Lumbar puncture with subsequent measurement of CSF core biomarkers (Aβ42, T-tau, and P-tau) was performed for all patients. Core biomarkers were analyzed with Innotest Kit enzyme-linked immunosorbent assays (Innogenetics, Ghent, Belgium) at the Department for Interdisciplinary Laboratory Medicine and Medical Biochemistry at Akershus University Hospital, Norway, which participates in the Alzheimer’s Association QC program for CSF biomarkers (Mattsson et al., 2011 (link)). Laboratory recommended cut-off values for a normal test were applied (Kalheim et al., 2018 (link)): Aβ42 above 700 pg/ml; P-tau below 80 pg/ml; and T-tau below 300 pg/ml for patients younger than 50 years, below 450 pg/ml for patients aged 50–69 years, and below 500 pg/ml for patients older than 70 years.
Innotest kit
Manufactured by Fujirebio
Sourced in Belgium
The Innotest kit is a laboratory diagnostic product manufactured by Fujirebio. It is designed to perform specialized tests for clinical and research applications. The core function of the Innotest kit is to provide a reliable and accurate testing solution, though its intended use and specific applications are not detailed here.
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11 protocols using innotest kit
1
CSF Biomarker Analysis for Alzheimer's Diagnosis
2
Alzheimer's Biomarkers in Spinal Fluid
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The two hospitals performed lumbar punctures and collection of spinal fluid according to an identical procedure. The lumbar punctures were carried out between 9 a.m. and 11 a.m. Spinal fluid was collected in cryotubes and, within thirty minutes of collection, centrifuged for ten minutes at 2000 G. All the samples were analyzed at the same laboratory at Akershus University Hospital (AHUS). Samples were either sent to the laboratory on the same day or frozen at −20 degrees Celsius and later sent in a frozen state. All samples were analyzed with the Innotest kit (Innogenetics, Ghent, Belgium) for all three biomarkers. The laboratory follows its own internal quality control program by using control samples in every batch of samples analyzed. It is also part of the Alzheimer’s Association QC program for CSF biomarkers [28 ]. The Clinical Neurochemistry Laboratory in Gothenburg, Sweden, is in charge of the program in collaboration with the Alzheimer’s Association. The laboratory at AHUS receives samples from this program for analysis three times a year. The cutoffs for the three biomarkers recommended from the laboratory were applied: amyloid-β (Aβ42): > 550, phospho tau (P-tau): < 80, and total tau (T-tau): < 300 for patients under 50 years, < 450 for patients ages 50 to 69, and <500 for those 70 years and older. Age-specific cut offs exist for total tau only.
3
APOE Genotyping and CSF Biomarkers in Alzheimer's Evaluation
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APOE genotyping was conducted using the Illumina Infinium OmniExpress v1.1 chip at deCODE Genetics, Reykjavik, Iceland, and the results were dichotomized based on APOE-ɛ4 status (carrier of at least one APOE-ɛ4 allele, or not).
Lumbar puncture with measures of AD biomarkers (amyloid-β [Aβ], phosphorylated tau [P-tau] and total tau [T-tau]) in the cerebrospinal fluid (CSF) was carried out in 60 of the 123 patients. The CSF examination was done in patients where more information was warranted to increase the etiological diagnostic precision, mostly in younger patients. All CSF samples were analyzed at Akershus University Hospital (AHUS) using ELISA technique with the Innotest kit (Innogenetics, Ghent, Belgium). As the analyses were carried out as part of the clinical routine, the samples were analyzed on different dates and with different batches. The laboratory is part of the Alzheimer’s Association quality-control program for CSF biomarkers through a collaboration with the Clinical Neurochemistry Laboratory in Gothenburg, Sweden [36 (link)]. Cut-offs developed at AHUS were used when dichotomizing the results into a pathological/nonpathological variable (normal references: Aβ 550–1200 ng/L, P-tau <80 ng/L, T-tau in patients with ages <50, <300ng/L; in patients 50–70 years, <450 ng/L; in patients with ages >70, <500 ng/L).
Lumbar puncture with measures of AD biomarkers (amyloid-β [Aβ], phosphorylated tau [P-tau] and total tau [T-tau]) in the cerebrospinal fluid (CSF) was carried out in 60 of the 123 patients. The CSF examination was done in patients where more information was warranted to increase the etiological diagnostic precision, mostly in younger patients. All CSF samples were analyzed at Akershus University Hospital (AHUS) using ELISA technique with the Innotest kit (Innogenetics, Ghent, Belgium). As the analyses were carried out as part of the clinical routine, the samples were analyzed on different dates and with different batches. The laboratory is part of the Alzheimer’s Association quality-control program for CSF biomarkers through a collaboration with the Clinical Neurochemistry Laboratory in Gothenburg, Sweden [36 (link)]. Cut-offs developed at AHUS were used when dichotomizing the results into a pathological/nonpathological variable (normal references: Aβ 550–1200 ng/L, P-tau <80 ng/L, T-tau in patients with ages <50, <300ng/L; in patients 50–70 years, <450 ng/L; in patients with ages >70, <500 ng/L).
4
CSF Biomarkers Measurement in Dementia
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A lumbar puncture with measurement of the CSF core biomarkers Aβ42, T-tau and P-tau using the ELISA technique with the Innotest kit (Innogenetics, Ghent, Belgium) was performed for all patients. The analysis was done at the laboratory at Akershus University Hospital, Norway. The laboratory is a part of the Alzheimer´s Association QC program for CSF biomarkers [22 ]. The recommended cut-off value for a normal test was until June 2017 >550 pg/mL for Aβ42. As of October 2018, when these data were collected, the recommended cut-off values for a normal test from the laboratory were Aβ42 >700 pg/mL [23 (link)], P-tau <80 pg/mL and T-tau <300 pg/mL for patients below 50 years, < 450 pg/mL for patients aged 50 to 69 years, and <500 pg/mL for patients older than 70 years. These cut-off values were used to support the clinical diagnoses that were made (see section below).
5
Cerebrospinal Fluid Biomarker Analysis
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Lumbar puncture with the examination of Aβ, t- and p-tau was conducted according to the Norwegian national guidelines (Braekhus et al., 2011 (link); Knapskog et al., 2017 (link)).
The lumbar punctures were carried out between 9 a.m. and 11 a.m. Spinal fluid was collected in cryotubes and was centrifuged for 10 min at 2,000 g within 30 min of collection. Samples were either sent to the laboratory on the same day or frozen at −20 degrees Celsius and later sent in a frozen state. They were analyzed using the Innotest kit (Innogenetics, Ghent, Belgium) for the three biomarkers (SeeTable 1 for absolute values and number of patients with values outside the reference values of the laboratory: Aβ42, t-tau, and p-tau). We also analyzed p-tau, but this value was not used in the regression due to its high correlation with t-tau. For more details regarding the biomarkers in CSF, see Knapskog et al. (2017 (link)).
The lumbar punctures were carried out between 9 a.m. and 11 a.m. Spinal fluid was collected in cryotubes and was centrifuged for 10 min at 2,000 g within 30 min of collection. Samples were either sent to the laboratory on the same day or frozen at −20 degrees Celsius and later sent in a frozen state. They were analyzed using the Innotest kit (Innogenetics, Ghent, Belgium) for the three biomarkers (See
6
Apolipoprotein E Genotyping and CSF Biomarkers
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Apolipoprotein E (APOE) genotyping was done in 143 patients by deCODE Genetics, Reykjavik, Iceland using the Illumina Infinium OmniExpress v1.1 chip. The result was dichotomized based on whether the patient carried at least one APOE-ε4 allele, or not.
Cerebrospinal fluid (CSF) biomarkers of amyloid-β-42 (Aβ), total tau (t-tau) and phosphorylated tau 181 (p-tau) were analyzed at Akershus University Hospital (AHUS) using ELISA technique with the Innotest kit (Innogenetics, Ghent, Belgium). Due to changes in analyzing methods, cut-offs have been changed during the inclusion period of this study and results were therefor dichotomized as "pathological" and "non-pathological" according to the applicable cut-offs. 18 F-Flutemetamol PET (amyloid PET) was carried out at the Department of Nuclear Medicine at OUH. CSF results and amyloid PET results were included in the study only in patients who had a pathological result any time prior to, or maximum 12 months after the MRI examination, or who had a non-pathological result maximum 12 months prior to or any time after the MRI examination. Thus, the Aβ status was available in 138 patients (109 CSF, 29 PET) and the p-tau and t-tau statuses were available in 107 patients.
Cerebrospinal fluid (CSF) biomarkers of amyloid-β-42 (Aβ), total tau (t-tau) and phosphorylated tau 181 (p-tau) were analyzed at Akershus University Hospital (AHUS) using ELISA technique with the Innotest kit (Innogenetics, Ghent, Belgium). Due to changes in analyzing methods, cut-offs have been changed during the inclusion period of this study and results were therefor dichotomized as "pathological" and "non-pathological" according to the applicable cut-offs. 18 F-Flutemetamol PET (amyloid PET) was carried out at the Department of Nuclear Medicine at OUH. CSF results and amyloid PET results were included in the study only in patients who had a pathological result any time prior to, or maximum 12 months after the MRI examination, or who had a non-pathological result maximum 12 months prior to or any time after the MRI examination. Thus, the Aβ status was available in 138 patients (109 CSF, 29 PET) and the p-tau and t-tau statuses were available in 107 patients.
7
Cerebrospinal Fluid Biomarker Sampling
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Cerebrospinal fluid collection samples were obtained immediately prior to saliva sampling by lumbar puncture using an atraumatic needle, after overnight fasting, and collected in polypropylene tubes. All CSF samples were collected between 9 and 12 a.m. CSF samples were centrifugated at 2500× g for 10 min at 4 °C and frozen at −80 °C until analysis. CSF concentrations of Aβ42, t-tau, and tau phosphorylated at 181 were evaluated by a commercial ELISA method with the Innotest Kit for diagnostic use (Fujirebio, catalog number 81574 for p-tau, catalog number 81572 for tau, and catalog number 81576 for Aβ42).
8
Alzheimer's Biomarker Quantification
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CSF Aβ42, Aβ40 and ttau were analysed using Euroimmun kits (EUROIMMUN AG, Lübeck, Germany) and ptau using INNOTEST kit (Fujirebio, Gent, Belgium). All measurements were performed by board‐certified laboratory technicians who were blinded to clinical data.
9
Biomarker Profiling for Alzheimer's Disease
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CSF was obtained through a lumbar puncture; the second and third milliliters were collected and centrifuged to prevent blood contamination. The supernatant was stored at − 80 °C until further analysis.
CSF core AD biomarkers (Aβ 42, Aβ 40, p-tau, and t-tau) were analyzed at the Department of Biochemistry at Lariboisiere University Hospital Paris, France, using commercially available INNOTEST® kits (Fujirebio Europe NV, Gent, Belgium) in a delay of 1 month after collection. CSF profiles were analyzed according to the following cut-offs: A+: Aβ42/Aβ40 ratio < 0.076; T+: p-tau > 58 pg/mL; N+: t-tau > 340 pg/mL [26 (link)]. Patients were classified as Aβ-positive and Aβ-negative according to the Aβ42/Aβ40 ratio.
CSF NRG1 concentration was measured using the Human NRG1 DuoSet ELISA kit (R&D Systems, Minneapolis, MN) as reported in Mouton-Liger et al. [24 (link)].
All the CSF synaptic markers were assessed at the Clinical Neurochemistry Laboratory at the Sahlgrenska University Hospital (Mölndal, Sweden). CSF neurogranin and CSF GAP-43 concentrations were measured using in-house developed ELISAs [8 (link), 10 (link)]. CSF SNAP-25 concentration was measured by immunoprecipitation mass spectrometry according to a validated method [9 (link), 11 (link)].
CSF core AD biomarkers (Aβ 42, Aβ 40, p-tau, and t-tau) were analyzed at the Department of Biochemistry at Lariboisiere University Hospital Paris, France, using commercially available INNOTEST® kits (Fujirebio Europe NV, Gent, Belgium) in a delay of 1 month after collection. CSF profiles were analyzed according to the following cut-offs: A+: Aβ42/Aβ40 ratio < 0.076; T+: p-tau > 58 pg/mL; N+: t-tau > 340 pg/mL [26 (link)]. Patients were classified as Aβ-positive and Aβ-negative according to the Aβ42/Aβ40 ratio.
CSF NRG1 concentration was measured using the Human NRG1 DuoSet ELISA kit (R&D Systems, Minneapolis, MN) as reported in Mouton-Liger et al. [24 (link)].
All the CSF synaptic markers were assessed at the Clinical Neurochemistry Laboratory at the Sahlgrenska University Hospital (Mölndal, Sweden). CSF neurogranin and CSF GAP-43 concentrations were measured using in-house developed ELISAs [8 (link), 10 (link)]. CSF SNAP-25 concentration was measured by immunoprecipitation mass spectrometry according to a validated method [9 (link), 11 (link)].
10
Comprehensive CSF Biomarker Analysis for AD
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CSF was obtained through a lumbar puncture; the second and third milliliterswere collected and centrifuged to prevent blood contamination. The supernatant was stored at - 80 °C until further analysis.
CSF core AD biomarkers (Aβ 42, Aβ 40, p-tau and t-tau) were analyzed at the Department of Biochemistry at Lariboisiere University Hospital Paris, France, using commercially available INNOTEST ® kits (Fujirebio Europe NV, Gent, Belgium). CSF pro les were analyzed according to the following cut-offs: A+: Aβ42/Aβ40 ratio <0.076; T+: p-tau > 58 pg/mL;N+:t-tau >340 pg/mL(26). Patients were classi ed as Aβpositive and Aβ-negative according to the Aβ42/Aβ40 ratio. CSF NRG1 concentrationwasmeasured using the Human NRG1 DuoSet ELISA kit (R&D Systems, Minneapolis, MN) as reported in Mouton-Liger et al (24) .
All the CSF synaptic markerswere assessedat the Clinical Neurochemistry Laboratory at the Sahlgrenska University Hospital (Mölndal, Sweden). CSF neurograninand CSF GAP-43 concentrations were measured using in-house developed ELISAs(8,10).CSF SNAP-25concentration was measured by immunoprecipitation mass spectrometry according to a validated method (9, 11) .
CSF core AD biomarkers (Aβ 42, Aβ 40, p-tau and t-tau) were analyzed at the Department of Biochemistry at Lariboisiere University Hospital Paris, France, using commercially available INNOTEST ® kits (Fujirebio Europe NV, Gent, Belgium). CSF pro les were analyzed according to the following cut-offs: A+: Aβ42/Aβ40 ratio <0.076; T+: p-tau > 58 pg/mL;N+:t-tau >340 pg/mL(26). Patients were classi ed as Aβpositive and Aβ-negative according to the Aβ42/Aβ40 ratio. CSF NRG1 concentrationwasmeasured using the Human NRG1 DuoSet ELISA kit (R&D Systems, Minneapolis, MN) as reported in Mouton-Liger et al (24) .
All the CSF synaptic markerswere assessedat the Clinical Neurochemistry Laboratory at the Sahlgrenska University Hospital (Mölndal, Sweden). CSF neurograninand CSF GAP-43 concentrations were measured using in-house developed ELISAs(8,10).CSF SNAP-25concentration was measured by immunoprecipitation mass spectrometry according to a validated method (9, 11) .
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