hours of ICU admission exhibited increased bleeding tendency but this was not associated with reduced platelet function.

Cerebrospinal fluid and peripheral blood proteomics in Traumatic Spinal Cord Injury: A prospective pilot study.

•Proteomics enable profiling of inflammatory responses after spinal cord injury.•Proteins are differentially expressed over time.•Proteins are differentially expressed in cerebrospinal fluid and peripheral blood.•A poor relationship exists between protein expression and neurological outcome.

The inflammatory response and blood-spinal cord barrier integrity in traumatic spinal cord injury: a prospective pilot study.

PURPOSE: Triggering of inflammatory responses and disruption of blood-spinal cord barrier (BSCB) integrity are considered pivotal events in the pathophysiology of traumatic spinal cord injury (TSCI). Yet, these events are poorly understood and described in humans. This study aims to describe inflammatory responses and BSCB integrity in human TSCI. METHODS: Fifteen TSCI patients and fifteen non-TSCI patients were prospectively recruited from Aarhus University Hospital, Denmark. Peripheral blood (PB) and cerebrospinal fluid (CSF) were collected at median day 0 [IQR: 1], median day 9 [IQR: 2], and median day 148 [IQR: 49] after injury. PB and CSF were analyzed for immune cells by flow cytometry, cytokines by multiplex immunoassay, and BSCB integrity by IgG Index. RESULTS: Eleven TSCI patients completed follow-up. Results showed alterations in innate and adaptive immune cell counts over time. TSCI patients had significantly increased cytokine concentrations in CSF at the first and second follow-up, while only concentrations of interleukin (IL)-4, IL-8, and tumor necrosis factor-α remained significantly increased at the third follow-up. In PB, TSCI patients had significantly increased IL-6, IL-8, and IL-10 concentrations and significantly decreased interferon-γ concentrations at the first follow-up. Results further showed increased IgG Index indicative of BSCB disruption in seven TSCI patients at the first follow-up, five TSCI patients at the second follow-up, and two patients at the third follow-up. CONCLUSIONS: Our results suggest that TSCI mainly triggers innate inflammatory responses that resolves over time, although with some degree of non-resolving inflammation, particularly in CSF. Our results cannot confirm BSCB disruption in all TSCI patients.

Serum GFAP - reference interval and preanalytical properties in Danish adults.

OBJECTIVES: Glial fibrillary acidic protein (GFAP) is a promising biomarker that could potentially contribute to diagnosis and prognosis in neurological diseases. The biomarker is approaching clinical use but the reference interval for serum GFAP remains to be established, and knowledge about the effect of preanalytical factors is also limited. METHODS: Serum samples from 371 apparently healthy reference subjects, 21-90 years of age, were measured by a single-molecule array (Simoa) assay. Continuous reference intervals were modelled using non-parametric quantile regression and compared with traditional age-partitioned non-parametric reference intervals established according to the Clinical and Laboratory Standards Institute (CLSI) guideline C28-A3. The following preanalytical conditions were also examined: stability in whole blood at room temperature (RT), stability in serum at RT and -20 °C, repeated freeze-thaw cycles, and haemolysis. RESULTS: The continuous reference interval showed good overall agreement with the traditional age-partitioned reference intervals of 25-136 ng/L, 34-242 ng/L, and 5-438 ng/L for the age groups 20-39, 40-64, and 65-90 years, respectively. Both types of reference intervals showed increasing levels and variability of serum GFAP with age. In the preanalytical tests, the mean changes from baseline were 2.3% (95% CI: -2.4%, 6.9%) in whole blood after 9 h at RT, 3.1% (95% CI: -4.5%, 10.7%) in serum after 7 days at RT, 10.4% (95% CI: -6.0%, 26.8%) in serum after 133 days at -20 °C, and 10.4% (95% CI: 9.5%, 11.4%) after three freeze-thaw cycles. CONCLUSIONS: The study establishes age-dependent reference ranges for serum GFAP in adults and demonstrates overall good stability of the biomarker.

Short-term biological variation of serum glial fibrillary acidic protein.

OBJECTIVES: Serum glial fibrillary acidic protein (GFAP) is an emerging biomarker for intracerebral diseases and is approved for clinical use in traumatic brain injury. GFAP is also being investigated for several other applications, where the GFAP changes are not always outstanding. It is thus essential for the interpretation of GFAP to distinguish clinical relevant changes from natural occurring biological variation. This study aimed at estimating the biological variation of serum GFAP. METHODS: Apparently healthy subjects (n=33) had blood sampled for three consecutive days. On the second day, blood was also drawn every third hour from 9 AM to 9 PM. Serum GFAP was measured by Single Molecule Array (Simoa™). Components of biological variation were estimated in a linear mixed-effects model. RESULTS: The overall median GFAP value was 92.5 pg/mL (range 34.4-260.3 pg/mL). The overall within- (CVI) and between-subject variations (CVG) were 9.7 and 39.5%. The reference change value was 36.9% for an increase. No day-to-day variation was observed, however semidiurnal variation was observed with increasing GFAP values between 9 AM and 12 PM (p<0.00001) and decreasing from 12 to 9 PM (p<0.001). CONCLUSIONS: Serum GFAP exhibits a relatively low CVI but a considerable CVG and a marked semidiurnal variation. This implies caution on the timing of blood sampling and when interpreting GFAP in relation to reference intervals, especially in conditions where only small GFAP differences are observed.

Biological variation of serum neurofilament light chain.

OBJECTIVES: The neurofilament light chain (NfL) has emerged as a versatile biomarker for CNS-diseases and is approaching clinical use. The observed changes in NfL levels are frequently of limited magnitude and in order to make clinical decisions based on NfL measurements, it is essential that biological variation is not confused with clinically relevant changes. The present study was designed to evaluate the biological variation of serum NfL. METHODS: Apparently healthy individuals (n=33) were submitted to blood draws for three days in a row. On the second day, blood draws were performed every third hour for 12 h. NfL was quantified in serum using the Simoa™ HD-1 platform. The within-subject variation (CVI) and between-subject variation (CVG) were calculated using linear mixed-effects models. RESULTS: The overall median value of NfL was 6.3 pg/mL (range 2.1-19.1). The CVI was 3.1% and the CVG was 35.6%. An increase in two serial measurements had to exceed 24.3% to be classified as significant at the 95% confidence level. Serum NfL levels remained stable during the day (p=0.40), whereas a minute variation (6.0-6.6 pg/mL) was observed from day-to-day (p=0.02). CONCLUSIONS: Serum NfL is subject to tight homeostatic regulation with none or neglectable semidiurnal and day-to-day variation, but considerable between-subject variation exists. This emphasizes serum NfL as a well-suited biomarker for disease monitoring, but warrants caution when interpreting NfL levels in relation to reference intervals in a diagnosis setting. Furthermore, NfL's tight regulation requires that the analytical variation is kept at a minimum.

Neurofilament Light Chain as A Biomarker for Brain Metastases.

BACKGROUND: Brain metastases are feared complications in cancer. Treatment by neurosurgical resection and stereotactic radiosurgery are only available when metastatic lesions are limited and early detection is warranted. The neurofilament light chain (NfL) is a sensitive neuron-specific biomarker released following neuronal decay. We explored serum NfL as a biomarker of brain metastases. METHODS: Serum was collected from 43 stage IV lung cancer patients with brain metastases and 25 stage I lung cancer patients. Serum was collected at time of cancer diagnosis and at time of brain metastasis diagnosis. In nine patients with brain metastases, additional samples were available between the two time points. NfL was quantified by Single Molecule Array (Simoa)™. RESULTS: The median NfL level was significantly higher in patients with brain metastases than in patients without (35 versus 16 pg/mL, p = 0.001) and separated patients with an area under the curve of 0.77 (0.66-0.89). An increase in NfL could be measured median 3 months (range: 1-5) before the brain metastasis diagnosis. Further, a high level of NfL at time of brain metastasis diagnosis correlated with an inferior survival (hazard ratio: 2.10 (95% confidence interval: 1.11-3.98)). CONCLUSIONS: This study implies that NfL could be a potential biomarker of brain metastases.

Reference interval and preanalytical properties of serum neurofilament light chain in Scandinavian adults.

The neurofilament light chain (NfL) is a promising biomarker of neuronal injury which is approaching routine clinical use. With the development of ultra-sensitive technologies, NfL has become measurable in the peripheral blood but the reference interval for serum NfL remains to be established. NfL was measured by a single-molecule array (Simoa™) analysis under internal and external quality control which is established for routine clinical use. Serum samples from 342 reference subjects, 18-87 years were analyzed. The age-partitioned reference interval was established according to Clinical and Laboratory Standards Institute guidelines, an approximation of the upper reference interval limit per 10-year age-groups was performed, and key pre-analytical properties were examined. Serum NfL levels increased 2.9% per year. The non-parametric reference interval for the age groups 18-40, 41-65, and >65 years were 2.8-9.7 ng/L, 4.6 - 21.4 ng/L, and 7.5-53.8 ng/L, respectively. The estimated upper reference interval limits per 10-year intervals corresponded well with the 90% confidence limits of the non-parametric reference interval. The recovery of serum NfL after seven days at room temperature or three freeze-thaw cycles were 93% (95% CI: 89%-97%) and 92% (95% CI: 83%-102%) and levels in serum were only slightly higher than levels in plasma (p < .0001). The study establishes the serum NfL reference interval, provide estimated upper reference intevral limits in 10-year intervals to increase the clinical applicability and uncover pre-analytical properties that make serum NfL feasible for clinical use.

Plasma neurofilament light chain is associated with mortality after spontaneous intracerebral hemorrhage.

Background Neurofilament light chain (NfL) is a neuron-specific biomarker with prognostic ability in several types of central nervous system injuries. This study investigates if plasma NfL (pNfL) is elevated early after spontaneous intracerebral hemorrhage (ICH) and whether such elevation reflects disease severity and day-30 outcome. Methods pNfL was quantified by single molecule array analysis in 103 reference subjects (RS) and in samples from 37 patients with ICH obtained on admission to hospital and at 24-h follow-up. The primary outcome was day-30 mortality. Clinical status on admission was evaluated by standardized scoring systems. Results Median pNfL among RS was 9.6 (interquartile range [IQR] 6.2) pg/mL. Upon admission, ICH patients had pNfL of 19.8 (IQR 30.7) pg/mL increasing to 35.9 (IQR 44.5) pg/mL at 24 h (all, p < 0.001). On admission, pNfL was higher among ICH non-survivors than survivors (119.2 [IQR 152.6] pg/mL vs. 15.7 [IQR 19.6] pg/mL, p < 0.01) and this difference was observed also on 24 h follow-up (195.1 [IQR 73.9] pg/mL vs. 31.3 [IQR 27.8] pg/mL, p < 0.01). The area under the receiver operating characteristic curve (ROC AUC) for discrimination of day-30 mortality was significant on admission (AUC = 0.83, 95% confidence interval [CI]: 0.56-1.0) and increased on 24-h follow-up (AUC = 0.93, 95% CI: 0.84-1.0). The odds ratio (OR) for death, by each quartile increase in pNfL was significant both on admission (OR = 4.52, 95% CI: 1.32-15.48) and after 24-h follow-up (OR = 9.52, 95% CI: 1.26-71.74). Conclusions PNfL is associated with day-30 mortality after spontaneous ICH when early after the ictus.

Plasma Neurofilament Light Chain Is Associated with Poor Functional Outcome and Mortality Rate After Spontaneous Subarachnoid Hemorrhage.

The initial clinical status after subarachnoid hemorrhage (SAH) is an important outcome predictor, but the mechanisms behind the early brain injury (EBI) remains incompletely understood. Elevated neurofilament levels in the cerebrospinal fluid at protracted stages after SAH are associated with poor outcome, but the potential association between plasma neurofilament (pNfL) levels during EBI, disease severity on admission, and poor outcome remains unaddressed. Plasma NfL (pNfL) was measured by single molecule array in 44 SAH patients on admission and 24 h after ictus, as well as in 44 controls. Disease severity on admission was assessed by validated scoring systems, and day 30 modified Rankin Scale (mRS) score was registered. Admission levels of pNfL correlated with clinical disease severity scores (rho = 0.43, p < 0.01 and rho = 0.48, p < 0.001) as well as day 30 mRS score (rho = 0.53, p < 0.001). Each quartile increase in pNfL was independently associated with poor functional status (mRS > 4) [odds ratio = 1.98, 95% confidence interval (CI): 1.01-3.88, p = 0.05]. Non-survivors had higher pNfL than survivors; on admission [17.6 pg/mL (IQR 11.4) vs. 8.4 pg/mL (IQR: 8.9), p < 0.01] and 24 h after ictus [29.9 pg/mL (IQR 90.4) vs 7.8 pg/mL (IQR 26.9), p = 0.01]. Each quartile increase in pNfL was independently associated with reduced survival rate [log-rank = 0.02, hazard ratio = 2.29 (95% CI): 1.15-4.57), p = 0.02]. PNfL levels are associated with disease severity during the EBI phase of SAH. Higher pNfL levels during EBI are associated with poor functional outcome on day 30 after ictus and increased mortality rate.