Biodiversity is one of the key principles of sustainability for food and agriculture. The Veneto region, in Northern Italy, is one of the regions with the highest number of local chicken breeds. Padovana and Polverara are tufted, white eggshell breeds, and different plumage varieties exist. Pepoi, Ermellinata di Rovigo, Robusta Maculata, and Robusta Lionata are dual-purpose and tinted eggshell breeds. The laying rate changed according to the breed. The eggs produced by these breeds differed in weight, eggshell traits , yolk-to-albumen ratio, Haugh units, and meat and blood spots. The consumer can choose a diversified egg quality according to their cooking purposes.p < 0.05) among breeds were observed for yield performance and egg quality. From 39 to 50 weeks of age, the hen-day egg production was higher in PA-C and RM than in RL, and PO-W and ER were intermediate; PA-G, PO-B, and PP were the lowest. The hen-day egg production changed according to the age of the hens. From 39 to 42 weeks of age, ER showed the highest hen-day egg production and PA-G the lowest; from 47 to 50 weeks, PA-C, PO-W, and RM were the highest and PP the lowest. The tinted eggshell breeds, with the exception of PP, had higher egg weights than white eggshell breeds. PP egg weight was similar to PO-B. As regards the tinted eggshell breeds, RM eggs had the highest eggshell a* and b*, and PP the lowest. PA-C had the most spherical eggs, and PO-B and ER had the most ovoid eggs. PO-W and RM had the highest eggshell thickness and ER had the lowest. The highest eggshell ratio was observed for PO-W and PO-B, and the lowest for ER. The yolk-to-albumen ratio was higher in the white eggshell breeds than in PP, ER, and RL. ER had the highest Haugh units and PA-G the lowest. PO-W, PO-B, PA-C, PA-G, and ER had the lowest egg inclusions, and RL and RM the highest. Tinted eggshell eggs differed from white eggshell eggs by having higher meat spots. Results indicated that the eggs produced by the eight local chicken breeds differed according to the laying rate and a wide range of physical external and internal characteristics which allow the consumer to distinguish them for their genetic origin by the eggshell shape and colour, and to use them for different purposes to valorise poultry biodiversity.The aim of this study was to compare yield performance (from 39 to 50 weeks of age) and egg physical characteristics (at 50 weeks of age) of eight autochthonous chicken breeds of the Veneto region . Four white eggshell breeds, namely Padovana Camosciata , Padovana Dorata , Polverara Bianca , and Polverara Nera , and four tinted eggshell breeds, namely Pepoi (PP), Ermellinata di Rovigo (ER), Robusta Maculata (RM), and Robusta Lionata (RL) from a conservation centre were considered in the trial. Significant differences ( Egg production for human consumption has deeply changed throughout the last century, from small retail points until 1950 to big markets, from eggs laid by hens belonging to local purebreds to eggs laid by hybrid hens . HybridsThe Veneto region, in Northern Italy, has a tradition for poultry production, given its position in the Po Valley, and it has historically produced a high quantity of maize and cereals, which are the main ingredients of the chicken diet. At the beginning of the last century, several breeds were reared in the farms of the region, but they were gradually lost to the increasingly intensive production systems based on hybrids . FortunaThe aim of this study is to compare the egg production and egg physical characteristics of eight local chicken breeds of the Veneto region.The hens included in the trial belonged to breeds of the Veneto region, Northern Italy. Padovana (PA) and Polverara (PO) breeds produce eggs with white eggshells, and two plumage varieties for each breed were studied: Padovana Camosciata , Padovana Dorata , Polverara Bianca , and Polverara Nera . Pepoi (PP), Ermellinata di Rovigo (ER), Robusta Maculata (RM) and Robusta Lionata (RL) lay eggs with tinted eggshells. The origins of these breeds and the main features of the hens are summarized in 2) and outdoor (5 m2/hen) spaces, divided by netting. In the outdoor space of each breed, the grass growth was not high, due to the previous absence of rain throughout the summer months and the prolonged walking activity of the birds throughout the next months, which did not allow for high regrowth. In the indoor spaces, the floor was covered by wood shavings. On the floor, wooden nests and perches were available to the hens. The birds were subjected to the same prophylaxis procedures, rearing conditions , and feeding from the time of hatching through the laying period. The animals were fed ad libitum different feeds: one throughout the growing period until the pubertal age, and one throughout the laying period (39 to 50 weeks of age) based on maize and soybean . The pubertal phase and the beginning of laying activity were at the end of summer through the beginning of autumn for PP, ER, RM, and RL, when the photoperiod and environmental temperature, at the latitude (45°02′53′’ N) of the Veneto region, were decreasing. After about 2.5 months of laying activity, their egg production stopped. From December to the end of the trial (March), the natural photoperiod was complimented by artificial light inside the rooms, where the hens spent the night, to obtain a photoperiod of 14L:10D. The PA and PO breeds started laying after the first weeks of artificially complimented photoperiod. The trial started in January (at 39 weeks of age) when the hens of all the breeds were in laying activity and lasted in March (at 50 weeks of age). The environmental temperature varied from 5 °C to 12 °C.Sixty birds per breed were reared under a free-range farming system from 8 weeks of age when the outdoor temperatures and relative humidity allowed the birds conditions promoting their wellbeing. The chicks hatched during the same period, in spring; during the first 4 weeks of life, the birds were kept indoors on litter, under infrared radiation lamps, at an environmental temperature decreasing from 32 to 24 °C. After 8 weeks in indoor conditions, the birds had free access to outdoor spaces. Each breed had access to indoor (4 hens/mThe eggs were collected according to European Regulations (EC No. 1/2005 and EC No. 1099/2009) on animal care and welfare. The sampling did not affect the welfare of the hens as it was carried out when the animals were not in the nests, thus avoiding their handling. The hen-day egg production (number of eggs/number of live hens × 100) was checked daily from January, when the hens were 39 weeks old, until March. At 49–50 weeks of age, throughout two weeks, the mean daily weight of the eggs  were checked for each breed. The hen-day egg mass was calculated as hen-day egg production (%) × daily egg weight (g).2) and volume (cm3) were calculated as [At 50 weeks of age, when all the breeds had been laying since about 12 weeks, on each daily collection of about 15 eggs, carried out throughout 4 days, the eggshell and internal traits were analysed, excluding the defective eggs . Given the hen-day egg production of the studied breeds and the number of hens per breed, the daily egg collection allowed a representative number of eggs for each breed for further analyses. All the measurements were carried out on a one-day old egg. On each egg, external and internal traits were measured to obtain an adequate number of observations per breed: external eggshell traits and weight of every single egg (60 observations: 15 eggs/day × 4 days), eggshell thickness (35 observations: 8–9 eggs/day × 4 days), egg components (25 observations: 6–7 eggs/day × 4 days), Haugh units (22 observations: 7–8 eggs/day × 3 days), yolk colour (9 observations: 4–5 eggs/day × 2 days), meat and blood spots (60 observations: 15 eggs/day × 4 days). Each egg was weighed, and the eggshell length  and width  were measured by callipers (0.01 mm). The eggshell colour was measured by a colorimeter Chroma meter CR 300  using the CIE scale [lated as :Surface The eggshell thickness was measured by means of a digital calliper (0.001 mm)  at the equatorial level of each egg. The yolk was manually separated from the albumen three times and weighed, and the albumen weight was calculated as the difference between the egg weight and the weight of the yolk and eggshell. Before weighing the eggshell, it was cleaned from residues of albumen and then dried for 12 h at 45 °C. The eggshell ratio (%), albumen ratio (%), and yolk ratio (%) were calculated as the ratio between each egg component and egg weight × 100. To evaluate the physical internal quality of albumen and yolk, each egg was broken, and the yolk and albumen were put on a glass plate to detect the inclusions (blood and meat spots) by means of a mirror placed under the glass. For measuring the albumen height, a micrometer (0.01 mm)  was used. The yolk colour was measured by the DSM yolk fan . Egg weight and albumen height were used to calculate the Haugh Units (HU) as measured by Haugh (1937) .p < 0.05.The effect of breed on the hen-day egg production was evaluated from 39 to 50 weeks of age and at three ages  using a factorial model 8 × 3. The data on the hen-day egg mass (49–50 weeks) and egg physical traits (50 weeks) were analysed by one-way ANOVA, considering the breed effect. The GLM procedure of SAS  was used for this purpose. Tukey’s post-hoc test was performed for testing statistical differences between the groups. For testing statistical differences for blood and meat spots, the chi-square test was used. Given that in the PA-C and PA-G, and the PO-W and PO-B, the meat and blood inclusions were very low, PA-C and PA-G, and PO-W and PO-B were considered as two breeds  when testing differences for meat and blood inclusions. Significance was set at p < 0.05). For the white eggshell hens, PA-C was higher than PA-G and PO-W was higher than PO-B (p < 0.05). For the tinted eggshell breeds, the hen-day egg production was higher in RM than in RL (p < 0.05), ER was intermediate, and PP was the lowest (p < 0.05). PA-C and RM showed the highest hen-day egg production, and PA-G, PO-B, and PP had lower egg production than the other breeds (p < 0.05).The hen-day egg production , from 39p < 0.05), and the other breeds were intermediate. PP and PA-G were the lowest (p < 0.05). From 43 to 46 weeks of age, the laying rate increased for all the breeds; extreme values were observed for RM and PA-G, as they showed the highest and the lowest laying rates, respectively (p < 0.05). PA-C, PO-W, and RL reached higher values than those of PA-G and PO-B (p < 0.05), whereas ER and PP were intermediate. Throughout the third period (47 to 50 weeks of age), the hens had divergent trends, according to the breeds. Most of them  increased the laying rate, even if with a tendential trend for some of them, whereas PP and RL decreased it. The hen-day egg productions of PA-C, RM, and PO-W were higher than those of PO-B and RL (p < 0.05), whereas PA-G and ER were intermediate; PP showed the lowest laying rate (p < 0.05).p < 0.05). For the white-eggshell breeds, PO-B was the lowest, and for the tinted eggshell breeds, the lowest were RL and PP (p < 0.05).The hen-day egg mass , which rp < 0.05). The PA-G egg weight was higher than that of PA-C (p < 0.05), and PO-B and PO-W egg weights were the lowest (p < 0.05). PP, followed by PO-B and PO-W, showed the lowest egg weight. As the frequency of the size class is concerned, the ER, RM, and RL hens laid eggs mainly of medium size (70%), followed by large size (23%) and small size (6%). Only RL laid very large size eggs, but at a low level (1.7%). The PP eggs were of small (88%) and medium size (12%).PA-C showed similar (50%) percentages of small (<53 g) and medium eggs (53–63 g), whereas for the PA-G breed the medium size was prevailing (85%), and a lower percentage was shown for the small size (15%). PO breeds gave a high percentage of small-size eggs (88% in PO-W and 80% in PO-B) and a lower percentage of medium-size eggs (10% in PO-W and 20% in PO-B). Only PO-W laid large-size eggs, but at a very low level (1.7%).p < 0.05); PP, PO-W, and PO-B were the lowest (p < 0.05). The egg width was higher in PA than in PO eggs (p < 0.05). The eggs of ER and RL showed the highest length (p < 0.05), followed by PA-G (p < 0.05), and those of RM were intermediate. PA-C and PO-B were lower than PA-G (p < 0.05), and PP was the lowest (p < 0.05). The shape index showed marked differences only between a few breeds: PA-C showed the highest (p < 0.05) shape index and PO-B and ER the lowest (p < 0.05). The other breeds were intermediate, being similar to PA-C , and PO-B and ER (RL and PA-G).p < 0.05), and ER, RM, and RL were the lowest (p < 0.05).The area-to-volume ratio was higher in PP, PO-W, and PO-B than in PA-C and PA-G (p < 0.05), whereas PO-W was intermediate. All the tinted eggshell breeds showed a lightness lower than that of the white eggshell breeds (p < 0.05). They showed different values, as PP was higher than RL, which was higher than ER, and RM was the lowest (p < 0.05). The a* index (redness) and the b* index (yellowness) differed among the tinted eggshell breeds, being higher in RM, followed by ER, RL, and PP (p < 0.05). The white eggshell breeds did not differ for a* and b* , which were lower than those of the tinted eggshell breeds (p < 0.05). The eggshell thickness was similar in RM and PO-W, which were higher than PA-C, and ER was the lowest (p < 0.05). The eggshell weight was higher in RM than in PA-C, ER, and PP (p < 0.05). The eggshell percentage was higher in PO-W and PO-B than in PA-C, PA-G, and RL (p < 0.05); ER was the lowest (p < 0.05).The lightness (L) differed among the white eggshell breeds, being higher in PA-G than in PA-C and PO-B (p < 0.05). PA-G was higher than PO-B (p < 0.05), which was the lowest (p < 0.05), and the other breeds were intermediate.p < 0.05). The lowest was PO-B (p < 0.05). ER was intermediate between RL and PP, and PA-G was intermediate between PA-C and PO. ER had the highest Haugh units, followed by PA-C, PO-W, and PO-B, and the lowest values were for PA-G (p < 0.05). PP was similar to ER, and RM and RL were more similar to the white eggshell breeds.The highest albumen ratio was observed in RL, followed by PP, then RM, PA-C, and PO-W (p < 0.05). The PA-G showed the highest yolk ratio, whereas the lowest was in the tinted eggshell breeds (p < 0.05), with the exception of RM, which was more similar to the white eggshell breeds. PO-B was intermediate between PA-G and PA-C. Similar results were observed for the yolk-to-albumen ratio. It was higher in PO-B than in PA-C and PO-W (p < 0.05). PP, ER, and RL showed the lowest yolk-to-albumen ratio (p < 0.05), and RM was similar to PA-C and PO-W.Yolk weight showed the highest values in RM and PA-G, and the lowest in PO-W, followed by PP (p < 0.05) from RL and RM, which were similar. PP was intermediate between the first three breeds (p < 0.05) and RL and RM (p < 0.05). Total inclusions (p < 0.05) between white and tinted eggshell breeds for a higher (p < 0.05) presence of meat spots in the tinted eggshell breeds, whereas blood spots were similar.clusions b differep < 0.05), whereas the other breeds were intermediate. The white eggshell breeds were more differentiated than the tinted eggshell breeds.In Gallus [This trial compared local breeds characterized by different genetic background, despite not well known in some cases. The white eggshell breeds seem to differ from the tinted eggshell breeds due to a different genetic origin based on different species of the wild Gallus ,29. The Gallus ,4,5. In Gallus . FurtherGallus . The onsGallus . AdequatGallus . FurtherGallus . The whiGallus . It is hGallus . As far Gallus , the oriGallus ,13, withGallus . For theGallus . DiffereEgg size is one of the traits which producers, transformers, and consumers are interested in. The PA, PO, and PP hens produced eggs of small and medium sizes. PO and PP produced mainly (80%) small-size eggs, whereas PA produced medium-size eggs, with differences between the plumages being chamois and golden, this last plumage with a higher value (85% vs. 50%). The ER, RM, and RL hens produced eggs classified into the first three size classes, with about 70% medium-size eggs, followed by large-size eggs (21%) and small-size eggs, especially ER (12%). RL hens gave eggs classified as very large size, even if at a low percentage (1.7%). The results indicate that more than 50% of the egg production of PA-C, PO, and PP are small-size eggs, generally not sold in the retail market, and only PA-G gave more than 80% medium-size eggs.Currently, egg weight, the rearing system, and special diet are indicated on the carton of table eggs, which the consumer considers in the retail market . GeneralThe egg weight mainly varies according to the genotype (body weight) and the age of the hen, both in hybrids and in purebreds ,5,34,35.Egg size, especially for the purebreds, may be considered, also, with other quality traits which could describe an egg quality profile for each breed and address it to different purposes and uses in cooking preparation.The eggshell traits are important both for the producer and the consumer, as they may affect many egg properties, and, when they are peculiar for the quality profile of each breed, may identify the breed of the egg-laying hen, also.The eggshell colour is a trait which may largely influence the first choice of the consumer , with diThe pigments found in the shells of domestic hens are protoporphyrin IX, biliverdin, coproporphyrin, and uroporphyrin, and their production depends on many factors . For theFor table eggs, many are the eggshell traits involved with the resistance of the egg and its internal quality during storage ,44. The CALB1 gene, which is associated with calcium transport in the intestine and uterus, and that of the MP4 gene, which codes for a protein used to bind calcium to the shell [The area-to-volume ratio, which is higher in an ovoid egg, is important during egg storage . An adeqhe shell . Besideshe shell . In thishe shell ,44. The he shell  and a cohe shell . Furtherhe shell . Given the shell .The eggshell ratio, as a consequence of its dimensions and mineral content, differed between the studied breeds. Any comparison with the results of previous trials is difficult as the changes in the eggshell weight and ratio are affected by the breed, age of the hens and the different environmental conditions studied ,44,50,51The eggs produced by these local breeds showed differences also in internal quality. Most of the internal egg quality traits showed differences according to eggshell colour, which seems to be the main factor identifying their genetic origin. The quantity of yolk and albumen in each egg depends not only on the egg weight but on the genotype of the hen, as the breeds showed a different ratio between yolk and albumen. In this trial, the white eggshell breeds showed a yolk-to-albumen ratio higher than 0.50, varying from 0.56 to 0.61. The tinted eggshell breeds PP, ER, and RL showed a yolk-to-albumen ratio of about 0.50, and only RM reached 0.56. The eggs of indigenous chicken breeds, without any genetic selection, addressed to changing the weight of the egg components, show a higher yolk-to-albumen ratio than that of other avian species, to supply more nutrients for young birds to survive independently . These dYolk and albumen spots, originating, respectively, during ovarian follicle rupture and during the albumen secretion by the oviduct , are undThe main properties of albumen are addressed to protect the embryo against pathogens; however, the proteins gave albumen also the characteristics of foaming, emulsifying, and gelling . For somThe yolk colour is another internal quality trait evaluated by the consumer. This evaluation often does not consider the yolk pigmenting properties in cooking preparations, only the healthiness which a not pale colour may suggest. The yolk colour differed among white eggshell breeds, whereas no differences were detected for the tinted eggshell breeds. PA-C breed showed a Roche scale value lower than PO-B and ER. The reason may be higher egg production and, thus, a dilution effect of the pigments. Also, differences in yolk composition and the vitelline membrane characteristics may have affected the perception of the colour.The results indicate that the laying rate changed according to breed, and eggs produced by the eight local breeds of the Veneto region differed in size and many eggshell traits. The consumer may consider not only the exterior aspect of the egg but also the interior quality, as the studied breeds showed differences, especially in the weight of albumen and yolk, the ratio between them, the meat and blood inclusions, and the Haugh units. Further studies are needed for these breeds reared outdoors, to investigate changes in the egg quality according to the age of the hens and the environmental conditions. Many of the studied egg traits allow the consumer to distinguish the eggs for their genetic origin through the eggshell shape and colour, and to use these table eggs for different purposes. For each breed, an egg quality profile should be drawn up and referred to the different stages of the laying cycle, with the purpose to indicate, on the egg carton, the eggshell, albumen, and yolk traits.


Doublecortin (DCX) and glypican-2 (GPC2) are neurodevelopmental proteins involved in the differentiation of neural stem/progenitor cells (NSPCs) to neurons, and are developmentally downregulated in neurons after birth. In this study, we investigated whether the concentrations of DCX and GPC2 in the cerebrospinal fluid (CSF) from human pediatric patients reflect this developmental process or are associated with cerebral damage or inflammatory markers.CSF was collected from pediatric patients requiring neurosurgical treatment. The concentrations of DCX, GPC2, neuron-specific enolase (NSE), S100 calcium-binding protein B (S100B), and cytokines  were measured using immunoassays.From March 2013 until October 2018, 63 CSF samples were collected from 38 pediatric patients ; the median term born-adjusted age was 3.27 years . The median concentration of DCX was 329 pg/ml  and that of GPC2 was 26 pg/ml . DCX and GPC2 concentrations independently significantly associated with age, and their concentration declined with advancing age, reaching undetectable levels at 0.3 years for DCX, and plateauing at 1.5 years for GPC2. Both DCX and GPC2 associated with hydrocephalus, NSE, IL-1β, IL-2, IL-8, IL-13. No relationship was found between sex, acute infection, S100B, IL-4, IL-6, IL-10, IFN-γ, TNF-α and DCX or GPC2, respectively.Concentrations of DCX and GPC2 in the CSF from pediatric patients are developmentally downregulated, with the highest concentrations measured at the earliest adjusted age, and reflect a neurodevelopmental stage rather than a particular disease state. It is a complex, timely, and spatially regulated process that involves multiple signaling pathways , and is The cerebrospinal fluid (CSF) is a brain-specific fluid whose molecular composition may offer a window into ongoing physiological or pathological processes of the central nervous system (CNS). Doublecortin (DCX) and glypican-2 (GPC2), two neurodevelopmental proteins, have been detected and quantified in rodent and human CSF, and proposed as potential CSF biomarkers of neurogenesis , 6. DCX The specific association of these two proteins in the CSF with ongoing developmental, early postnatal, or adult- neurogenesis in the brain tissue remains nevertheless difficult to unambiguously establish, as CNS injuries/disorders or physiological development are accompanied by substantial cell death that may contribute to their presence in the CSF. Indeed, our own research in a rat model of neonatal hypoxia-ischemia (HI) indicated that CSF-DCX increased significantly 3 days after the brain HI injury, but this latter finding correlated with both HI-induced neurogenesis and infarct severity, thereby leaving unresolved whether a direct link exists between CSF-DCX and endogenous neurogenesis . Accordihttps://www.strobe-statement.org/) were followed to enhance the quality and standardization for the reporting of observational studies.This observational study was performed at the University of Basel Children’s Hospital and the University Hospital Basel, a Swiss tertiary academic medical care center. The STROBE guidelines . Written informed consent was given by the legal custodian of all patients.Patients less than 18 years old requiring a neurosurgical procedure involving the lumbar or cranial CSF compartment, performed at the Division of Pediatric Neurosurgery, University Children’s Hospital of Basel, were eligible for this study. Patients were not included if the neurosurgical procedure by itself either did not involve the CSF compartment or if no CSF was collected during the neurosurgical procedure. CSF could be collected repeatedly per patient if they required multiple neurosurgical procedures, with each procedure requiring a new hospital admission. In one premature patient with extradural drainage due to ventricular hemorrhage, CSF was repeatedly collected from this neurosurgical procedure.st February 2013 and 30th November 2017, and kept at 4°C until further processing. CSF was then centrifuged at 4°C at 2000 g for 10 min, and the supernatant was aliquoted and stored at -80°C until analysis. CSF was classified as clear, xanthochrome (yellow) or bloody (reddish) after centrifugation by eye estimation.Lumbar or ventricular CSF was collected periprocedurally between 1Clinical data  were extracted from individual patients’ records at the time point corresponding to CSF collection. In prematurely born patients, adjusted age was calculated from chronological age and expected due date . In this study, adjusted age is mentioned, unless stated otherwise. Main diagnoses, indications for neurosurgical intervention, and type of intervention are summarized in The two immunoassays were developed on the Meso Scale Discovery (MSD) platform. For the DCX assay, an MSD 96-well streptavidin-coated plate was blocked with 150 μl/well of blocking buffer  for 2 hours at room temperature without shaking. Plates were then coated overnight at 4°C with 25 μl of a biotinylated anti-DCX antibody  in assay buffer , and for two additional hours at room temperature on a rotational shaker (600 rpm) the next day. Plates were then washed three times with 150 μl/well of wash buffer , and 50 μl of recombinant DCX (range between 0.61 to 10’000 pg/ml) or CSF samples (1:2 dilution) were incubated with the ruthenium-labelled m83 anti-DCX antibody  on a rotational shaker (600 rpm) for 4 hours at room temperature. After disposal of samples and 3 washes, 150 μl/well of reading buffer (MSD read buffer T with surfactant) was added and the plate was immediately imaged on an MSD Sector Imager 6000. For the GPC2 assay, the same procedure was followed using biotinylated and ruthenium labelled anti-GPC2 antibodies  and recombinant GPC2 . The mean limit of detection (LOD) for each assay was 5.8 and 0.05 pg/ml for DCX and GPC2, respectively.The Meso Scale Discovery assay V-Plex (Cat# K15210G-1) was used to measure levels of cytokines and chemokines, namely IFN-γ, IL-10, IL-13, IL-1β, IL-2, IL-4, IL-6, IL-8 and TNF-α according to the manufacturer’s instructions. The corresponding LODs in pg/ml were 0.97, 0.13, 0.66, 0.04, 0.14, 0.06, 0.16, 0.1 and 0.26 respectively.Human Neuron-specific Enolase (NSE) and S100B were measured using commercially available assays  and performed according to the manufacturer’s instructions. The LODs in pg/ml were 97 and 2.7 respectively.The primary outcome was the association of DCX, or GPC2 in the CSF with patients’ age. Secondary outcomes were the association of DCX with GPC2 and the association of DCX or GPC2 with CSF markers for CNS damage or inflammation, or clinical features .To summarize the baseline characteristics data of our study population, we used the total number and percentage of the total for categorical variables, and median and first- and third quartiles  for continuous variables. In the main manuscript, the summary statistics provided for continuous variables are calculated using values at first measurement, to avoid misrepresenting the population by giving too much weight to the data from patients with repeated CSF sampling. It is noteworthy that for the assessment of the relationships between CSF-DCX, CSF-GPC2 and all other parameters, all values from each patient are taken into account, and not just values at first measurements.In the analysis, we used the fully probabilistic (Bayesian) approach to use all the information of our small dataset containing censored data  while taking into account the hierarchical nature of the data. The details of the models  are provided in To assess the relationship between DCX and GPC2 with age, we fitted an asymptotic mixed-effects regression model . The asyTo assess the relationship between DCX and GPC2 with conventional markers of brain damage  and inflammation , we fitted for each marker a univariable linear mixed-effects model with a random intercept to account for the multiple measurements per patient.We fitted a univariable linear mixed-effects model with a random intercept to account for the repeated measurements per patient. In all models including measurements of DCX, we had measurements below the LOD of the assay. Using the fully probabilistic Bayesian approach, it is not necessary to ignore or to impute these censored values; instead, they can be incorporated into the model, where they are eventually integrated out of the posterior distribution . For betHemolysis is a very well-documented confounder of NSE determination . Thus, iAll analyses were performed on the complete cases dataset, i.e., data entries with missing data (due to unavailability of CSF sample) were excluded. All the measured concentrations were log-transformed . Data analysis was performed using R Version 3.6.1 . To fit the models, we used the R package Bayesian Regression Models using Stan (brms) . AdditioA total of 63 CSF samples were collected from 38 patients. Among the patients, 21 had a single CSF sample collection, and 17 underwent repeated CSF sampling from multiple neurosurgical procedures, totalling up to 41 CSF samples. Baseline characteristics of patients are shown in DCX concentration was measured in the 63 CSF samples from all 38 patients, with a median concentration for the first available measurements of 329 pg/ml . CSF-DCX was below the LOD of the assay in 40 samples (63.5%) from 30 patients. The majority of samples below the LOD were from patients older than ~4 months. Only two patients older than 4 months had detectable DCX in the CSF: a 5-year-old male with a grade III astrocytoma  and a 9-year-old male with a teratoma of the III ventricle with obstructive hydrocephalus .For the measurement of GPC2, all 59 available CSF samples from 38 patients were above the LOD. The median concentration of GPC2 for the first available measurements was 26 pg/ml .All other analytes quantified are displayed together with DCX and GPC2 in After censoring the patients with CSF-DCX below the LOD, the association between log-transformed concentrations of DCX and age could be fitted in an asymptotic regression model, with the highest concentrations observed in the youngest patients, followed by a steep decline in DCX concentrations with advancing age .The relation between log-transformed concentrations of GPC2 and age could also be modelled with an asymptotic statistical fit . The higThe relation between CSF-DCX/GPC2 and age was then examined in the 17 patients who underwent repeated CSF sampling. Overall, the age-related pattern in individual patients was consistent with that observed in the whole patient’s cohort, namely a steep decline in CSF concentrations during prenatal/early postnatal stages followed by a stabilization of values, with either undetectable (for DCX) or very low levels (for GPC2) in older patients . PatientThere was a linear relationship between CSF-DCX and GPC2 .Significant, positive associations were found for both CSF-DCX and CSF-GPC2 respectively, with hydrocephalus, NSE, IL-2, IL-8, IL-13, and IL-1β. No associations were found for sex, acute infection, IL-4, IL-6, IL-10, IFN-γ, TNF-α, S100B, for both CSF-DCX and CSF-GPC2 . The detOf note, the median age at CSF collection was significantly different among patients diagnosed or not with hydrocephalus, namely 0.455 years (~5 months) for CSF samples from hydrocephalus patients  versus 8.32 years for CSF samples from patients with different diagnoses  .We also further examined the association between CSF-DCX/GPC2 and NSE, in particular because hemolysis is a very well-documented confounder of NSE determination . After rA question raised by the association between CSF-DCX/GPC2 and the CSF concentrations of NSE, IL-2, IL-8, IL-13, and IL-1β is whether the latter also decrease asymptotically with age. The relation between log-transformed CSF concentrations of IL-2 and age could be fitted in such asymptotic regression curve . HoweverThe concentration of DCX and GPC2 in the CSF from pediatric patients correlated with age. The highest CSF concentrations of DCX and GPC2 were measured at birth and declined thereafter. CSF-DCX and CSF-GPC2 also correlated with hydrocephalus, the neuronal marker NSE, but not with the astrocyte marker S100B, acute infection or sex. CSF-DCX and CSF-GPC2 also associated with individual cytokines, but not with others, independently of their classical pro- or anti-inflammatory categorization.+ young migrating neurons that gives rise to a heterogeneous population of neurons in the anterior forebrain, persists in the human infant brain during at least the first 5 months of life Reviewers' comments:Reviewer's Responses to Questions  Comments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?  The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Partly**********  2. Has the statistical analysis been performed appropriately and rigorously?   Reviewer #1: Yes**********  3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.  The Reviewer #1: No**********  4. Is the manuscript presented in an intelligible fashion and written in standard English?  PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: Yes**********  5. Review Comments to the Author  Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: Neurogenesis plays a key role on brain development and homeostasis but there are not currently good indicators of the neurogenic state in a living animal. The cerebrospinal fluid, CSF, may reflect the physiological state of the central nervous system, as shown by the latest findings reporting cognitive benefits to adult mice after infusion of CSF from young mice.Doublecortin (DCX) and Glypican-2 (GPC2) are key neurodevelopmental proteins involved in neurogenesis in mice, and have been proposed as potential reporters of neurogenesis. DCX and GPC2 have peak expression in the CSF perinatally and decline subsequently in early postnatal stages in rodents, but there are few or no reports on the CSF expression of those markers in humans.Data for human patients is key to understanding human development and disease, but ethical experimental constraints limit their availability. This highlights the relevance of the present study by Guzman and colleagues, an approach to systematically investigate DCX and GPC2 expression in infant CSF. The same constraints affect this study, as data provided belongs to pediatric patients and is, therefore, partial and reflects infants requiring neurosurgery. But the information provided, together with future similar studies will help understand human neurogenesis.The biggest strength of the study is the observation that human CSF expression of both factors reflects experimental data from rodents. The manuscript is well written and worthy of publication in PLOS ONE, with the authors nicely acknowledging the weak points of the study in the discussion. The reviewer would nevertheless suggest a few minor modifications to provide all available data to the public. In the reviewer’s view, this would improve the impact of the manuscript.MajorPoints.1. Full details of the clinical baseline characteristics for each patient are accounted for in the Supplementary Table 1, yet the information regarding the individual patient’s values for the DCX and GPC2, as well as the values for cytokines and markers for brain damage are not provided. In view of the reviewer, the information regarding the patient diagnosis accompanied by the individual measurements of DCX, GPC2 and the relationship with the individual values of the rest of molecules tested could provide a better understanding of their role.2. Similarly, some of the patients were subject to repeated measures, but the respective measurements are not accounted for in the presented manuscript. In the reviewer’s opinion, this longitudinal information should be provided, as it would allow for better characterization of the dynamics of doublecortin and glypican-2 during human development, and in disease.3. The reviewer wonders whether attributing the DCX values below the detection limits to the mean values of the lower detection limit to DCX concentration is generating an artifact to the data. Indeed, to the reviewer’s eye, the DCX data matches better a linear regression than an asymptote once the values that are below the detection limits of the assay (or data from patients with diseases of proliferative nature) are left out of the analysis. Would a randomized attribution of the values of DCX concentration –from 0 pg/ml to the lower detection limit– alter the interpretation of the data? Alternatively, a visual indication of the actual measurement should be included in the graph.  , IL-2, IL-8 and IL-13. To the reviewer’s eye, these show that maximum expression levels appear perinatally to later stabilize. On the other hand, the authors do not display the same data for the remaining interleukins and the marker S100B. In view of the reviewer, these graphs should also be provided.4. The researchers found positive correlation between the CSF levels of DCX and GPC-2 and the cytokines IL-1beta, IL-2, IL-8 and IL-13, and the marker for brain damage NSE. The authors provide a graph showing that log-transformed measurements of NSE do not show an asymptotic decline with age. The authors state that a visual assessment of the relation between log-transformed concentrations of cytokines with age indicated an absence of relation between those parameters. The authors provide the graphs for IL-1�Minor Points.1. A below-detection limit value is present in the figure-1 graph.2. S2 table is labelled as “S1”. S4 tables are labelled as “S3”.**********  6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.  Reviewer #1: No**********https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,  7 Sep 2022Thank you for allowing us to submit a revised version of our manuscript. The valuable insights of the reviewer have significantly improved our paper.Our point-by-point response to the reviewer is presented in the file “Response to Reviewers”.AttachmentResponse to Reviewers.docxSubmitted filename: Click here for additional data file. 6 Dec 2022Doublecortin and Glypican-2 concentrations in the cerebrospinal fluid from infants are developmentally downregulatedPONE-D-22-09668R1Dear Dr. Guzman,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Robert BlumAcademic EditorPLOS ONEAdditional Editor Comments :Dear authors,I took over the mansucript as academic editor. There was some delay due to a lack of response by the reviewer. Anyhow, the reviewer gave great comments.Indeed, this paper is important and provides a clear message. Data handling is transparent and you answered all reviewer comments.For these reasons, I decided to terminate the review process and decided to recommend your work for publication in PLOSone.Kind regardsRobert Blum (academic editor)Reviewers' comments: 5 Jan 2023PONE-D-22-09668R1 Doublecortin and Glypican-2 concentrations in the cerebrospinal fluid from infants are developmentally downregulated Dear Dr. Guzman:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staffon behalf ofPD Dr. Robert Blum Academic EditorPLOS ONE


Objectives: This study aimed to assess the spatial distribution and determinant factors of handwashing practice using essential handwashing agents (soap and water) among households in Ethiopia.Methods: A two-stage stratified cluster sampling technique was used. Mixed-effect logistic regression analysis was also used to identify determinants of handwashing practice with essential agents.Results: In Ethiopia, household handwashing practices with essential agents had spatial variation . The Amhara and Somali regions were identified as significant hotspots with low handwashing practice using essential agents.Conclusion: In Ethiopia, handwashing practice with essential agents showed spatial variation across the country with a very low rate. Areas with low handwashing practice with essential agents need high priority in the allocation of resources to ensure communities’ access to fixed and portable handwashing facilities, soap, and reliable water supplies. Households with low access to improved sanitation facilities, low wealth status, and low educational status should be targeted for the intervention. Handwashing is defined as the brief, vigorous rubbing together of all surfaces of lathered hands followed by rinsing under a stream of water. It is the act of cleansing hands using soap and water or using an antiseptic hand rub for the removal of transient microorganisms from the hands. Hand hygiene is a simple, easily implemented, and effective practice, and is a significant contributor to the prevention and control of infectious diseases, particularly water- and foodborne and feco–orally transmitted diseases –3.Handwashing with soap and water  is one of the most effective measures against infectious diseases. Handwashing removes pathogenic agents and reduces the number of microbes on the hands, which could minimize the spread of germs and thereby prevent diarrhea, acute respiratory infections like influenza, and skin infections , 5. HandGlobally, 2.3 billion people lack basic sanitation . About 8Reducing the number of child deaths in those aged 5 years and below is one focus of Sustainable Development Goals (SDGs)  To this Inadequate and inefficient infrastructures for water, sanitation, housing, and personal hygiene facilities are responsible for such an unacceptable burden of diseases and deaths . ExistinThe Government of Ethiopia, with support from several development partners and Non-Governmental Organizations (NGOs), launched the One WASH National Program in 2013 . The govFurthermore, hand hygiene practices, including important handwashing agents, vary by region –26, and The Ethiopian Demography and Health Survey (EDHS) 2016 was used to conduct secondary data analysis. Every 5 years, the EDHS is conducted in Ethiopia’s nine regional states  as well as two administrative cities (Addis Ababa and Dire-Dawa) . Ethiopiwww.measuredhs.com, after permission was granted via an online application that explained the study’s aim. The EDHS 2016 household data (HR) set was used. Between 18 January and 27 June 2016, Ethiopia conducted the 2016 Ethiopian Demographic and Health Survey (EDHS) [Data for this study, including geographic coordinates (longitude and latitude coordinates), were obtained from the official database of the Demography Health Survey (DHS) program, Using the 2007 Population and Housing Census as a sampling frame, the EDHS 2016 used a stratified two-stage cluster sampling technique. In the first stage, 645 enumeration areas (EAs) were carefully selected with a probability proportional to the EA size and an independent selection in each sampling stratum . In the second stage, 28 households were systematically chosen. The full EDHS 2016 report included a detailed sampling technique . The totThe dependent variable for this study was the “presence of handwashing practice with essential agents in the households.” In 2016 EDHS, to obtain handwashing information, interviewers asked to see the place where members of the household most often wash their hands. Soap and water, the essential handwashing agents, were observed in households. The dependent variable was then divided into two groups: “Yes” if a household had experienced handwashing with essential agents and “No” if a household did not experience handwashing practices with essential agents within the study period. The explanatory variables included in the study were residence, the number of household members, the sex of the household head, improved water source, educational status of household head, improved sanitation facility, region, wealth quintile, and place where household members wash their hands.Handwashing practice with essential agents: this was defined as the brief, vigorous rubbing together of all surfaces of lathered hands with soap and water. Soap and water are essential handwashing agents .Improved sanitation facilities: this was defined as one that hygienically separates human excreta from human contact. This includes flush or pour-flush toilets flowing to a piped sewer system, septic tank, or latrine, ventilated pit latrine, pit latrine with slab, and composting toilet. The type of sanitation facility used by each household was classified as having or not having an improved sanitation facility , 31.Improved water sources: this was defined as whether the household used piped water , tube well/borehole, protected well, protected spring and rainwater collection for drinking purposes. If bottled water was used, the households must have had to use any of the improved water sources listed above for other purposes, such as cooking and washing hands, to be considered as using improved water sources , 32.Before any statistical analysis, the data were weighted to restore the generalizability of the survey and take into account the sampling design, and get reliable statistical estimates. We used STATA 14 software to show descriptive statistics and summary statistics in the form of text and tables.Exploring the spatial distribution, global spatial autocorrelation, and identifying major hotspot areas of handwashing practices with essential agents were carried out using ArcGIS version 10.3 and SaTScan version 9.6 statistical software.p < 0.05) can lead to rejection of the null hypothesis  and show the presence of spatial autocorrelation.The presence of spatial autocorrelation was determined using the global Moran’s index (Moran’s I). Moran’s I values range from −1 to 1 . A value<50% of the population was used.Using Kuldorff’s SaTScan software, a Bernoulli-based model was fitted to find statistically significant spatial clusters of handwashing practice with essential agents. Households with experience of handwashing with essential agents was taken as cases and those with no experience of handwashing practice with essential agents were considered as controls to fit the Bernoulli model. The default maximum spatial cluster size of For each potential cluster, a Likelihood Ratio (LR) test statistic and the p-value were applied to identify if the number of observed cases within the potential cluster was significantly higher than expected or not. The scanning window with maximum likelihood was the most probable performing cluster, and the p-value was assigned to each cluster using Monte Carlo hypothesis testing by comparing the rank of the maximum likelihood from the real data with the maximum likelihood from the random data sets. Based on 999 Monte Carlo replications, the most performing cluster and the secondary clusters were ranked by using their likelihood ratio test .According to EDHS data, households in one cluster may be more similar to one other than households in another cluster. This contradicts the assumptions of observational independence and equal variance across clusters. This implies the use of advanced models to account for between-cluster heterogeneity. Due to the dichotomous nature of the response variable, binary logistic regression and a Generalized Linear Mixed Model were fitted. Deviance Information Criteria were used to compare the models (DIC). A mixed-effect model with the lowest DIC was chosen. The Intra-class Correlation Coefficient (ICC) value was 0.2, which told us to select the Generalized Linear Mixed Model over the binary logistic regression model.After adjusting the effect of other variables, variables with p-values ≤0.2 in the bi-variable analysis were fitted in the multivariable analysis to quantify the effect of each variable. Adjusted Odds Ratio (AOR) with a 95% Confidence Interval (CI) and p-value < 0.05 in the multivariable model was declared as determinant factors of handwashing practice with essential agents. A variance inflation factor (VIF) was also used to assess multi-collinearity.This study used a weighted sample of 9,966 households. The percentage of households living in rural areas was 72.55%. The household wealth of 29.14% of the household was in the two poor wealth quintiles, 17.98% were in the middle and 52.86% were in the two upper wealth quintiles. The majority of the households (70.25%) were using an improved water source, but only 19.46% had an improved sanitation facility .p < 0.001). A cluster of high rates of handwashing practice with essential agents was observed over the study area. The outputs were automatically generated keys on the right and left sides of each panel. A z-score of 20.49 indicated that there is less than a 1% likelihood that this clustered pattern could be the result of chance. The end tails are bright red and blue, indicating a higher level of significance.  households practice handwashing with essential agents. The highest prevalence of household handwashing practice with essential agents was identified in Addis Ababa, Tigray, Afar, Dire Dawa, SNNPR, and Gambella, while the Amhara and Somali regions had the lowest prevalence of household handwashing practice with essential agents .p < 0.001), and 7 clusters were modest performing clusters . Addis Ababa, SNNPR, Tigray, Beneshangul, and Afar regions had the highest performing clusters of handwashing practice with essential agents (Most likely (primary clusters) and secondary clusters of handwashing practice with essential agents were identified. In EDHS 2016, spatial scan statistics identified a total of 61 high and modest performing spatial clusters of handwashing practice with essential agents. Of these, 54 clusters were high performing clusters  compared to households where household heads had no education.Households where household heads had higher educational status were two times more likely to wash their hands  compared to households where household heads had no education.Households with richer wealth quintiles were 1.94 times more likely  to wash their hands compared with households in the lowest wealth quintiles. Households in the richest wealth quintiles were 2.45 times more likely to wash their hands with essential agents  compared with households in the lowest wealth quintiles. Households that do not use improved sanitation facilities were less likely to wash their hands with essential agents .The odds of washing hands with essential agents were decreased by 81% among households with mobile places where household members wash their hands  compared with households with fixed places where household members wash their hands. Compared to households in the Tigray region, households in Addis Ababa  were more likely to wash their hands with essential agents. Compared to households in the Tigray region, households in Amhara  and Somali  were less likely to wash their hands with essential agents .ArcGIS- and SaTScan-based spatial statistical techniques provide an opportunity to clarify and identify household handwashing practice with essential agents within a country and upcoming investigations into predictors responsible for increased handwashing practice with essential agents. Geographic understanding of handwashing practice with essential agents in Ethiopia using these tools was important to target interventions in high-risk areas.p < 0.001) indicated that there was a significant clustering of handwashing practice with essential agents in the study area. Our findings showed that only 13.03% of the households practiced handwashing with essential agents in Ethiopia.In the current study, the distribution of handwashing practice with essential agents varied from country to country. A Global Moran’s value of 0.62  in the Somali region. In the Somali region, the use of improved water sources is low, and the average water transportation time (roundtrip) is significant . The othAnother factor that affects handwashing practice with essential agents was the wealth quintile; the odds of handwashing with essential agents were higher among households in the richer and richest wealth quintiles. Similar studies were done in Vietnam  and IndoHouseholds not using improved sanitation facilities were less likely to wash their hands. This finding is consistent with those from other settings elsewhere . This coThe educational status of the household head was found to be a predictor of handwashing with essential agents in this study. Household heads with secondary and higher educational levels were more likely to wash their hands with essential agents as compared to household heads with no education. Similar studies have also shown a direct relationship between household head educational status and handwashing practice with essential agents , 39, 40.The current studies showed that handwashing with essential agents was decreased by 81% among households with mobile places where household members wash their hands as compared with households with fixed places where household members wash their hands. The possible explanation could be households with fixed places where household members wash their hands would be more conscious about handwashing practice with essential agents. The study’s representativeness at the national and regional levels was one of its strengths. Furthermore, statistical analysis using GIS and SaTScan helped to detect statistically high-risk clusters of handwashing practice with essential agents. However, the study did not show the particular locations for data confidentiality reasons. Because the EDHS data were secondary, some potentially important predictors, such as social, behavioral, and cultural factors of handwashing with essential agents, were not included.Handwashing practice with essential agents was found to be spatially clustered; the Addis Ababa, SNNPR, Tigray, Beneshangul, and Afar regions had the highest practicing rates, and the Amhara and Somali regions had the lowest practicing rates in this study. Educational status, improved sanitation facility, region, wealth quintile, and place where household members wash their hands were significantly associated predictors of households with handwashing practice with essential agents. Therefore, areas with a low rate of handwashing with essential agents are of high priority for the allocation of resources to ensure communities’ access to fixed and portable handwashing facilities, soap, and reliable water supplies. Households with reduced access to improved sanitation facilities, low wealth status, and low educational status should be targeted for the intervention.


Neuroinflammation may contribute to psychiatric symptoms in older people, in particular in the context of Alzheimer’s disease (AD). We sought to identify systemic and central nervous system (CNS) inflammatory alterations associated with neuropsychiatric symptoms (NPS); and to investigate their relationships with AD pathology and clinical disease progression.We quantified a panel of 38 neuroinflammation and vascular injury markers in paired serum and cerebrospinal fluid (CSF) samples in a cohort of cognitively normal and impaired older subjects. We performed neuropsychiatric and cognitive evaluations and measured CSF biomarkers of AD pathology. Multivariate analysis determined serum and CSF neuroinflammatory alterations associated with NPS, considering cognitive status, AD pathology, and cognitive decline at follow-up visits.NPS were associated with distinct inflammatory profiles in serum, involving eotaxin-3, interleukin (IL)-6 and C-reactive protein (CRP); and in CSF, including soluble intracellular cell adhesion molecule-1 (sICAM-1), IL-8, 10-kDa interferon-γ-induced protein, and CRP. AD pathology interacted with CSF sICAM-1 in association with NPS. Presenting NPS was associated with subsequent cognitive decline which was mediated by CSF sICAM-1.Distinct systemic and CNS inflammatory processes are involved in the pathophysiology of NPS in older people. Neuroinflammation may explain the link between NPS and more rapid clinical disease progression.The online version contains supplementary material available at 10.1186/s12974-022-02473-3. Neuropsychiatric symptoms (NPS) are frequent in older people and may represent the clinical manifestation of a variety of acute and chronic cerebral pathologies. NPS are particularly common among patients with Alzheimer’s disease (AD) , 2, and Increased neuroinflammatory activity and altered cytokine levels in the CNS have been related to psychiatric and behavioral syndromes. The activation of the central innate immune response by cytokines initiates a behavioral response known as sickness behavior  which isIn this study we analyzed a large panel of neuroinflammatory markers at systemic and CNS levels, and hypothesized that specific circulating blood and CSF neuroinflammatory marker profiles are associated with the occurrence of NPS in general and of individual syndromes such as depression, anxiety and apathy in older subjects with normal cognition or with beginning cognitive decline. A further objective was to determine how these inflammatory profiles change depending on the presence of cerebral AD pathology and/or cognitive impairment. Finally, we verified if the identified inflammatory signatures were associated with clinical disease progression at follow-up visits.We enrolled 87 community dwelling individuals, aged 55 or older, from a brain aging study conducted in the Department of Psychiatry and the Department of Clinical Neurosciences, University Hospital of Lausanne, Switzerland. Cognitively impaired participants were recruited among memory clinic outpatients and displayed no major psychiatric or neurological disorders, nor substance abuse or severe or unstable physical illness that may contribute to cognitive impairment, including history or clinical signs of inflammation. All subjects in this group had clinical diagnoses of either MCI or mild dementia, and a Clinical Dementia Rating (CDR ) score ≥The neuropsychological assessment and the administration of informant questionnaires to the participant’s proxy was previously described . BrieflyLumbar and venous punctures were conducted during the same visit between 8:30 and 10 a.m. after an overnight fast yielding 10–12 ml of CSF and 40 ml of blood, respectively, were performed, spun down at 4 °C, immediately aliquoted, and snap frozen at − 80 °C until assayed . Study pAPOE) genotype were determined as previously described [The CSF albumin index  as a marker of blood–brain barrier (BBB) function along with the Apolipoprotein E (escribed .1–42 ratio > 0.0779 was defined as an AD CSF profile as previously described [CSF beta-amyloid 1–42 (Aβ1–42), total-tau (Tau), and tau phosphorylated at threonine 181 (pTau181) concentrations were measured using commercially available ELISA kits . A pTau181/Aβescribed . Brieflyescribed .n-transformed prior to correlation and regression analyses to approach Gaussian distribution. Normal distribution was subsequently assessed using the Kolmogorov–Smirnov test that identified 6 neuroinflammatory biomarkers in CSF  and 5 in serum  not normally distributed.Quantitative analysis of 38 neuroinflammatory biomarkers in both CSF and serum was achieved using a sandwich immunoassay  as previously described . Biomarkt-tests comparing participants with and without NPS for continuous variables and Chi-square tests for categorical variables. Correlations between NPI-Q total score and neuroinflammatory markers were assessed with Spearman’s rho. Independence of variables used in regression models was tested with variance inflation factor (VIF). No variable entered in these models had VIF above 7, with a majority below 3, demonstrating absence of multicollinearity and allowing regression models to be used. Benjamini–Hochberg correction of P-value for multiple testing was applied for all analyses using a false-discovery rate of 0.25. Statistical data analysis was performed with IBM SPSS Statistics software version 25. All statistical models performed in this study were verified for possible overfitting using the Hosmer–Lemeshow test for goodness-of-fit. Models with a Hosmer–Lemeshow Chi-squared value yielding a P-value > 0.05 were rejected and the previous iteration was considered instead.Descriptive statistics for the cohort were performed using 1–42 ratio) and BBB function  by entering them into the model before considering inflammatory marker concentrations. All models used a forward selection method based on the significance of the score statistic to select the smallest possible number of molecules associated with the occurrence of NPS in each case.In order to identify neuroinflammatory marker combinations in CSF or serum associated with NPS, we used binary regression models with NPI-Q > 0 or NPI-Q = 0 as dependent variable while entering either all CSF or all serum markers in the models. We explored the effect of the following confounders: cognitive status (CDR = 0 or CDR > 0), CSF AD biomarker profiles  linked to sickness behavior and associated with neuroinflammation in the literature, i.e., depression, apathy and anxiety, we used binary logistic regression models corrected for cognitive status (CDR = 0 or CDR > 0) as above. These models used a forward selection method based on the significance of the score statistic to select the smallest number of molecules associated with each individual symptom.1–42 ratio × neuroinflammatory marker measurements. This was then used in a binary regression model with NPS occurrence as dependent variable. This approach tested whether a positive CSF biomarker profile for AD modified the effect of each individual neuroinflammatory on the occurrence of NPS.The interactions between an AD CSF profile, neuroinflammatory markers and the presence of NPS were tested by creating and interaction variable: pTau181/AβNPI-Q scores at baseline were correlated with progression of disease severity as measured by changes in CDR-SoB at follow-up clinical assessments at 18 and 36 months in the whole cohort and in participants with a positive NPI-Q score using Spearman rho’s. Linear regression was used to test associations between CSF neuroinflammatory markers associated with NPS, and cognitive decline computed by change in CDR-SoB over time (CDR-SoB difference at follow-up/time between baseline and follow-up) corrected for baseline CDR-SoB assessment. Finally, we tested the association of the interaction between NPI-Q total score and sICAM-1 on CDR-SoB change using a binary logistic regression model using cognitive decline at 18 or 36 months  as dependent variable. We entered NPI-Q total score, sICAM-1 concentration and the interaction between NPI-Q score and sICAM-1 concentration (NPI-Q score × sICAM-1 levels) as variables into this model.Demographic, clinical, and biological characteristics of participants with or without NPS (NPI-Q = 0 or NPI-Q > 0) are given in Table The measured concentrations of all individual CSF and serum neuroinflammatory markers are shown in Additional file 1–42 ratio was also associated with NPS in this context . Each of the selected individual CSF neuroinflammatory markers was also associated with the volume of a specific subset of brain regions  remained associated with the presence of NPS; and both CRP , and VEGF-D  were associated with the presence of NPS when considering the CSF AD biomarker profile. In parallel, we also investigated the effects of BBB function on these associations by correcting our model for Qalb. In this model, the CSF inflammatory markers associated with the occurrence of NPS were CRP, IP-10 and sICAM-1. In serum, MIP-1α, VEGF were associated with the occurrence of NPS (data not shown). When added to a reference model built using APOE status, sex, age, years of education and cognitive status at baseline .Using binary regression models, we identified a combination of four neuroinflammatory markers in CSF that best predicted the occurrence of NPS: CRP, IP-10, sICAM-1 and IL-8 , we found that only the interaction between the pTau181/Aβ1–42 ratio and CSF sICAM-1 was associated with the presence of NPS .Since the CSF concentrations of CRP, IP-10, sICAM-1, and a positive CSF biomarker profile for AD at baseline were associated with a positive NPI-Q score, we tested whether the interaction between neuroinflammation markers and a positive CSF biomarker profile for AD was also associated with NPS. Using binomial regression models with interaction variables . These regions have been associated with both neurodegeneration in AD and inflammation in previous works , 53. In This is to our knowledge the first study to assess a large panel of inflammatory markers in paired serum and CSF samples to determine inflammatory signatures of NPS in older people while considering cognitive status and biomarkers of AD pathology. The size of our cohort does not allow for stratification into subgroups according to the A/T/(N) classification framework, limiting further exploration of the relationship between the identified neuroinflammatory marker profiles and AD pathology and how neuroinflammation could play differential roles in different pathological presentations. A further limitation of this study is related to the fact that subjects included in this cohort had no psychiatric disorder or severe psychiatric symptoms that could interfere with cognition, and that candidates with more marked neuropsychiatric symptoms were not considered. However, both elderly subjects with normal cognition and with early stages of cognitive decline were considered, allowing to evaluate the relevance for clinical disease progression over time. Apathy had a relatively low frequency in our cohort and some relationships with this syndrome could have been missed. Furthermore, we did not include subjects with more advanced stages of dementia, where the pathogenesis of NPS may differ from early stages of cognitive decline. Whether the identified signatures can be used in clinical practice as markers of neuroinflammation-related NPS and as targets for intervention needs to be further investigated in independent samples.We have identified specific CSF and serum inflammatory signatures associated with NPS that indicate distinct contributions of systemic and CNS inflammation to the pathophysiology of NPS. This study is the first to relate inflammation markers to NPS in older people with normal cognition or with cognitive impairment while considering AD pathology as indicated by CSF biomarkers. While the CSF signature appears to indicate inflammatory processes that originate within the CNS and are independent of the core AD pathology, the serum signature may represent systemic inflammatory activity which may contribute to pathological processes in the CNS that clinically manifest as NPS. The inflammatory signatures of NPS indicate syndrome-specific underlying processes opening the perspective of targeted interventions to reduce NPS and their long-term consequences.Additional file 1: Table S1. Neuropsychiatric symptom distribution within the study cohort. Table S2. Longitudinal distribution of cognitive and neuropsychiatric status within the cohort. Table S3. Concentrations (in pg/ml) of neuroinflammatory markers in CSF. Table S4. Concentrations (in pg/ml) of neuroinflammatory markers in serum. Table S5. Associations of volumetric data with neuroinflammatory marker concentration. Table S6. Reference model. Figure S1. Concentrations and correlations of neuroinflammatory markers with NPI-Q score in the whole cohort. Figure S2. Predictive model of the presence of neuropsychiatric symptoms.


Cerebrospinal fluid (CSF) analysis is of utmost importance for diagnosis anddifferential diagnosis of patients with suspected multiple sclerosis (MS).Evidence of intrathecal immunoglobulin G (IgG) synthesis proves the inflammatorynature of the disease, increases diagnostic certainty and substitutes fordissemination in time according to current diagnostic criteria. The goldstandard to determine intrathecal IgG synthesis is the detection ofCSF-restricted oligoclonal bands (OCBs). However, advances in laboratory methodsbrought up κ-free light chains (FLCs) as a new biomarker, which are produced inexcess over intact immunoglobulins and accumulate in CSF in the case of centralnervous system-derived inflammation. Overwhelming evidence showed a highdiagnostic accuracy of intrathecal κ-FLC synthesis in MS with sensitivity andspecificity of approximately 90% similar to OCB. κ-FLCs have advantages as itsdetection is fast, easy, cost-effective, reliable, rater-independent andreturning quantitative results which might also improve the value of predictingMS disease activity. An international panel of experts in MS and CSF diagnosticsdeveloped a consensus of all participants. Six recommendations are given forestablishing standard CSF evaluation in patients suspected of having MS. Thepanel recommended to include intrathecal κ-FLC synthesis in the next revision ofMS diagnostic criteria as an additional tool to measure intrathecalimmunoglobulin synthesis. Evidence of intrathecal immunoglobulin G (IgG) synthesis in the CSF, althoughnot specific for MS, substitutes for dissemination in time according to current diagnostic criteria and increases diagnostic certainty in the appropriate clinical setting. The gold standard to prove intrathecal IgG synthesis is the detection ofCSF-restricted oligoclonal bands (OCBs).Diagnosis of multiple sclerosis (MS) requires the combination of clinical signs andsymptoms with para-clinical findings obtained by magnetic resonance imaging andcerebrospinal fluid (CSF) analysis.6 Similar to immunoglobulins,free light chains (FLCs) accumulate in the CSF in the case of chronic inflammatorydiseases of the central nervous system such as MS. FLCs were discovered long ago; however, their quantitative detection withhigh sensitivity and, thus, in low-level compartments such as CSF was not possibleuntil technological advances at the beginning of the century. The breakthrough was achieved by producing detection antibodies directedagainst unique FLC epitopes.B cells produce intact immunoglobulins by assembling light chains and heavy chainsvia disulfide bonds and non-covalent interactions, but B cells also produce lightchains in excess of 10%–40% over heavy chains and secrete them as free forms .5,6 Simi and the detection of κ-FLC has considerable methodological advantagescompared to the detection of OCB.10 However, a strong consensuson the role of κ-FLC as a biomarker in MS is still lacking. This might be due to acertain heterogeneity between published studies ranging from different patientpopulations, assays, different κ-FLC measures (e.g. κ-FLC index versus absolute CSFκ-FLC concentration) and cut-off values.A multitude of studies have shown a high diagnostic accuracy of the κ-FLC isotype inthe CSF to discriminate patients with MS from other neurological diseases;In an effort to evaluate and recommend the type of CSF analysis that yields thegreatest diagnostic sensitivity and specificity for the diagnosis of MS consideringnew technologies in the last two decades, a working group was formed. The aim was toproduce a report for neurologists and laboratory medicine specialists on what wouldbe considered as ‘standard’ for the evaluation of CSF in patients with suspected MSand to provide consensus recommendations on the use of κ-FLC in routine diagnosticwork-up.In October 2021, an international panel convened in Vienna, Austria, to discuss theuse of CSF and, in particular, the applicability of κ-FLC in the CSF for routinediagnostic purposes in patients with suspected MS. The panel was composed of expertsin the diagnosis and management of MS patients and/or CSF analysis, includingneurologists and laboratory medicine specialists from 11 institutions across eightcountries and three continents. All participants are listed as authors of thismanuscript. This meeting was endorsed by the European Committee for Treatment andResearch in Multiple Sclerosis (ECTRIMS).).The panel presented and discussed data from research published in English about CSFanalysis and, in particular, about κ-FLC in patients with clinically isolatedsyndrome (CIS) and MS. While specific recommendations for the implementation ofκ-FLC in the diagnostic process for patients with suspected MS were developed duringthe meeting, the panel set out to create the whole consensus document afterwards.Furthermore, the panel decided to perform a systematic review and meta-analysis toprovide a summary of the diagnostic value of κ-FLC in patients with CIS and MS andto compare its performance to OCB  was required. If agreement on arecommendation was not achieved, it was discussed again, and a new proposalre-circulated, until a final agreement was achieved.Qalb) and intrathecal Ig synthesiscontributes to the diagnosis of MS and the exclusion of other causes of CNSinflammation mimicking MS, for example, vasculitis, chronic infection or otheracquired demyelinating disorders, such as neuromyelitis optica spectrumdisorders (NMOSD) and myelin oligodendrocyte glycoprotein-associated disorders (MOGAD). A recent, comprehensive study on routine CSF parameters in more than 500patients with CIS and MS applying the McDonald criteria 2017 revealed thatapproximately 50% of patients show CSF pleocytosis with a WBC count up to 40/µL(95th percentile), that lymphocytes followed by monocytes are the predominantcell type in CSF cytology, that the blood-CSF-barrier function is abnormal inless than 10% of cases with Qalb up to approximately10 (95th percentile), and that intrathecal IgG synthesis as determined byCSF-restricted OCB are present in up to 95% of patients. CSF findings slightly differ between the different MS disease courses;for example, patients with progressive MS show lower WBC counts and lessfrequently CSF pleocytosis; and CSF-restricted OCBs are found in approximately80% of CIS patients but in up to 95% of MS patients with lower prevalence in areas of lower geographic latitude. Relevant differential diagnoses frequently show higher WBC counts,different WBC subpopulations (e.g. relevant percentages of neutrophils) andhigher Qalb and OCB infrequently (only up to10%–20%).14 For details on CSF collection and analysis, we refer topreviously published consensus guidelines.15The full spectrum of routine CSF parameters including white blood cell (WBC)count, differential cell profile ,albumin quotient , which should be interpreted in the context of clinical and imagingfindings.Different methods are available for the detection of intrathecal immunoglobulinsynthesis in patients with suspected MS, each with certain strengths andlimitations. Reiber or Auer et al. formulae referring patients’ individual values to a predefinedupper normal limit. This approach shows a moderate diagnosticsensitivity of approximately 70% in MS patients.Quantitative intrathecal IgG synthesis: The concentration of total IgG inCSF and serum are determined followed by the calculation of certainformulae such as IgG index,4 This techniquecompares paired CSF and serum samples of each individual patient.Intrathecal IgG synthesis is demonstrated if OCBs are present in CSFwithout corresponding bands in the serum (patterns II and III) .Qualitative intrathecal IgG synthesis: The detection of oligoclonal IgGbands (OCB) by isoelectric focussing (IEF) followed by immuno-detectionis currently the gold standard method.and III) .4 Thisκ-FLC), where againpatients’ individual values are compared to a predefined upper normallimit. An overview of the different κ-FLC measures investigated in MS isprovided in Quantitative intrathecal κ-FLC synthesis: κ-FLC concentrations aremeasured in CSF and serum followed by calculation of either the κ-FLCindex or an intrathecal κ-FLC fraction  andspecificity from 69% to 100% (89%). OCB had a diagnostic sensitivity of 37% to100% (85%) and a specificity of 74% to 100% (92%). The comparison of these twoparameters by bivariate mixed model – considering between-study and within-studyheterogeneity and having a statistical power of 99% – clearly showed that thediagnostic accuracy of κ-FLC index and OCB are similar.κ-FLC, or the CSF κ-FLC concentration, also achieved adiagnostic accuracy, which was similar to OCB. However, due to the low number ofstudies the statistical power for these comparisons was smaller than 80% and,thus, insufficient to interpret non-statistically significant results with asmall enough Type II error. For detailed analyses please refer to Hegen et al.The other parameters previously used to determine intrathecal κ-FLC, for example,IFThe single most informative analysis in MS, although not disease-specific, isthe assessment of intrathecal immunoglobulin synthesis, either byqualitative detection of a CSF-unique IgG fraction (using IEF followed byimmunodetection), or a quantitative CSF κ-FLC fraction (using nephelometryor turbidimetry). Conceptually, it seems necessary to determine the locally synthesizedκ-FLC fraction separate from the blood-derived fraction as done for total IgG.The majority of studies used the κ-FLC index2555 orIFκ-FLC.385558 Both approaches considerQalb which is an established marker of theblood-CSF-barrier function and correct for the absolute serum κ-FLC concentration. Few studies usedQκ-FLC.51 Other authors determinedthe absolute CSF κ-FLC concentrations only.5961 As the intra-thecaloriginated κ-FLC fraction is greater than 80% in most CIS/MS patients,29 one mightargue that the contribution of blood-derived κ-FLC to the total CSF κ-FLCconcentration is negligible in cases with intrathecal synthesis. One studyreported that around 15% of CIS/MS patients showed even higher absolute κ-FLCconcentrations in CSF than in serum that proves an intrathecal synthesis per se.κ-FLC in the CSF – similar to immunoglobulins or other proteins – originateeither from the blood by diffusion across the blood-CSF-barrier, or byintra-thecal production under pathological conditions.κ-FLC and CSF κ-FLC concentration and observed a similardiagnostic accuracy. However, the statistical power was below 80% and, thus,insufficient to interpret non-statistically significant results. Therefore, thesuperiority of, for example, κ-FLC index over CSF κ-FLC concentration cannot be excluded. There is evidence of one recent study that specifically addressed thisquestion, separated patients into low and high CSF κ-FLC categories and observedthat κ-FLC index, IFκ-FLC, Qκ-FLC and CSFκ-FLC concentration showed similar diagnostic performance in the high category,but not in the low category with the inferiority of CSF κ-FLC and to some extentalso of Qκ-FLC. One might conclude that the impact of serum κ-FLC andQalb is indeed negligible in patients with highintrathecal κ-FLC synthesis, but probably not in patients with only low ormodest intrathecal κ-FLC production. A very recent large multicenter studyincluding more than 1600 patients also reported that κ-FLC index andIFκ-FLC performed slightly better than absolute CSF κ-FLC concentration. Further studies are required to compare the different κ-FLC measures inpatients with varying degrees of intrathecal B-cell activity, varyingblood-CSF-barrier function and varying serum κ-FLC concentrations, as the impactof grossly elevated serum FLC levels or elevatedQalb has not been sufficiently investigated.This might be of interest also in terms of differential diagnosis, as, forexample, NMOSD sometimes shows considerable blood-CSF-barrier dysfunction, which might lead to higher diffusion of κ-FLC from the blood into CSF andpossibly to false-positive results if only absolute CSF κ-FLC concentrations aremeasured.The above-mentioned meta-analysis compared the performance of κ-FLC index,IFMethods considering CSF/serum κ-FLC concentration and CSF/serum albuminquotient, for example, the κ-FLC index, show a good overall agreement withCSF κ-FLC concentrations, but seem to be superior in cases with low ormodest intrathecal κ-FLC production. or monoclonal  detection antibodies.For the determination of κ-FLC in CSF and serum, nephelometry or turbidimetry iswidely used and have, compared to, for example, enzyme-linked immunoassays, theadvantage that automated single measurements are feasible. It was the perceptionof the panel that nephelometry or turbidimetry is widely accessible. With regardto the assays, κ-FLC can be measured by use of either polyclonal  In contrast, monoclonal assays contain a cocktail of multiple monoclonalantibodies which are each produced in hybrid cell lines and finally mixed together. Due to these differences in the production, certain characteristics inassay performance arise, for example, a higher lot-to-lot variation inpolyclonal assays.65Polyclonal assays contain anti-κ-FLC antibodies that are raised in sheep afterimmunization with a pool of human κ-FLC followed by an adsorption step againstintact immunoglobulin (containing bound light chains), so that onlyκ-FLC-specific antibodies remain in the final antisera.6668 as well as by concordancerates .68 However,these studies contained samples from patients with, for example, monoclonalgammopathy covering κ-FLC measurements ranging up to thousands mg/L.68 Thedifferences observed between the methods occurred mainly at the upper end of theanalytical range.68 These extremely elevated concentrations found in theserum of monoclonal gammopathy patients are not relevant in MS patients withoutthese co-morbidities.There are a few studies that compared the polyclonal (Freelite) and monoclonal (NLatex) assay in serum and found a moderate agreement as determined bycorrelation analyses (with a coefficient of > 0.9) or Passing–Bablokregression (with a slope between 0.74 and 0.99) and κ-FLC index  with moderate agreement.Scarce evidence on assay comparison exists for absolute CSF κ-FLC concentration and reference intervals; however, there are no studies addressing the impact on CSF κ-FLC.The choice of the platform has an impact on serum κ-FLC measurement However, this has to be investigated by further research.Considering the information provided on the platform and assay-specificvariations, it is plausible to assume that κ-FLC index might be less prone tolaboratory variations compared to absolute CSF κ-FLC concentration (due to theuse of CSF/serum κ-FLC ratio resulting in a dimensionless variable). Theabove-mentioned meta-analysis did not show a statistically significantdifference between κ-FLC index across the methods employing monoclonal orpolyclonal antibodies as reagents in multiple platforms clinically available. formula, intrathecal κ-FLC synthesis was still not affected. Anexplanation is probably the small molecular size of κ-FLC of approximately 24kDa compared to the larger size of immunoglobulins, for example, of 150 kDa forIgG.72 Smaller molecules show a higher CSF/serum ratio, and inthe case of artificial blood contamination, the relative increase of CSFconcentration is lower. With regard to OCB, blood contamination reduces thechance of detecting faint bands and might lead to false negative results.κ-FLC in the CSF are by far less susceptible to blood contamination, which canoccur due to traumatic lumbar puncture, as compared to other CSF proteins, forexample, immunoglobulins. A study showed that even in case of a bloodcontamination that led to false-positive intrathecal IgG synthesis in almost 90%of patients as determined by the Reiber Studies investigating CSF stability are missing too.Free light chains are stable in serum samples frozen at −20°C for a storageduration of at least 1 year. Whether thereafter is a relevant change has notbeen investigated yet. Most studies on the biological variation of serum κ-FLC apart from anunderlying disease revealed a small within-subject variationof <10%.76 Whether this small within-subject variation can also beextrapolated to CSF levels in MS has to be determined. Recently, it has beenshown that high-dose corticosteroids resulted in lower serum FLC concentrations;however, CSF levels and, more importantly, the FLC index were not affected.Patient-related factors might impact κ-FLC concentration. κ-FLC in serum mightdepend on renal clearance, that is, higher serum FLC concentrations were foundin older patients with decreased renal function. or if a cut-off is determined to discriminate patients with MS from otherinflammatory neurological diseases. Cut-off points might also vary depending onwhether the focus is to increase diagnostic sensitivity or diagnostic specificity. The impact of laboratory methods on κ-FLC measurements, which again mightinfluence cut-off points, has been discussed above.Cut-off points might depend on the clinical question, that is, whether an upperreference limit is determined in a non-inflammatory control population,Qκ-FLC as well as different formulae to defineQlim κ-FLC23 for calculation ofIFκ-FLC compared CIS/MS patients to heterogeneous controlpopulations, and only the minority used pure non-inflammatory disease controls.Furthermore, most of these studies applied a discriminatory cut-off rather thana cut-off that maximizes either diagnostic sensitivity or diagnostic specificity.The vast majority of studies using κ-FLC index, CSF κ-FLC concentration, Even though this cut-off is in line with those identified by severallarge – partly multicenter – studies,47 it has to be clearlystated that this was an exploratory analysis.For the κ-FLC index, reported cut-off values ranged from 2.4 to 20. In theabove-mentioned meta-analysis, a mean cut-off of 6.1 could be determined. With regard to the different non-linear formulae,23 the smallnumber of studies did not allow any between-study comparisons. There is only one study that compared the performance of all threeformulae within an independent cohort reporting similar diagnostic sensitivitiesranging from 96% to 98% in MS patients and 40% to 44% in CIS patients. A comparison in terms of specificity is still lacking. Due to the smallnumber of studies, no cut-off for QFLC could be determined. At this point, we would like to state that besides laboratory variations,also handling non-detectable CSF values might have an impact on κ-FLC indexvalues. This means that reported cut-off values might be biased by this issue.For a detailed discussion, we refer to Hegen et al.For CSF κ-FLC concentration, a mean discriminatory cut-off to differentiateCIS/MS patients from controls at 0.96 mg/L was observed. Here again, thiscut-off is the result of exploratory analysis and is based only on a limitednumber of studies.There is extensive data on quite similar cut-off values for κ-FLC index.However, multicenter studies using different platforms and assays should beperformed to definitively confirm these cut-offs, and certified referencematerials should be developed.Determination of intrathecal κ-FLC synthesis and CSF-restricted OCB has certainstrengths and limitations . κ-FLC is measured by nephelometry or turbidimetry which is – in contrastto the detection of OCB – an easy, reliable, labour-saving, cost-efficient andrater-independent method.10 The intrathecal κ-FLCsynthesis, for example, by determination of the κ-FLC index, returns a metric result, while OCB status is dichotomous returning either a positive or negative result. The quantitative result of κ-FLC might gain additional utility in theprediction of disease activity in early MS (discussed below).As outlined above, the intrathecal κ-FLC synthesis shows a high diagnosticaccuracy to discriminate patients with CIS and MS from other neurologicaldiseases with a sensitivity and specificity of approximately 90%, similar to OCB.80 or inpatients with the CNS involvement of systemic diseases .However, it cannot be differentiated whether increased κ-FLC measures are theconsequence of an intrathecal IgA, IgM and/ or IgG synthesis. CSF κ-FLC levelsdo not provide information on the clonality of immunoglobulin production anddifferentiation between systemic inflammation with an additional intrathecalinflammation (OCB Pattern III), or an isolated intrathecal inflammation (OCBPattern II) is not possible. This additional information provided by OCB can behelpful in some clinical situations, for example, in patients with monoclonalgammopathies and suspected CNS involvement (i.e. Bing Neel syndrome),82 prior determination ofIgG concentration is recommended so that the appropriate dilution of samples canbe performed and, thus, the same amount of IgG applied for the IEF run.Determination of κ-FLC in the CSF and serum requires a sample volume of at least200 µL (due to the dead volume in the cuvette placed in the nephelometer orturbidimeter). Even though OCB testing requires only a minimum of approximately10–20 µL (placed on the gel for IEF),Determination of intrathecal κ-FLC synthesis might be used as a first-linescreening test in MS. The reflex approach applies two cut-off points and reportsresults in case of clearly negative or clearly positive values. In the case ofvalues between the two cut-off points ( ‘grey zone’), OCB detection shouldfollow as a second step . Discrepancies between intrathecal κ-FLC synthesis and OCB might alsoarise from different cut-offs defining OCB positivity, which is differentnumbers of CSF-restricted bands. Of interest, there are studies reporting that relevant differentialdiagnoses of MS, for example, NMOSD, show lower κ-FLC levels which might fallinto a grey zone . Further studies are needed to investigate patients with low intrathecalκ-FLC levels.The low cut-off should ensure that patients with a negative result have indeed nosigns of intrathecal B-cell activity, while the second higher cut-off shouldunequivocally identify patients with intrathecal B-cell activity. While in themajority of CIS and MS patients the extent of intrathecal κ-FLC synthesis ishigh and the interpretation as a positive result clear, in case of lowpositives, several issues should be considered including analytic and biologicalvariation. The low cut-off will label a certain proportion of samples asκ-FLC-positive in the absence of OCB in CSF. Some cases will be explained byintrathecal IgA or IgM synthesis.60Studies evaluating the financial aspect of a reflex approach found the sequentialuse of κ-FLC as a screening test and if needed OCB as a confirmation test beingless expensive, in terms of reagents, material and personnel as compared to OCBdetection. Further advantages of the reflex approach still include a reductionin turn-around times and faster reporting of results. These cut-off points might also be the rationale for further studies.However, as evidence defining the ‘grey zone’ is still lacking, one mightsuggest, at least for the κ-FLC index, using the lowest and highest cut-offpoints that have been published, that is 2.4 and 20 for the κ-FLC index, respectively.If results on intrathecal κ-FLC are borderline, an evaluation by OCB can helpto clarify the presence of an intrathecal immunoglobulin synthesis, or viceversa. However, until evidence defining borderline intrathecal κ-FLCsynthesis is established, the combination of both tests might be the bestoption at this moment. The majority of studies reported that intrathecal κ-FLC synthesis isassociated with conversion from CIS to MS83 and that the extentof intrathecal inflammation as reflected by the κ-FLC index predicted thetime to conversion to MS as well as disability progression.84 Tworecent studies showed in a multivariate approach considering other alreadyknown risk factors such as baseline MRI lesions that in patients with afirst CNS demyelinating event, high κ-FLC index is an independent riskfactor for early second clinical attack85 and fulfilment of2017 McDonald criteria. These findings fit to previous studies that reported a predictivevalue of IgG index87 and the number of OCB,8789 that is, the extentof intrathecal inflammation, with future MS disease activity. In clinicalpractice, intrathecal κ-FLC synthesis might serve as a predictive biomarkerin MS, that is, it could – together with other surrogate markers such as MRI– identify patients in need of early, highly efficacious disease-modifyingtreatment and, thus, facilitate treatment decision-making.There are only a few studies on the predictive value of κ-FLC index in MS. Anoverview is given in Hegen et al.Determination of intrathecal κ-FLC synthesis should be included into the nextrevision of MS diagnostic criteria as an additional tool to measureintrathecal immunoglobulin synthesis .