Biochemical markers have a central position in the diagnosis and management of patients in clinical medicine, and also in clinical research and drug development, also for brain disorders, such as Alzheimer’s disease. The enzyme-linked immunosorbent assay (ELISA) is frequently used for measurement of low-abundance biomarkers. However, the quality of ELISA methods varies, which may introduce both systematic and random errors. This urges the need for more rigorous control of assay performance, regardless of its use in a research setting, in clinical routine, or drug development. The aim of a method validation is to present objective evidence that a method fulfills the requirements for its intended use. Although much has been published on which parameters to investigate in a method validation, less is available on a detailed level on how to perform the corresponding experiments. To remedy this, standard operating procedures (SOPs) with step-by-step instructions for a number of different validation parameters is included in the present work together with a validation report template, which allow for a well-ordered presentation of the results. Even though the SOPs were developed with the intended use for immunochemical methods and to be used for multicenter evaluations, most of them are generic and can be used for other technologies as well.

A growing body of evidence suggests that the plasma concentration of the neurofilament light chain (NfL) might be considered a plasma biomarker for the screening of neurodegeneration in Alzheimer’s disease (AD). With a single molecule array method (Simoa, Quanterix), plasma NfL concentrations were measured in 99 subjects with AD at the stage of mild cognitive impairment (MCI-AD; n = 25) or at the stage of early dementia (ADD; n = 33), and in nondemented controls (n = 41); in all patients, the clinical diagnoses were in accordance with the results of the four core cerebrospinal fluid (CSF) biomarkers (amyloid β (Aβ)1–42, Aβ42/40, Tau, and pTau181), interpreted according to the Erlangen Score algorithm. The influence of preanalytical storage procedures on the NfL in plasma was tested on samples exposed to six different conditions. NfL concentrations significantly increased in the samples exposed to more than one freezing/thawing cycle, and in those stored for 5 days at room temperature or at 4 °C. Compared with the control group of nondemented subjects (22.0 ± 12.4 pg/mL), the unadjusted plasma NfL concentration was highly significantly higher in the MCI-AD group (38.1 ± 15.9 pg/mL, p < 0.005) and even further elevated in the ADD group (49.1 ± 28.4 pg/mL; p < 0.001). A significant association between NfL and age (ρ = 0.65, p < 0.001) was observed; after correcting for age, the difference in NfL concentrations between AD and controls remained significant (p = 0.044). At the cutoff value of 25.7 pg/mL, unconditional sensitivity, specificity, and accuracy were 0.84, 0.78, and 0.82, respectively. Unadjusted correlation between plasma NfL and Mini Mental State Examination (MMSE) across all patients was moderate but significant (r = −0.49, p < 0.001). We observed an overall significant correlation between plasma NfL and the CSF biomarkers, but this correlation was not observed within the diagnostic groups. This study confirms increased concentrations of plasma NfL in patients with Alzheimer’s disease compared with nondemented controls.

Introduction: Synaptic dysfunction and degeneration are central events in Alzheimer’s disease (AD) pathophysiology that are thought to occur early in disease progression. Synaptic pathology may be studied by examining protein biomarkers specific for different synaptic elements. We recently showed that the dendritic protein neurogranin (Ng), including the endogenous Ng peptide 48 to 76 (Ng_48–76), is markedly increased in cerebrospinal fluid (CSF) in AD and that Ng_48–76 is the dominant peptide in human brain tissue. The aim of this study was to characterize Ng in plasma and CSF using mass spectrometry and to investigate the performance of plasma Ng as an AD biomarker. Methods: Paired plasma and CSF samples from patients with AD (n = 25) and healthy controls (n = 20) were analyzed in parallel using an immunoassay developed in-house on the Meso Scale Discovery platform and hybrid immunoaffinity-mass spectrometry (HI-MS). A second plasma material from patients with AD (n = 13) and healthy controls (n = 17) was also analyzed with HI-MS. High-resolution mass spectrometry was used for identification of endogenous plasma Ng peptides. Results: Ng in human plasma is present as several endogenous peptides. Of the 16 endogenous Ng peptides identified, seven were unique for plasma and not detectable in CSF. However, Ng_48–76 was not present in plasma. CSF Ng was significantly increased in AD compared with controls (P < 0.0001), whereas the plasma Ng levels were similar between the groups in both studies. Plasma and CSF Ng levels showed no correlation. CSF Ng was stable during storage at −20°C for up to 2 days, and no de novo generation of peptides were detected. Conclusions: For the first time, to our knowledge, we have identified several endogenous Ng peptides in human plasma. In agreement with previous studies, we show that CSF Ng is significantly increased in AD as compared with healthy controls. The origin of Ng in plasma and its possible use as a biomarker need to be further investigated. The results suggest that CSF Ng, in particular Ng_48–76, might reflect the neurodegenerative processes within the brain, indicating a role for Ng as a potential novel clinical biomarker for synaptic function in AD.

Introduction: Neuroinflammation and synaptic degeneration are major neuropathological hallmarks in Alzheimer’s disease (AD). Neurogranin and YKL-40 in cerebrospinal fluid (CSF) are newly discovered markers indicating synaptic damage and microglial activation, respectively. Methods: CSF samples from 95 individuals including 39 patients with AD dementia (AD-D), 13 with mild cognitive impairment (MCI) due to AD (MCI-AD), 29 with MCI not due to AD (MCI-o) and 14 patients with non-AD dementias (non-AD-D) were analyzed for neurogranin and YKL-40. Results: Patients with dementia or MCI due to AD showed elevated levels of CSF neurogranin (p < 0.001 for AD-D and p < 0.05 for MCI-AD) and YKL-40 (p < 0.05 for AD-D and p = 0.15 for MCI-AD) compared to mildly cognitively impaired subjects not diagnosed with AD. CSF levels of neurogranin and YKL-40 did not differ between MCI not due to AD and non-AD dementias. In AD subjects no correlation between YKL-40 and neurogranin was found. The CSF neurogranin levels correlated moderately with tau and p-tau but not with Aβ₄₂ or the MMSE in AD samples. No relevant associations between YKL-40 and MMSE or the core AD biomarkers, Aβ₄₂, t-tau and p-tau were found in AD subjects. Conclusions: Neurogranin and YKL-40 are promising AD biomarkers, independent of and complementary to the established core AD biomarkers, reflecting additional pathological changes in the course of AD.

Because of the emerging intersections of HIV infection and Alzheimer's disease, we examined cerebrospinal fluid (CSF) biomarkers related of amyloid and tau metabolism in HIV-infected patients. In this cross-sectional study we measured soluble amyloid precursor proteins alpha and beta (sAPPα and sAPPβ), amyloid beta fragment 1-42 (Aβ_1-42), and total and hyperphosphorylated tau (t-tau and p-tau) in CSF of 86 HIV-infected (HIV+) subjects, including 21 with AIDS dementia complex (ADC), 25 with central nervous system (CNS) opportunistic infections and 40 without neurological symptoms and signs. We also measured these CSF biomarkers in 64 uninfected (HIV-) subjects, including 21 with Alzheimer's disease, and both younger and older controls without neurological disease. CSF sAPPα and sAPPβ concentrations were highly correlated and reduced in patients with ADC and opportunistic infections compared to the other groups. The opportunistic infection group but not the ADC patients had lower CSF Aβ_1-42 in comparison to the other HIV+ subjects. CSF t-tau levels were high in some ADC patients, but did not differ significantly from the HIV+ neuroasymptomatic group, while CSF p-tau was not increased in any of the HIV+ groups. Together, CSF amyloid and tau markers segregated the ADC patients from both HIV+ and HIV- neuroasymptomatics and from Alzheimer's disease patients, but not from those with opportunistic infections. Parallel reductions of CSF sAPPα and sAPPβ in ADC and CNS opportunistic infections suggest an effect of CNS immune activation or inflammation on neuronal amyloid synthesis or processing. Elevation of CSF t-tau in some ADC and CNS infection patients without concomitant increase in p-tau indicates neural injury without preferential accumulation of hyperphosphorylated tau as found in Alzheimer's disease. These biomarker changes define pathogenetic pathways to brain injury in ADC that differ from those of Alzheimer's disease.