Forensic DNA phenotyping (FDP) provides the ability to predict the human external traits from unknown sample donors, directly from minute amounts of DNA found at the crime scene. We developed a MPS multiplex assay, with the aim of genotyping all 41 DNA markers included in the HIrisPlex-S system for simultaneous prediction of eye, hair and skin colours. Forensic samples such as blood, skeletal remains, touch DNA, saliva swab, artificially degraded samples together with individuals with known phenotypes and a set of 2800 M control DNA were sequenced on the Ion Torrent platform in order to evaluate the concordance testing results and the forensic suitability of the 41-plex MPS assay. The panel was evaluated by testing a different number of PCR cycles and the volume of reagents for library preparation. The study demonstrated that full and reliable profiles were obtained with 0.1–5 ng, even with high degraded DNA. The increment of the number of PCR cycles results in an improvement of correctly genotyping and phenotyping for samples with low amounts of degraded DNA but higher frequencies of artefacts were found. The high DNA degradation level did not influence the correct genotyping and phenotyping and the critical parameter affecting the result is the quantity of input DNA. Eye and hair colour was predicted in 92.60% of individuals and skin colour in 85.15% of individuals. The results suggest that this MPS assay is robust, highly sensitive and useful for human pigmentation prediction in the forensic genetic field. Forensic DNA phenotyping (FDP) is a set of innovative genetic tools that allows the age, appearance and biogeographical ancestry (BGA) prediction of unknown perpetrators from various biological traces found at the crime scene. In recent years, this new branch of forensic genetics has gained a great deal of importance for its potential applications as a supplementary investigative tool whenever conventional DNA profiling fails to provide a match with any reference profiles or genetic profiles entered in criminal databases. The FDP outcomes are provided in terms of “prediction”, i.e., are probabilistic, and they can only infer a specific phenotypic feature to a certain degree of probability (probabilistic inference). It must be kept in mind that FDP is not an identification tool but rather an investigative tool that helps identify unknown suspects or to help with non-criminal missing person cases. DNA phenotyping is indicated as a “biological witness” because it acts as a witness that describes the appearance of a person of interest, potentially providing even more accurate information than the human eyewitness does, which is known to be unreliable. One branch of the FDP tool, which is particularly advanced and studied, is the prediction of the appearance, i.e., the externally visible characteristics (EVCs); both because these phenotypic characteristics are determined by a relatively low number of genes, making these the least complex genetic trait to analyse  and becaCurrently, the 41 DNA polymorphisms of the HIrisPlex-S system  represenMost of these studies ,11,12,14In the present study, we describe the development and validation of an MPS multiplex assay, based on Ion Torrent technology, targeting all the 41 SNPs included in HIrisPlex-S system. The MPS panel was designed to maintain the size of the amplicons below 180 bp, in order to investigate the usefulness of the panel with a broad kind of forensic casework sample, including different biological matrices and containing different levels of DNA quantity and quality. n = 5), touch DNAs (n = 5) together buccal swab (n = 5) of same donor used as reference, blood samples (n = 5), skeletal remains  and artificially degraded DNAs (n = 2), produced with a method based on aqueous hydrolysis of the DNA in vitro following the published protocols in [To investigate the effectiveness of the designed panel, a selection of forensic samples  consistiocols in . Regardiocols in ,21,22,232 254 nm for 12 h. The powder for DNA extraction was produced using a Dremel drill applying the lowest speed and collected into vials. The weight of the obtained powder was 0.1 g. The powder was incubated rotating for 24 to 48 h at 37 °C in 1 mL of extraction buffer—Urea in EDTA 0.5 M and 10 µL of Proteinase K 20 mg/mL [The samples were processed in the Centre of Molecular Anthropology for Ancient DNA studies, University of Rome Tor Vergata , set specifically for ancient DNA recovery and processing, whose access is strictly restricted. The samples were UV-irradiated at 6 J/cmThe Centre of Molecular Anthropology for Ancient DNA studies  provided also three blood samples consisting of DNA from an Amhara donor from Asela, Oromia Region, central Ethiopia (AM11) ; one froFor the other selected samples, different DNA extraction and quantification methods were performed. The details of the tested samples are given in Sensitivity study was performed by using the 2800 M  as control DNA, diluted to concentrations of 5 ng/µL, 1 ng/µL, 500 pg/µL, 100 pg/µL, 50 pg/µL and 25 pg/µL.https://ampliseq.com/ (accessed on 7 March 2019)), using the FFPE DNA type option (125–175 bp amplicon range) in order to allow analysis of degraded DNA. Two primer pools were designed to amplify 35 amplicons covering the 41 SNPs.The 41 SNPs of HirisPlex-S systems  were anaThe MPS assay design was evaluated by testing different numbers of PCR cycles  and different reagents volume were used in the library preparation steps. Forty-eight libraries were sequenced in this study, seven of which were prepared in half volume test, i.e., the volume of all reagents used, and the library building was half of that recommended in the protocol. The amount of input DNA, number of PCR cycles and other information about MPS libraries are reported in TM Library Quantification kit  on a Rotor-Gene Corbett 6000 (Qiagen) following the manufacturer recommendations. The MPS libraries were prepared using the Precision ID Library Kit (TFS) according to Ion AmpliSeq DNA and RNA library preparation user guide . The amplification of each primer pool was performed in 10 μL PCR reactions and combined after target amplification to yield a total volume of 20 μL. Thermal cycling was performed on the Veriti™ 96-Well Thermal Cycler  using the following conditions: enzyme activation for 2 min at 99 °C, followed by 99 °C for 15 s and 60 °C for 4 min. After PCR reactions, 2 μL FuPa Reagent was added to partially digest primer sequences (TFS) and incubated for 10 min at 50 °C, for 10 min at 55 °C, for 20 min at 60 °C. An amount of 4 µL Switch Solution, 2 µL DNA Ligase, 2 µL diluted Ion Xpress™ Barcode Adapters were added into 22 µL digested PCR reaction for ligation of libraries with adaptors, and the mix was then incubated for 30 min at 22 °C, 5 min at 68 °C, 5 min at 72 °C. Then the libraries were purified with Agencourt™ AMPure™ XP Reagent  and the final concentration of each barcoded library  was deteTM System (TFS) were used for sequencing the forty-eight libraries of the present study. Thirty-eight barcoded libraries were diluted to 100 pM, pooled in equal volume aliquots and then submitted to emulsion PCR to generate template positive Ion Sphere™ Particle (ISPs) containing clonally amplified DNA. Emulsion PCR (emPCR) was performed in the Ion OneTouch™ 2 Instrument  with the Ion PGM™ Hi- Q™ View OT2 Kit . The template-positive ISPs were enriched on Ion OneTouch™ ES (TFS) and sequenced on Ion Personal Genome Machine™ (PGM™) System by using Ion PGM™ Hi- Q™ Sequencing Kit , two Ion 318™ Chip v2  types and 200 read mode. The remaining ten libraries were sequenced on Ion Gene Studio S5TM , performing the libraries enrichment and chip loading steps using the Ion Chef™  and the Ion 510™ and Ion 520™ and Ion 530™ Kit . An appropriate volume of each manually prepared library, adjusted to 30 pM concentrations, was combined to create an approximately equimolar pooled library for the Ion Chef™  and it was loaded on Ion 520 Chip. Two different Ion Torrent platforms, Ion Personal Genome Machine™ (PGM™) System and Ion Gene Studio S5All raw data were processed by the Torrent Suite (v. 5.0.4/5.10.1) and the reads were aligned against human reference genome (GRCh37/hg19). Coverage analysis was carried out by Coverage Analysis (v.5.0.4.0/v.5.10.0.3) plugin, which provided information about mapped reads, on-target percentage and mean depth of coverage downloadable for each sample library . SequencCertainly, the applied threshold for PMDtool should be considered stringent, preventing obtaining the 50× coverage aimed to obtain a proper “locus call”. Indeed, this value is hard to obtain, whichever is the ancient DNA sample, as the DNA preservation for archaeological specimens is usually mined by taphonomy and the chemical–physical processes occurring in the deposition environment, which is detrimental to the proper preservation of ancient DNA . So far,For performance evaluation of the designed panel, the relative depth of coverage (rDoC) across all target sequences was calculated as the ratio of depth of coverage (DoC) at single amplicon to total DoC of the sample.https://hirisplex.erasmusmc.nl/ (accessed on 15 April 2022), to generate individual predictions of eye, hair and skin colours. Notably, this software is able to generate additional predictions of hair colour shades (light/dark). The model is based on 9466 individuals for eye colour, 1878 and 854 individuals for hair colour and hair colour shades (light/dark) prediction, respectively, and lastly 1423 for skin colour. Prediction results were reported as predictive probability values  for each category. The eye colour prediction result consists of three categories: blue, intermediate and brown; the highest p-value was considered as the predicted eye colour. This tool provides a high discrimination level between blue and brown colour categories, but accuracy is lower for intermediate colour prediction. The hair colour prediction result consists of four categories: blond, brown, red and black. Finally, the skin colour prediction result consists of five categories, based on a dermatological established Fitzpatrick scale [Genotype data of the 41 DNA variants can be uploaded to the easy-to-use DNA Phenotyping web tool found at ck scale  for skinck scale , which aThe main parameter of the two Ion 318™ Chip v2 and Ion 520 Chip run in this study are reported in MC1R gene. The amplicons in this panel were designed to be relatively short (size ranging between 124–174 bp), which are especially advantageous for the analysis of challenging forensic samples. The MPS results , which is the ratio between the coverage of each locus and the overall coverage of the sample, was assessed separately for reference samples and degraded DNA samples across all loci. A uniform distribution in all 41 DNA markers both in reference samples and in degraded DNA samples, regardless of the amplicon size, was found. To evaluate if the input DNA amount can affect the panel performance, the rDoC for non-degraded DNA samples  was evaluated after pooling the samples in two groups according to the DNA input used for amplification, named as follows: DNA input > 0.5 ng and DNA input < 0.5 ng. All loci displayed a good uniformity regardless of amplicon length and DNA input used for amplification , except The trend of rDoC distribution in degraded samples remains quite homogeneous in all loci , except To understand the reasons for the irregular rDoC values shown by the amplicon AMPL7160226302, an in-depth analysis of sequencing data was conducted by Integrative Genome Viewer (IGV) software  comparinThe generated data were processed with HID SNP Genotyper  a software plugin that analyses barcoded samples, and then finds the genotype at positions specified in the bed file. The plugin applies a locus-level quality checks filter to flag possible incorrect data due to one of the following: coverage (COV) is less than twice the standard deviation compared to the mean; percentage positive coverage (PPC), the ratio of coverage from positive strand to negative strand is <30% or >70%; major allele frequency (MAF) ratio of major allele coverage to total coverage is <35% or >65% for heterozygotes and >95% for homozygotes; genotype not valid (NOC) observed when an “NN” genotype is called. It must be highlighted that a flag does not necessarily mean that there is an error but that a detailed revision of that call is required . FurtherThe decreasing amount of 2800 M Control DNA (Promega), ranging from 5 ng to 25 pg, was used as template DNA to test the sensitivity of the designed panel. The scalar amounts of control DNA were amplified at 21, 23 and 25 PCR cycles and, as expected, a reduction in reading depth was observed with the decreasing DNA input. The HID SNP Genotyper plugin (v.4.3.2) was used for the allelic caller and results showed that complete and reliable genotypes could be obtained with an amount of DNA up to 0.1 ng at 21 and 23 PCR cycles . When usp-value 0.836), dark brown/black hair  and lighter intermediate skin . For the libraries prepared with a half reaction volume, the results are consistent with those prepared with a standard volume, except for one no-call in samples with 0.1 ng. However, the no-call involved the amplicon AMPL7160226302, which showed in this assay critical results with less than 100 pg of DNA. Based on these results, for this panel we used in the remaining samples 23 PCR cycles, with the sensitivity threshold set to 100 pg. The prediction of pigmentation traits of 2800 M Control DNA (Promega) was performed using the prediction guide recommendations ,46 and dAll DNA samples were subjected to MPS sequencing using the Ion Torrent technology. The phenotype prediction was performed using the HIrisPlex-S web tool, which allows a simultaneous prediction of eye, hair and skin colour. The appearance prediction was easily obtained for the samples showing complete genotyping profiles, while different considerations should be made for the interpretation of the remaining 26.92% of partial profiles. Notably, the prediction accuracy in case of partial profile is strongly affected by missing genotyped markers and the lack of data could be detrimental to the final prediction, resulting in the loss of AUC  .MC1R variants for hair colour prediction and the HERC2-SLC45A2-IRF4 gene set for eye and hair colour prediction [The HIrisPlex-S system does not exclude the possibility of predicting eye, hair or skin colour for partial profile, unless the missing genotypes are all ediction . p-value of 0.393 for pale skin and for 0.544 for intermediate skin. Likewise, for the sample FG, we expected a dark skin colour but the webtool predicted a dark to Black category . However, these discrepancies in skin colour could depend on the subjective judgment of the researcher who recorded the phenotypic categories of the individual samples at the time of collection. The phenotype prediction is reported in Five buccal samples of individuals with known phenotypes were analysed to test all predictable categories for eye, hair and skin colours obtainable by HIriSplex-S system webtool. The five individuals with known phenotypes were amplified with 5 ng of DNA input and using 25 PCR cycles. The mean amplicons coverage values observed in these samples ranged between 2451 and 6499 . The covIn the present study, we evaluated two artificially degraded DNA together with their non-degraded sample reference. One sample (TS29) with high degraded DI  was amplified with approximately 1 ng of input DNA using 21 PCR cycles, as well as its reference sample (TS26). A full genotype concordant with the reference sample profile was obtained. The subject presents blue eyes, blond/dark blond hair and pale skin. The other artificially degraded DNA sample (TS22) which showed DI not calculable and a low amount of DNA  was amplified using 21 PCR cycles. Partial profiles were obtained with only a few loci correctly genotyped (6/41 SNPs) but with coverage lower than genotyping threshold fixed and therefore no predictions were possible. These results obtained by artificially degraded samples were very relevant, confirming that the high degradation index did not influence the correct genotyping and the critical parameter that affects the result is the quantity of DNA input. In this study, three blood samples from three different modern populations were evaluated. The sample named RC544 belongs to a donor from the Ecuadorian community of African ancestry living along the Rio Cayapas in the Esmeraldas province ,27,28,29The maximum amount allowed by the protocol, in terms of ul, was amplified using 23 PCR cycles because the samples showed inhibitors and a low amount of DNA upon quantification. In detail, the samples AM11 and RC544 were amplified with 1.8 ng and 4 ng of input DNA, respectively, using 23 PCR cycles, while the PK48 sample was amplified with 0.1 ng of input DNA. The coverage obtained for these samples reflected the amount of DNA used in PCR reactions; indeed, the coverage of PK48 samples was 438.3, and unlike the other two samples, had only 42% reads on target and a partial profile was obtained (38/41 SNPs). Despite the lack of three SNPs , the HIrisPlex-S webtool allowed a complete phenotype prediction which for this sample showed an individual with brown eyes, dark brown/black hair and intermediate skin as reported in Six ancient DNA samples were included in this study, even if after DNA extraction and quantification only two samples displayed a DNA amount suitable for library preparation .The first ancient DNA sample, BAS539, was found in the Necropolis Collatina, not far from the centre of Rome and roughly dated between the 1st and the 3rd cent. CE . The MPSThe second archaeological sample, FIDENE, belongs to a hydrocephalic child from a 1995 excavation of a cemetery close to suburban estates close to the ancient town of Fidenae and is dated between the end of the first century and the beginning of the second century DC. The MPS library was prepared by amplifying 55 pg of input DNA using 23 PCR cycles. The mean amplicon coverage was 247.6 and partial profile with five no called loci was obtained for this samples.After MPS sequencing, the sample presented a partial profile with 36/41 SNPs genotyped, the five missing SNPs  were related only to skin category prediction. Despite these missing loci, intermediate skin colour was predicted for this sample, with an AUC loss of 0.014 . An incop-value < 0.5 was observed for each phenotype category. We collected four casework samples, whose identity was already known together with their appearance. The DNA amount used for library preparation ranged between 53 pg to 338 pg and only one sample showed a complete SNPs profile. This sample, named 6916, belonged to a female subject found 18 months after its disappearance in the Po River  in an advanced stage of decomposition. The designed panel was able to predict a subject with blue eyes, and dark blond/brown hair; however, an inconclusive result was obtained for the skin category because a EXOC2 and rs6119471 on gene ASIP were lost. These two missing SNPs affect the hair and skin prediction category; however, the resulting AUC loss was irrelevant for the determination of phenotypic categories (ranging between 0.001–0.002 for the hair category and 0.001 for the skin category). The phenotype predicted agreed with the description of the woman as a subject with brown eyes, and dark brown/black hair, but also for this sample an inconclusive result for skin phenotype was obtained. The remaining three samples displayed partial profiles. The 5880 sample, belonging to a missing woman found after 5 months on the bottom of Lake Garda, showed a partial profile in which the genotypes of two polymorphisms, rs4959270 on gene HERC2 gene) as described in [HERC2 gene), rs16891982 (SLC45A2) and rs12203592 (IRF4) was detected as described in [We observed an unexpected result for sample 5672, belonging to a man found 17 days after his disappearance, whose semi-carbonized corpse was buried in the countryside. For this sample, only a partial profile was obtained, with nineteen SNPs typed despite the library being prepared with 338 pg of input DNA. Moreover, library concentration was low (5.095 pM) and final MPS results showed a low mean depth value of 192.8 and only 23% of the reads on target. The previous STR typing performed on the same DNA sample showed a partial STR profile, displaying a gently slope down, suggesting a mild degradation status for this DNA sample. As the SNPs panel in this study was designed with amplicons sized below 174 bp, the DNA degradation does not seem to be the cause of the partial SNPs profile. Therefore, some not well-identified problems during library preparation were hypothesized for this sample. No predictions for eye colour were obtained because missing the input for rs12913832  that affects the skin prediction category and produced an AUC loss of 0.001. The resulting phenotypes prediction for this sample were brown eyes, black hair and dark skin. Remarkably, for all these samples the predicted phenotypes fully agreed with the expected ones. The last casework sample belongs to a young 15-year-old woman of Bengali descent whose bones were found eight years after her disappearance. This sample showed a very low DNA concentration (0.008 ng/µL) and therefore the maximum DNA amount available for library amplification was 53 pg. As our sensitivity studies clearly showed an increase in allelic drop-out when using less than 100 pg of input DNA. We performed two replicates of library and MPS, in order to consolidate the resulting genotypes. The consensus profile consists of 40/41 SNPs, with no-call at rs6119471 , which would compromise the genetic appearance prediction results. The touch DNA samples showed a DNA concentration ranging from 0.075 ng/µL to 5.487 ng/µL with a degradation index ranging between 0.70 and 2.3 ; in addiAll the samples showed a full profile with concordant genotyping and phenotyping results, except for the reference buccal swab TO06. Despite the DNA input used for the amplification being 2.1 ng and the library quantification being comparable to that of other successfully sequenced samples, after sequencing only 11.5% of reads on target with 43.6% uniformity were found. These results suggest an imbalance between the two primer pools used for amplification. The sample showed a partial profile with 30/41 SNPs correct genotyping, these missing polymorphisms affect all prediction categories with AUC loss ranging between 0.010–0.040 for eye prediction, 0.004–0.019 for hair prediction, 0.005 for shade prediction and 0.005–0.016 for skin prediction . Nonethep-value for the blond and brown categories, 0.416 and 0.452, respectively [Interestingly, the samples TO17 and TO18 showed hair darkening during childhood with an equal ectively . This study aims to develop a multiplex MPS assay to genotype all the 41 SNPs included in the HIrisPlex-S system for simultaneous prediction of eye, hair and skin colour. Several types of challenging forensic samples, different for biological matrix and degradation status, were used to evaluate the performance of the MPS-designed panel. The panel was evaluated by testing different numbers of PCR cycles  and reagent volumes used in the PCR for library building . Note that the use of half volumes for the PCR and library preparation did not compromise the quality of sequencing results, which is comparable to that of libraries prepared according to recommended volumes, amplifying the same amount of DNA.The MPS assay showed a very good performance in terms of sequence quality and coverage. The 41 loci have been uniformly amplified and sequenced in all different types of samples, without any difference between reference and degraded DNAs and regardless of the amplicon’s size. From the analysis of rDoc one amplicon, AMPL7160226302 with rs6119471 displayed a remarkable increase in rDoC value both in amplified reference samples with low input DNA and in degraded samples. After an in-depth review by IGV software, we considered that the high number of reads found is due to a high number of dimers that are formed during the amplification reaction, given the quality and quantity of DNA for these samples. Furthermore, though this locus is repeatedly flagged (COV) in several samples, IGV inspection confirms the genotyping obtained by the plugin in all samples.As assessed by sensitivity test, reliable genotype profiles were observed up to 100 pg DNA input and with 23 PCR cycles. Drop-out started to occur at 50 pg and at 25 pg input DNA, more drops out events occurred for the amplified samples at 23 and 25 PCR cycles. It is interesting to note that the increment of the number of PCR cycles results in improvement of sensitivity or in typing results, but an increase in artefacts was also observed. https://hirisplex.erasmusmc.nl/ (accessed on 15 April 2022)). Eye and hair colour was predicted in 92.60% of individuals and skin colour in 85.15% of individuals. The most frequently missing genotypes were observed for the rs6119471 on ASIP gene, which has an impact on skin colour prediction category for differentiation between African versus non-African. Interpretation of the results of eye, hair and skin colour obtained by the HIrisPlex-S Webtool was performed using the prediction guide recommendations. Brown eye colour was predicted in the majority of cases , blue in eight samples (33%); however, inconclusive prediction (probabilities for each phenotype <0.7) was noted in one sample. For hair colour prediction, the majority of samples showed a dark brown–black colour (52%), followed by blond or dark blond category (28%). In one single sample, red hair colour (4%) was predicted, as well as dark blond/brown and dark brown/brown categories. Interestingly, for one individual we noted a hair darkening during childhood, with equal p-value for the blond and brown category, 0.416 and 0.452, respectively. The skin colour prediction algorithm predicted most individuals  to fall between II, III and II/III  on Fitzpatrick’s Scale. Four individuals (22.22%) belonged to V Fitzpatrick’s category (dark to Black skin prediction). Finally, three samples showed an inconclusive result (highest prediction probability lower than <0.5) based on the skin prediction guide proposed by Chaitanya and colleges.The phenotyping prediction was obtained using the HIrisPlex-S webtool (In conclusion, the study demonstrated the panel’s ability to process low-level or degraded DNA typically found in forensic casework. The results show that full and reliable profiles were obtained with 0.1–5 ng, even with degraded DNA. The tests performed in this study show that the use of half volumes reagent for library preparation is applicable for good quality reference samples, but nothing can be inferred for challenging casework samples, as no tests were performed on this sample’s type.Based on our results, the DNA degradation status did not influence the correct genotyping and phenotyping and the critical parameter affecting the result is the quantity of input DNA. However, we recommend carefully setting the analytical threshold for locus calling and proceeding to a manual review of the results for samples with low DNA quantity and/or quality, preferring, where possible, a replication of the samples in order to avoid data loss.


Araucaria bidwillii, a conifer native to South‐East Queensland, Australia. They are one of the 19 species of Araucaria family found around the world, with the nuts from South America being the most commonly consumed. They are traditionally eaten boiled or roasted. This study aims to profile the sensory properties of bunya nuts with chestnut as a comparator. Since chestnuts do not come from a conifer tree, it is expected that there will be differences. Different methods of preparation are also expected to change the sensory attributes. Representative samples were collected from a variety of locations in South‐East Queensland, prepared and presented to a panel of 14 experienced tasters applying conventional sensory descriptive profiling.Bunya nuts are the seeds of hardness on the first bite than chestnuts and, when chewed, became more crumbly, dry, and grainy. They had a savory aroma and flavor, and roasted samples exhibited a roasted aroma. Bunya nut samples were less sweet than chestnut samples. Differences in the sensory properties due to method of preparation were also observed. Boiled bunya nuts were softer and moister, with lower scores for crumbly and grainy. This research is foundational in providing technical information on the sensory profile of this important Indigenous Australian nut and provides a strong basis to support novel food sector opportunities for the bunya nut as a reemerging food source not only in Australia, but also South America.During training, the panel developed a lexicon of 23 sensory attributes together with definitions and reference. Profiles of the boiled and roasted bunya nuts revealed higher scores for There is an increase demand for local, sustainable, and natural foods. Bunya nuts are native to Australia and are part of the Araucaria family, which includes 19 species that can be found around the world. To the best of our knowledge there is no study characterizing Araucaria nuts in terms of sensory attributes. This study builds a lexicon for bunya nuts and compares to chestnuts. It also shows how different preparation methods affect its sensory attributes, as well as possible future uses in product development. The outcomes might provide information to support studies on Araucaria nuts in other countries. The tree produces cones that fall once a year—during summer—and break open, releasing the nuts  and Chile (Araucaria araucana) also produce edible nut‐like seeds  and starch content   of bunya nuts  in comparison to chestnuts and, second, to explore the sensory impact of two different processing and preparation methods to produce roasted and boiled nuts. Changes in sensory attributes are expected due to different moisture contents of boiled and roasted nuts and also due to Maillard reactions when the nuts are roasted. Conventional descriptive analysis was applied using an experienced trained sensory panel to provide a comprehensive and informative qualitative and quantitative evaluation of the bunya nuts  and during two different seasons . The nuts were washed, dried at room temperature for 24 h, hand selected to remove damaged nuts, sealed in polyethylene bags under vacuum (each bag containing approximately 400 g), and stored (−18°C) until required. A total of 3 kg of chestnuts, equivalent to approximately 200 units, were purchased from Rocklea Markets, Queensland, Australia, and were similarly sealed and stored (−18°C), with each bag also containing approximately 400 g. The bunya nut samples were collected from different regions and seasons to ensure that the natural variation could be captured in this sensory study. A list of samples collected for evaluation is given in Table 2.2The nuts were prepared following typical preparation methods of Araucaria nuts  and the nuts (∼200 g) were brought to boil in 1.8 L of water in a 2.5 L stainless steel pot on a gas cook‐top (Fisher & Paykel). Once boiling, the heat was lowered to obtain a gentle simmer with lid on (slightly ajar) (45 min). Subsequently, the nuts were cooled in cold running tap water (1 min), peeled, directly distributed into coded (three‐digit blinding code) polypropylene sample cups (45 ml) with lids, and stored (4°C) until required for sensory evaluation.2.2.2The bunya nuts were cut in half and the chestnuts were cut in a shallow cross format through the flat side of the husk. This method prevented the nuts from overheating and bursting while cooking. After preheating the UNOX convection oven model XF135 (180°C) for 10 min, the nuts were placed on an aluminum tray lined with baking paper and subsequently heated in the oven (15 min) under humidity (40%). The trays were removed and the nuts were cooled to room temperature, peeled, directly distributed into coded (3‐digit blinding code) polypropylene sample cups (45 ml) with lids, and stored (4°C) until required for sensory evaluation.2.2.3All roasted or boiled nuts were evaluated by the trained panel within 15 h of cooking. Approximately 5 g of cooked nut was presented per sample representing two cut halves for bunya and two nut halves for chestnuts. Samples were removed from cold storage (4°C) 45 min prior to session and served at room temperature. Samples were presented to panelists for assessment on white trays. During formal evaluation, the 12 samples were assessed in quadruplicate and no more than 16 samples were assessed within a 2 h period.2.3Physicochemical analysis of the boiled and roasted bunya nut kernels was performed at an accredited laboratory . The following analyses were performed according to Association of official analytical collaboration (AOAC) methods: protein (AOAC 990.03 and 992.15), crude fat and oil (AOAC 960.39), moisture (AOAC 925.10 & 950.46), ash (AOAC 923.03 and 920.153), sugar profile by high performance liquid chromatography, dietary fiber (AOAC 985.29 & 991.42), and starch (AOAC 996.11 & AACC 76.13). The results are presented as the average of duplicates. The standard deviation was not provided by Symbio Alliance, although this value is always within 10% of variation between repeats as a requirement in this laboratory.2.4Prior to formal panel evaluation, a bench‐top tasting involving five experienced panelists was conducted in order to determine the suitability of samples for presentation to the trained panel, to identify relevant attributes, and to develop sample presentation protocols including palate cleansers.2.4.1The sensory panel was selected externally based on availability of individuals from a pool of panelists who had been previously tested for sensory acuity , dry , and hard to clear   with 16 samples presented per session 2 h) on separate days such that data for one and a third replicates were collected per session over 3 days. During sessions, a minimum break of 45 s was maintained between each sample. Attribute scores were collected using Redjade Sensory Software . Scales (0−100) were used with anchors “none” (0) to “high” (100) for all attributes except for  h on sep2.5Data were exported from Redjade to Microsoft Excel and XLSTAT  was used for product characterization, multivariate data analysis, and to analyze panelist performance. For all sensory attribute scores, minimum, maximum, mean, standard deviation, and coefficient of variation were calculated. Factors and interaction effects were analyzed using mixed model analysis of variance applied on the raw data set for each attribute to determine significant differences. Analysis of variance (ANOVA) was also performed on the sensory scores provided by each panelist to test discrimination power and repeatability of the panelists. A Pearson correlation coefficient was performed for each of the sample sets. Finally, a principal component analysis (PCA) was conducted on the mean scores for all samples to visually observe sample grouping, differentiation and to explore the samples sensory profiles.3The results obtained include the characterization of bunya nuts as well as an analysis of panel performance and robustness of data. Differences in sensory characteristics found between bunya nuts and chestnuts and between methods of preparation are also discussed.3.1The composition of boiled and roasted bunya nuts and the typical composition of chestnuts , coefficient of variation (CV %), and standard error of the mean (SEM) for each of the sensory attributes were calculated to determine panel performance in terms of how the samples were distributed and how the scales were used. This information can also be found in the Supporting Information Table . The 23 F ratios and significance for effects of the 12 samples. Aroma attribute sweet note and flavor attribute savory had the lowest contribution to the discrimination. This could be an indication that the panelists did not perceive much difference among samples for these attributes.Table hardness, dry, and grainy were clear as the scale was broadly used. Except for savory, the flavor attributes were also well differentiated. Differences in aftertaste and mouthfeel were more meaningful for sweet linger, where the scale was used very broadly. However, all of them showed a degree of variation.Most of the samples had high scores for aroma intensity. In terms of texture, differences in There were differences among panelists for each attribute Table , indicathardness, sweetness, and sweet linger, which means the replicates were similar among themselves and the method of preparation was well followed. Hardness depends largely on the roasting method. If the sample is slightly smaller, it will dry faster and probably be harder to bite. Sweetness and sweet linger are related with each other and also varied among replicates. The sensory panel performance and robustness of the sensory data were considered satisfactory and suitable for further data evaluation. Comparison among samples for each of these attributes is showed in the next section of the paper.The rating for each replicate was not statistically significant for most of the attributes Table , except 3.3X and Y axis explain 72% of the variation in the data. The first principal component (X axis) was driven by texture and aftertaste with numbing, drying, hard to clear, crumbly, and hardness having high positive loadings on PC1 and floury negative loadings. Flavor intensity, sweetness, and sweet linger also had high negative loadings on PC1. Chemical, earthy, and herbal aromas and flavors, and earthy aftertaste were all combined with high positive loadings on PC2, whereas savory flavor and aroma, and roasted aroma had negative high loadings on PC2.The PCA bi‐plot with the different attributes and the 12 samples is shown in Figure sweetness, sweet linger, sweet note, flavor intensity, and floury. Bunya nut samples collected in 2020 in Toowomba formed a second group across PC2 with high positive loadings for PC2 and correlation to herbal, chemical, and earthy aromas and flavors. In general bunya nut samples correlated to savory and roasted. Roasted bunya nut samples had slightly positive loadings for PC1 and high correlations to dry, grainy, crumbly, and hard, while boiled bunya nuts had almost zero loadings.The PCA plot suggests that four groups could be formed. Chestnuts were separated from bunya nut samples across PC1, correlating strongly to The third principal component Figure  explaineBunya nuts collected from different regions as indicated by: Blackbutt (BTT); Landsborough (LBH); Bunya Mountains (BMS); Toowomba (TW). Chestnut samples (CH). Processing types indicated as boiled (B) and roasted (R). Year of collection indicated (2019 ad 2020).Bunya nuts are hard at first bite, slightly less hard than Brazil nuts and, during mastication, they may feel crumbly and grainy, similarly to macadamias, although dry, possibly because of its high starch content. They have a savory aroma and flavor, similar to cooked potato, as well as a roasted aroma, similar to toasted nuts and popcorn. Bunya nuts may taste and smell slightly sweet and develop sweet linger when swallowed. Some nuts can show a combination of slightly chemical, herbal , and earthy (as root vegetables) aroma and flavors, ending with an earthy aftertaste. They are slightly hard to clear and leave a drying and numbing, almost metallic, sensation after swallowing.3.4The cob‐web plot in Figure numbing was very low or nonexistent.Both chestnuts and bunya nuts had a sweet note aroma, but chestnuts were much sweeter in flavor (68 vs. 22) and left a strong sweet linger sensation (61 vs. 18) after swallowed. The intensity of flavors and aromas in bunya nuts were not as strong as in chestnuts. In terms of texture, chestnuts were slightly more floury. In contrast, bunya nuts were harder to bite (54 vs. 7), and grainier (49 vs. 8). Bunya nuts were also more difficult to clear from the mouth and teeth and, after swallowing, they left a slightly numbing sensation, differently from chestnuts, where sweetness when compared to sucrose . When the nuts are roasted, Maillard reactions may occur and these reactions are often related to a roasted and sweet aroma, for example chicken and caramel , but rather would be considered a wild‐harvested product, with inherent variation, even when coming from the same tree, forest, or same region.4savory (aroma and flavor), roasted, grainy, crumbly, hard, dry, numbing, hard to clear, and drying, with subtle earthy, herbal, and chemical flavor and aroma notes, related especially to the late harvest samples from 2020 (Toowoomba origin), which may have been caused by the time of harvest. Bunya nuts are less intense in terms of aroma and flavor than chestnuts, and were significantly less sweet in aroma, flavor, and aftertaste. Texturally bunya nuts were less floury than chestnuts but were scored higher for hardness, graininess, drying, and hard to clear. The attribute numbing mouthfeel and earthy aftertaste were also subtle characteristic of bunya nuts that differentiated them from chestnuts. A comparison between roasted and boiled samples suggested that the main differences were related to texture. Roasted samples were scored higher for hardness, dry and drying mouthfeel, crumbly, and graininess. Roasted bunya nuts also scored higher for roasted (aroma) and sweet linger aftertaste.A lexicon containing 23 attributes was established to describe Australian Araucaria nuts (Bunya nuts) in terms of flavor, aroma, texture, and aftertaste. Overall, bunya nuts were profiled sensorially as intensely A. angustifolia and A. araucana species, and also the study on the sensory properties of bunya nuts after being subjected to different processing techniques, such as fermentation and drying and grinding to produce flour. Bunya nut flour may also present unique sensory characteristics and can be further utilized to produce bakery products and extruded snacks. Chemical analysis of volatile compounds and mechanical measurements of texture could also be studied in the future and related to the sensory results found in this study.Bunya nuts can be used as a suitable food source with an interesting flavor and aroma profile as a whole nut. They can be prepared using the two different methods aforementioned. In the future, products developed with bunya nuts can take into consideration the different aspects discussed in this study. Further research may include the comparison to nuts from Jaqueline Moura Nadolny: Data curation; formal analysis; investigation; methodology; validation; visualization; writing – original draft. Odette Best: Investigation; supervision; writing – review & editing. Emma Hassall: Formal analysis; resources; software.The authors declare no conflict of interest.n = 12 samples x 4 replicates x 14 panelists)Table S1 Summary of panelist performance, discrimination power and repeatability determined from an ANOVA model of the sensory data obtained for samples. (n = 12 samples x 4 replicates x 14 panelists).Table S2 Descriptive analysis (Click here for additional data file.


Moreover, as aresult of the antifouling capabilities of the incorporated zwitterions,this enhancement is preserved in bovine serum albumin (BSA) with ahigh protein concentration. The presence of zwitterion facilitatesthe transport of dopamine in the direction of the electrode by intermolecularinteractions such as cation−π and hydrogen bonds. Onthe other hand, polydopamine units attached to the surface form molecularpockets driven by hydrogen bonds and π–π interactions.As a result, the intermediate state of dopamine–analyte oxidationis stabilized, leading to the enhancement of the sensing properties.The importance of neurotransmitter sensing in the diagnosisandtreatment of many psychological illnesses and neurodegenerative diseasesis non-negotiable. For electrochemical sensors to become widespreadand accurate, a long journey must be undertaken for each device, fromunderstanding the materials at the molecular level to real applicationsin biological fluids. We report a modification of diamondized boron-dopedcarbon nanowalls (BCNWs) with an electropolymerized polydopamine/polyzwitterion(PDA|PZ) coating revealing tunable mechanical and electrochemicalproperties. Zwitterions are codeposited with PDA and noncovalentlyincorporated into a structure. This approach causes a specific separationof the diffusion fields generated by each nanowall during electrochemicalreactions, thus increasing the contribution of the steady-state currentsin the amperometric response. This phenomenon has a profound effecton the sensing properties, leading to a 4-fold enhancement of thesensitivity (3.1 to 14.3 μA cm Many of these diseasesare associated with dysfunctions in dopaminergic transmission centersin the brain. In particular, a deficiency in the substantia nigrais typical in Parkinson’s disease and leads to motor dysfunctionssuch as tremors.5 The most potent advantage of theelectrochemical sensors of neurotransmitters and neuromodulators istheir very high temporal (time) resolution compared to fluorescentsensors.5 Moreover, this method of sensingdoes not require the introduction of carefully engineered viral vectors capable of expressing the desired fluorescentmarkers into the tissue.Engineering directedmedical therapies as well as understandingthe neurological and metabolic background of those diseases requiresprecise and inexpensive tools for in situ measurements of dopamineconcentrations in the synaptic clefts and interstitial fluid of neuraltissue.5 which unfortunately does not allow precise imaging of single-neurontransmission and modulation. A potential solution to this problemis the fabrication of sensitive microelectrodes that are able to accomplishthis task.6 Therefore, it is essentialto fabricate miniaturizable electrode materials with a very high affinitytoward sensing a specific molecule, i.e., high sensitivity and selectivity,low limit of detection, and the ability to resist biofouling.However, the most critical drawbackof electrochemical sensorsis their relatively low temporal  resolution,7 They are well-known due to their antifoulingproperties,12 i.e., their ability to resist the adsorption of various biomoleculesand to prevent the creation of biofilm on their surface. The antifoulingmechanisms are generally complicated. Briefly, due to dipole–dipoleand dipole–multipole interactions, the zwitterion-coated surfacehas a high level of hydration with a low ion concentration in thedouble layer. In this case, the adsorption of a protein is thermodynamicallyunfavorable, because there is neither entropic gain through counterionrelease nor enthalpic gain through reduction of the contact surfacearea.12 Therefore, polyzwitterions are potent candidatesfor coatings for biosensors.Polyzwitterions (PZs) are polymers possessing two ionized functionalgroups in each structural unit: one positive and one negative.17 The structure of the resulting polydopamine (PDA)consists of several chemically different structural units arrangedin covalently or noncovalently cross-linked chains.19 Among these, there are single-ringed dopamine (DA) and dopaminequinone (DQ) units containing the primary amine group as well as double-ringeddihydroxyindole (DHI) and indole quinone (IQ) units with aromaticcarbon atoms capable of forming aryl–aryl cross-links.20 PDA has a set of very useful properties due to its capability offorming strong hydrogen bonds and π–π and π–cationinteractions with various molecules.21 It makes PDAa superior adhesive promoting agent,22 complexationagent for chelating metal ions,24 template for molecularimprinting,25 and a functional ingredientof stimuli  responsivecoatings26 and supercapacitors.27Dopamine can be polymerized onvarious surfaces using oxidativepolymerization or electropolymerization.28 and thermoresponsive hydrogels29 by means of oxidative polymerization. However,according to our best knowledge, the synthesis of hybrid PDA|PZ thinfilms by means of electropolymerization has not yet been reported.The platform electrodes for modifications are chemical vapor deposition(CVD)-synthesized boron-doped carbon nanowalls (BCNWs). They constitutea fractal structure30 of diamondized, verticallyoriented sheets of graphene.31 The structuralfeatures of the bare BCNW and functionalized BCNW|PDA|PZ electrodesare investigated via scanning electron microscopy (SEM), Fourier transforminfrared (FTIR) spectroscopy, and X-ray photoelectron spectroscopy(XPS) as well as nanomechanical testing. We have manifested the significantpotency of BCNW electrodes in electrochemical sensing,33 attributed to the fast charge transfer kinetics and surface conductivityresulting from the interplay between sp3 and sp2 phases.In this work, zwitterionic sulfobetainemethacrylate (SBMA) isincorporated into the PDA matrix utilizing a one-step electropolymerizationstrategy. Analogous chemistry was applied for the synthesis of antifoulingmembranes35 Electrochemical investigations supported by density functional theory(DFT) computations show that pristine the BCNW exhibits Case 4 behavior,but application of the PDA|PZ coating shifts it to Case 3. Finally,we show that this phenomenon is responsible for enhancement of theelectrode sensing properties in a neutral environment and in bovineserum solution.BCNWs can be thought of as an array of micro(nano)electrodes.There are several cases describing the electrochemical response dependingon the topology of the diffusion fields. In particular, Case 3 isan array with partially overlapping diffusion fields and Case 4, withcompletely overlapping diffusion fields resulting in a response almostidentical to that of an ideally planar electrode.36 Boron doping allowsfor the formation of sharp-edged, flat, and up to 3 μm longcarbon walls31 with superior electrochemicalperformance (k0 = 1.1 × 10–2 cm s–1 and ΔE = 85 mV).On the contrary, undoped CNW samples do not present highly developedmaze-like structures with high surface area impairing fast kineticsat the multiple redox centers (k0 = 3.7× 10–3 cm s–1). PristineCNWs revealed a significantly narrower electrochemical potential windowwith irreversible oxidation (+0.83 V) and reduction peaks (−0.50V).31The B:CNW growth mechanism is based on the joint chemistry of manyspecies in plasma, not only limited to CN–, HyCNHx, BH–x,and CH+x radicals.37 show that the peaks correspond to the redoxcouples as follows: the far cathodic peak to dihydroxyindole (DHI)–indolequinone (IQ), the middle one to leukodopaminechrome (LDC)–dopaminechrome(DC), and the last anodic one to dopamine (DA)–dopamine quinone(DQ), respectively. The current densities of each peak gradually decreasewith consecutive cyclic voltammetry (CV) scans until very small currents are reached. However, when zwitterionic monomeris introduced to the electrolyte , thedeposition characteristics are altered. First, only two redox couplescan be seen at −0.15 and +0.35 V, and second, they are shiftedwith respect to those in pure PDA deposition voltammograms.Both the PDA and PDA|PZ coatingswere electropolymerized in argon-purged 1× Tris buffer (pH 7.4)containing 5 mM dopamine with or without an excess of 500 mM SBMAmonomer. Significant differences between the deposition processescan be observed on cyclic voltammograms depending on the presenceof the additional zwitterionic monomer 1A,B. In 3)4 groups can interact with the aromatic ringsof the PDA, which leads to the withdrawal of the electron cloud fromthe ring.38 Therefore, further oxidationof DHI to IQ is energetically more favorable, and the peak shiftsfrom −0.25 to −0.15 V. Partial justification of thosephenomena are provided with the FT-IR spectra and ESP calculations(see further answers). It is foreseen that zwitterions could act catalyticallyfor the DC isomerization to DHI. However, the detailed mechanism ofthis catalysis is unknown to us. Presumably, the intermolecular interactionbetween PDA and PZ leads to the stabilization of the quinone–methideintermediate, which occurs in living organisms (as described in ref (40 (DFTB3LYP). This hypothesis would also explain the lack of the middleCV peaks corresponding to the LDC/DC pair in comparison to the purePDA electropolymerization as all the DC would quickly isomerize toDHI.Intermolecular electrostatic and π–cationinteractionsbetween PZ and PDA are responsible for the shift of the leftmost DHI/IQpeak toward more anodic potentials. Electrostatically positive N(CHd in ref ) and als41 or from an external solution) in the region between zwitterionicstructural units.42Moreover, the current magnitudes are two times smallerbut do notdecay as fast with consecutive scans. This observation indicates thatthe hybrid coating is more conductive than PDA, which has the utmostimportance in sensing applications. Higher conductivity could originatefrom the increased concentration of ionic charge carriers  strongly resemble pristinePDA curves. In those cases, three pairs of redox peaks are presentand the current gradually diminishes with consecutive cycling until50 μA cm–2 is reached. However, for higherPZ contents starting from a 5:100 ratio , currents do not plummet to very small values after50 cycles. Simultaneously, the second oxidation peak correspondingto the LDC → DC reaction is smaller in comparison to others.It presumably originates from the higher contribution of π–cationinteractions catalyzing chemical steps in the mechanism. Nevertheless,both CV curves and electrochemical impedance spectroscopy (EIS) spectraclearly indicate that, after deposition, the electrochemical activityis higher with increasing PZ content . Low-rate CVs for the 5:100 PDA/PZ ratio show both oxidation andreduction peaks, but the oxidation peaks are flattened for higherrates. Moreover, Randles slope (H) rises with increasingPZ content, but the linearity is weaker for lower ratios. From thoseresults, it can be inferred that the kinetics of charge transfer isgenerally slower at lower PZ contents. Therefore, choosing the highestpossible PZ content limited by solubility (5:500) is optimal for maximizingthe sensing performance of the material.Additionally,a further study of the electrodeposition CV curvesfor varying PZ content was performed to verify the effect of PZ onthe electropolymerization mechanism. At the lowest PZ content, they. Then, several topography scans were conducted on the BCNW areaof the specimen and on the BCNW|PDA or BCNW|PDA|PZ area. Each pointon the histograms  correspondsto the average of the single topography scan. When one calculatesthe average of those points for pristine and coated BCNW and subtractsthe values, the thickness of the hybrid PDA|PZ coating could be obtained,and it is equal to 51 nm. The thickness of the PDA solely was equalto 6 nm; however, this value is in the experimental error and, therefore,gives only a rough approximation.The thickness of thecoating was measured using AFM recorded atthe edge of pristine and coated BCNW  and aliphatic(3000–2800 cm–1) C–H bonds of thePDA44 and SBMA units.45 Third, a band at 1720 cm–1 correspondsto C=O stretching vibrations of carbonyls in quinone-rich unitsof PDA46 or SBMA methacrylate parts.45 The low intensity band at 1650 cm–1 could correspond to C=C stretching modes in some indole ringsin the PDA47 or unreacted adsorbed methacrylates.48 The small band at 1450 cm–1 is likely to be due to O–H deformation or C–N stretchingvibrations.The chemical structure ofthe coatings was investigated by FTIRattenuated total reflection (ATR) spectroscopy 1C. While–1 corresponding to S–O stretchingand one at 1030 cm–1 related to C–O stretchingmodes.45 The signals in this range aresignificantly higher than in the case of pure PDA. The most probableorigins of the variety of small bands in the range of 720–960cm–1 are different modes of C–H groups suchas bending, scissoring, and possibly C–S stretching.49 Those fingerprint bands occur only in the hybridcoating and therefore should come only from zwitterionic componentsof the coating. A similar set of bands, although slightly shifted,was observed in our previous work in a hybrid Nafion-co–PZcoating for glucose sensing applications.50The evidence supporting the idea of the zwitterionbeing incorporatedinto the coating includes two typical strong bands: a relatively broadone at 1160 cmFigure S1). Generally, there are no substantialdifferences in the whole range of the spectrum except the C–Hbond region (inset). Several C–H bands in the range between2750 and 3150 cm–1 are diminished and shifted afterdeposition of the PZ. Considering that most of the C–H bondsare present in PZ, not PDA, these results suggest noncovalent π–cationinteractions between tetraalkylammonium groups of PZ and aromaticrings of the PDA. Moreover, those changes support the fact that PDAundergoes cyclization to the indolequinone double-ringed units.These bands are also visible on the sample of the pristinePZ coating, C–O andC–N bonds (285 eV), C=O bonds (287–288 eV), anda broad signal of π–π satellite distortions at289–292 eV.The pristine BCNW does not contain sulfurcompounds. However, XPSinvestigations show that PDA contains some number of sulfates, mostprobably originating from trace amounts of sulfates in the Tris buffer.In the case of PDA|PZ, those signals are amplified due to the presenceof sulfonate groups in the SBMA monomers incorporated into the structure.29 in the PDA|PSBMA composite, wherePDA served as a noncovalent cross-linker. In the next section, nanomechanicalstudies are conducted to verify this hypothesis more rigorously.Several conclusions were drawn from the FTIR and XPS investigations.First, polydopamine is deposited onto the surface of the nanowalls,and SBMA zwitterions are incorporated into the PDA structure, mostprobably through noncovalent bonding involving π–cationinteractions. A similar structure was proposed recently by Zhang etal.Table S1). PurePDA increases the Young’s modulus ca. 2 times, while hybridPDA|PZ increases it up to 7.3 times. Moreover, the slope of the time–deformationcurves is the lowest among the tested electrodes, indicating the increaseof the rigidity of the nanowalls by PDA|PZ. Lastly, the final depthof the indenter in this experiment is the smallest, i.e., 376 nm incomparison to 698 nm for pristine BCNW, and the contribution of plasticdeformation during the indent is the highest for the hybrid coating.These observations strongly suggest that the hybrid coating has morecross-links in the polydopamine backbone than PDA. They are presumablycaused by electrostatic interactions between amphiphilic PZ moleculesand π–cation interactions. It stands in agreement withthe anticipated method of the incorporation of zwitterionic monomersinto the coating during electropolymerization.28The AFM-estimated thicknessof the PDA|PZ coating is equal to 51 nm and that of the PDA coatingis only 6 nm. However, the second value is in the range of the experimentalerror and, therefore, is only approximate. For a better understandingof the relationships between the chemistry and surface propertiesof the functionalized BCNW, nanoindentation studies were performedin static, scratch, and impact modes 3. The lo60 This feature is beneficial for biosensors workingin fluid-flow conditions, where abrasion and wear through fluid frictioncould cause damage over time.The effect of increased hardness is also manifestedin scratchtests 3C–D.For61 A significantlydifferent impact response is manifested by the functionalized BCNW.Instead of the swelling, there are oscillations around the axis ofzero deformation regardless of the number of impacts. The interpretationof this behavior is that after, coating the BCNW, the inner spacesbetween the walls are smaller or diminished completely so that thereis no place for swelling. In other words, the nanowalls are forcedto oscillate between their equilibrium positions because the coatingchanged the geometry and precluded the swelling. This observationprovides further confirmation that PDA and PZ cause a profound alterationof BCNW surface properties, which is also revealed in the electrochemicalperformance.Impact testing reveals severalimportant correlations between themechanical properties and structure of the functionalized BCNW electrodes.The bare BCNW exhibits a swelling behavior after each impact, whichis seen in –2) are present in this range. The PDA-functionalized electrode exhibitsoxidation and reduction waves originating from redox reactions ofcatechol/quinone pairs that are abundant in most of the structuralunits of PDA.62 However, those currents are significantlysmaller for the BCNW|PDA|PZ electrode. This observation stands inagreement with the FTIR and XPS investigations, because a large numberof molecules in the PDA|PZ coating are zwitterionic units, not PDA,and they do not possess reactive catechol/quinone redox pairs.Cyclic voltammograms (CVs)of the pristine and functionalized BCNW electrodes in neutral (1×Tris) electrolyte are shown in 2 current densitiescan be observed. The high electrochemical activity stems from thestructure of nanowalls and was thoroughly studied in our previousworks.36 Briefly, due to boron doping, they havehigh surface conductivity, and due to the interplay between the sp2 and sp3 phases, high charge transfer rates areachieved.Investigations of the processes occurring at theelectrode/electrolyteinterface were performed by measurements in a solution containinga 5 mM ferrocyanide redox pair. CV curves for pristine and functionalizedBCNW electrodes are given in 2, presumably due to adsorption ofnonconductive products of dopamine oxidation. Interestingly, whena hybrid PDA|PZ coating is deposited, the decrease in the redox responseof the ferroxyanides is smaller (40% of the bare BCNWs) and two redoxpeaks are preserved. This supports the hypothesis posed in previoussections that PDA|PZ results in higher surface conductivity than purePDA.After electrodeposition of PDA solely, there is asignificant lossin the redox response of ferrocyanides; current densities plummetto less than 0.1 mA/cmTable S2. In the case of pure PDA, both the realand imaginary impedances are significantly higher with respect tothe pure BCNW. More precisely, there is a huge (80×) increasein charge transfer resistance Rct andWarburg element magnitude Aw (14×),indicating the formation of a coating with low conductivity. However,in the case of the hybrid coating, the changes in impedance data arerather mild (3× increase of charge transfer resistance and 2×increase of the Warburg element magnitude), indicating the formationof the coating, but have less blocking than PDA solely. The kineticconstant k0 can be calculated on the basisof the charge transfer resistance from the equation:63R is the gas constant, T is the temperature, F is the Faradayconstant, n is the number of electrons involved inthe reaction (n = 1), α is the charge transfercoefficient, kred and kox are kinetic constants of the reduction and oxidationsemireactions in the redox couple, and finally Cox (0) and Cred (0) are the surfaceconcentrations of the redox active species. The simplification isbased on the fact that α ≈ 1/2, Cox (0) ≈ Cred (0), and kred ≈ kox due to the high degree of symmetry between oxidation and reduction.Similarly, the apparent diffusion coefficient D canbe calculated from the value of the Warburg element according to 63Dox and Dred are diffusion constants of the oxidatedand reduced agent of the redox (ferrocyanide) couple. The simplificationis based again on the symmetry between reduction and oxidation masstransport so that Cox (0) ≈ Cred (0) and Dox ≈ Dred ≈ D. Lastly, theaveraged double layer capacitance Cdl canbe computed using the Brug formula , butthe overall reversibility is increased. Nevertheless, it is stilla mixed diffusion/kinetics-controlled process. The hypothesis explainingthis behavior is as follows. The PDA|PZ coating decreases the activesurface area of the electrode and induces specific changes in thesurface geometry. As a result, the shape of the diffusion field aroundthe nanowalls is shifted from heavily overlapped to moderately overlappeddiffusion layers. In other words, the behavior of the BCNW after modificationchanges toward a microelectrode array, leading to the creation ofa steady-state response.35CVs of the bare BCNW and BCNW|PDA|PZ recordedwith different scanrates are shown in 65  and the same values calculated via fitting accordingto the Randles-Ševčík formula . The component B associatedwith the surface-bound reactions is close to zero and negligible inthis case.Another piece of information about the structure ofdiffusionalfields can be drawn from the nonlinear fitting of the current densityvs scan rate dependence 4I. Two f formula 4G–H (0.365 This reduction of peak separation is attributedto the fact that the hexaammineruthenium couple has a lower reorganizationenergy than the ferrocyanide couple and therefore can be treated asan OSET (outer sphere electron transfer) redox pair.70 The Randles-Ševčík slope obtainedfrom the oxidation peaks of the CV curves is equal to 35.5 μAcm–2 mV–1/2 s1/2 forpristine BCNW and 39.5 μA cm–2 mV–1/2 s1/2 for BCNW|PDA|PZ. Considering the almost pure diffusioncontrol (in contrast to the mixed control of ferrocyanides), it isanother suggestion that the surface of BCNW|PDA|PZ provides a moremicroelectrode-like geometry of diffusional fields for redox reactions,resulting in higher current densities. Asymmetries between the oxidationand reduction slopes can be attributed to the presence of nonzerobackground currents at the cathodic part of the potential window  after modification, which is a similar behavior to the ferrocyanidecouple. These observations strongly suggest that dopamine–dopaminequinone redox reactions are inner-sphere electron transfer (ISET),confirming the well-described mechanism.72 Lastly, log–log plots of the current density vs scan ratewere calculated according to 73A similar methodology was applied to verify the interactionsofdopamine with the surface of the pristine and the functionalized BCNWelectrodes.  couples 5I,J. The25 e.g., in the detectionof proteins,74 nitroaromatic explosives75 and insecticides.76Considering a high potency of BCNW towardsensing and the specific electrochemical properties acquired aftermodification with PDA|PZ, a set of dopamine sensing experiments wasconducted. This particular choice of analyte was motivated by thefact that the polydopamine moieties linked to the carbon surface couldincrease the selectivity via the creation of hydrogen and electrostaticinteractions with a chemically similar analyte, dopamine. This mechanismis commonly utilized for molecular imprinting applications with PDAas the template,65 However, while a pristineBCNW electrode exhibits a symmetric current pulse, BCNW|PDA|PZ increasedthe current plateau at potentials lower than the pulse. In other words,zwitterionic modification reduces the onset of the dopamine oxidationpotential. Simultaneously, the slope of the calibration curve is 4.6times higher, while the limit of detection decreases from 505 to 89nM. Moreover, the linear range is slightly widened in both lower andhigher conentrations .95 As a result, the chemical moietybeing sensed in BSA is not pure DA but DA–albumin. Accordingto the standard theory of voltammetry, the current–concentrationslope depends on the electroactive surface area, diffusion coefficients,and kinetic parameters.65 Therefore, thesensitivity decreases with higher dopamine concentrations either dueto blocking of the surface by DA–albumin or due to transportinhibition by the albumin adjoint. Regardless of the mechanism change,at a lower concentration range, the limit of detection remains verylow, 195 nM.Furtherinvestigations include dopamine sensing in an environmentreflecting body fluid conditions, particularly in bovine serum witha high albumin concentration (2 wt %). Dopamine oxidation DPV peaksare still present and increase with the concentration of dopamine.However, the prepeak current plateau plummets in contrast to thatof the 1× Tris buffer solution, and the calibration curve hasa different shape 6H. Two l–2) and started to decrease after the sixth day. This behavior canbe ascribed to the protective role of zwitterion preventing the adsorptionof albumins.59Dopamine sensing was tested for interference byseveral other electroactive,physiologically abundant compounds such as glucose, glycine, ascorbicacid, and paracetamol. Interferents were subsequently added to theelectrolyte in 3 times higher amounts than dopamine, i.e., 60 μM.Despite the significant excess of interferent, dopamine oxidationpulses were observed 6I in a sThis section is devoted to the elucidation of the intermolecularinteractions between the zwitterion (SBMA) incorporated into the PDAstructure and the analyte, dopamine (DA). Particular focus is putinto finding the molecular mechanism explaining the experimentallyobserved shifts in the diffusion fields after application of the PDA|PZcoating. Therefore, a series of geometry optimizations was performedfor different spatial arrangements along the zwitterion molecule andelectrostatic surface potential maps (ESPs) are plotted for each step.The idea was to simulate the transport of dopamine analyte towardthe electrode in the presence of zwitterion 7.96 Eitherway, a translation toward the polyzwitterion alkyl chain is thermodynamicallyfavored.The initial contact 7A of the97 At the last step, as the dopamine is translatedfurther, another hydrogen bond is formed between the catechol andmethacrylate oxygens with a length of 2.84 Å . The 6 h process resultedin thick vertically aligned carbon nanowall surfaces (d ∼ 3 μm) with a high content of the diamond phase.30BCNWelectrodes were manufactured on p-type (100) Si wafer plates utilizinga microwave plasma-enhanced chemical vapor deposition system  as reported elsewhere.50 voltammetry cycles were used for all electropolymerization experimentswith a 20 mV/s scan rate and at the potential range from −0.5to +1.0 V vs. Ag|AgCl|3 M KCl. In the case of pure PDA deposition,the solution consisted of 1× Tris and 5 mM dopamine and was purgedwith argon 15–20 min before starting the deposition. In thecase of coelectropolymerization, 500 mM SBMA monomer was also added.The pH for deposition was adjusted to 7.2 by adding 1 M NaOH. An MP-103hand-held potentiometric pH meter was used to control the pH value.


A ewe’s diet in the last period of gestation can modify the immunoglobulin G composition of the colostrum and consequently the health status of lambs. This study aims to determine the role of hazelnut skin on the immunological colostrum quality and the passive immunity transfer in newborns. The results show that hazelnut skin supplementation in the diet positively affects the immunoglobulin G composition of the colostrum. The experimental trial underlines that the use of by-products in livestock feeding provides a paramount opportunity to create a circular economy system with health benefit on farmed animals.Passive immunity transfer has a pivotal role in newborn lambs, where the colostrum represents the primary source of immunoglobulins. This study hypothesized that the high content in polyphenolic compounds, mono- and poly-unsaturated fatty acids, and vitamin E of hazelnut skin affects blood and colostrum immunoglobulin G (IgG) concentration and related gamma-glutamyl-transferase (GGT) and lactate dehydrogenase (LDH) levels in sheep and their lambs. In the last 45 days of pregnancy, ewes were divided into a control (CTR) and a hazelnut skin supplemented group (HZN). Blood and colostrum were collected from ewes and lambs before the first suckling, at 24 and 48 h after birth, then IgG concentration, GGT and LDH activity levels were measured. IgG concentration in the colostrum and in lamb’s serum were significantly greater in HZN than CTR. No significant difference was detected for ewe’s blood. A significant positive correlation was found between IgG and GGT in lambs’ serum and colostrum, between IgG and LDH, as well as between GGT and LDH in lambs’ serum and colostrum. Our results suggest that hazelnut skin supplementation influences IgG colostrum concentration, with improved immune passive transfer to the suckling lambs. The transfer of maternal derived immune factors is confirmed by the GGT and LDH enzyme activity levels. Sheep farming for meat production represents a significant economic opportunity for the livestock sector. The lambs of meat sheep breeds  are exclusively fed with maternal milk until weaning at 60–90 days of life. As a result, they are characterized by higher birth weight and better growth performances than dairy sheep lambs. In addition, the natural suckling lamb feeding system ensures better animal welfare and meat quality compared to the artificial milk feeding regime . The lamThe acquisition of passive immunity by early ingestion of maternal immunoglobulins from the colostrum is critical for newborn survival. The ingestion of the colostrum should occur within the first 12–24 h after birth, as intestinal absorption of the immunoglobulins rapidly decreases . InadequThe immunoglobulins G (IgG) are the primary markers for evaluating passive immunity transfer to newborns ,9,10. ThSeveral studies have investigated the relationship between colostrum-mediated immunity and the expression of specific blood serum enzymes, such as gamma-glutamyl-transferase (GGT) and lactate dehydrogenase (LDH) ,19,20,21The quantity and quality of the colostrum is influenced by the health status and diet of ewes during the gestational period . Some auCurrent evidence shows that by-products of fruit and vegetable processing can be used in farm animal nutrition as functional feed ingredients to improve food product quality . The usepost-partum period. Specifically, this work analysed IgG, GGT and LDH levels in both the serum and colostrum of ewes and lambs following different diets (with and without hazelnut skin inclusion). The second objective was to investigate the relationship between IgG concentration and GGT and LDH activity levels in blood and colostrum to assess their use as predictors of passive immunity.This study aimed to evaluate the effect of hazelnut skin on the immunological colostrum quality and immunity status of Biellese ewes and lambs during the The project was approved by the Italian Ministry of Health, General Directorate of Animal Health and Veterinary Pharmaceuticals, Office VI .2  pens and kept under standard management and environmental conditions, in compliance with the European Directive (Council Directive 98/58/EC) on the minimum standards for the protection of animals bred or kept for farming purposes. Animal health status was monitored throughout the experimental period. Only singleton delivery sheep were selected. Sheep with clinical symptoms in the peri-partum period were excluded from the study. At the end of the lambing time, each group included eight ewes and eight lambs.The study was carried out at the Teaching Farm of the Department of Veterinary Science . Twenty multiparous Biellese ewes at second and third parity, aged 18–36 months, with an average live weight of 75–80 kg and body conditions score (BCS) equal to 2.75–3.25 were estrus-synchronized using 60 mg medroxyprogesterone acetate intravaginal sponges  for 14 days, followed by an intramuscular injection of 125 µg cloprostenol sodium  at sponge removal ,47. The ®, Norel & Nature Nutrition—Madrid, Spain). The hazelnut skin group (HZN) received 1.0 kg of the same mixed hay and 0.5 kg of a second experimental pelleted concentrate, in which the rumen protected fat was replaced by hazelnut skin. This byproduct was collected from a local producer  and it was obtained from the kernel’s separation after the roasting process. The concentrate contained (%): barley (27.3), maize (27.3), hazelnut skin (27.3), and soybean meal (13.6). Both experimental concentrates also included (%): calcium carbonate (2.0), sodium chloride (1.0), precipitated dicalcium phosphate dihydrate (0.5), sodium bicarbonate (0.3), magnesium oxide (0.2), and mineral-vitamin premix (0.5). The proximate compositions of experimental feedstuffs are presented in The experimental diets were formulated following NRC recommendations . During The experimental concentrates and hay were offered individually, since all ewes were kept at individual feeders, twice a day (at 8.00 a.m. and 4.00 p.m.). The established amounts of hay and concentrate were weighed before being distributed and were completely consumed by all of the animals.L) and was estimated according to National Research Council equations [Feed ingredients were representatively sampled and stored until analysis. The samples were ground with a cutting mill to pass a 1-mm screen sieve . AOAC procedures were used to determine dry matter (DM), ash, crude protein (CP), acid detergent fiber, and acid detergent lignin (ADF and ADL) . Ether equations .The FA composition of feedstuffs was assessed using a combined direct transesterification and solid-phase extraction method as described by Alves et al. . A detaiFA daily intake (g/head) of individuals and groups was estimated considering the daily dry matter intake (DMI) and the analytically determined FA composition of each feedstuff .The contents of total extractable phenols (TEP), non-tannin phenols (NTP) and condensed tannins (CT) in hazelnut skin were determined according to Iussig et al. . Total tAll analyses were performed in duplicate.g for 10 min. The serum was transferred into Eppendorf tubes and frozen at −20 °C until analysis.Newborn lambs were allowed to be licked and dried by their dams and then the lambs were weighed, sexed, and ear-tagged within 1 h after birth. Blood samples were collected in ewes and newborn lambs before suckling at birth (A), and then at 24 and 48 h after birth . Lambs were also blood sampled at 10 days after birth (D). Blood samples were collected by jugular vein-puncture and transferred into a 9-mL blood tube . Blood samples were maintained at room temperature for 30 min for blood clotting and then centrifuged at 1500× Colostrum samples were obtained within 1 (A), 24 (B), and 48 h (C) after parturition. Samples were collected by hand-milking into 20-mL plastic tubes 10-mL from each udder, inverted 8–10 times to mix for thorough and accurate homogenization, and stored at −20 °C until analysis.g for 15 min to remove fat and sediments. The supernatant was then centrifuged at 18,000× g for 1 h and the intermediate layer was analysed [Serum and colostrum samples were labelled with the animal identification number and date of collection and transferred to the “Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia ermediate layer was aRomagna”. Colostrum samples were thawed and centrifuged at 4000× analysed . Serum sBlood serum electrophoresis was carried out with a HYDRAGEL 30 Beta1-Beta2 protein electrophoresis kit  using the HYDRASIS and PHORESIS software  ,59.Colostrum total protein was determined using the biuret method . ProteinThe GGT and LDH activity levels  were determined by a biochemical automatic analyzer IL650 .All statistical analyses were performed using R software version 4.0.3 (R Core Team 2020). Differences in dry matter, fatty acids and polyphenols intake between the two experimental diets were analyzed using a mixed model for repeated measures over time considering the effect of dietary treatment, the effect of sampling date, and their interaction. Results are reported as LSMeans.p < 0.05.A descriptive analysis of IgG, LDH, and GGT concentration in serum and colostrum was calculated using means and standard deviation. The differences in IgG, LDH, and GGT by the CTRL and HZN group were graphically evaluated through strip charts, using the ggplot R package . A multihttps://CRAN.R-project.org/package=PerformanceAnalytics; accessed on 4 May 2021).A correlation analysis was carried out to evaluate the relationship among IgG, LDH, and GGT concentration in serum and colostrum, using the PerformanceAnalytics R package (p < 0.001) between the two groups of ewes in the two periods for the intake of all individuals and groups of FA from the diets. In particular, the HZN group showed a higher intake of monounsaturated FA  and polyunsaturated FA , as well as lower intakes of saturated FA  when compared to the CTR group.The effects of dietary hazelnut skin in ewes’ diet on DM and FA intake during gestation and lactation periods are shown in The amounts of TEP, NTP, TT, CT, and HT ingested daily by the ewes of the HZN group were equal to 48.07, 19.03, 28.24, 3.29, and 24.94 g/head × day, respectively (data not reported in the table).The interaction between dietary treatment and sampling date (DT × SD) was not significant for the considered parameters.IgG concentration (g/L) in sheep and lamb’s blood and in the colostrum at each sampling time are reported in The trend of IgG concentration in sheep was constant across sampling times in both groups A. HoweveThe pattern of IgG concentrations in lambs’ serum is comparable between the two groups B. The loConsidering the colostrum, IgG concentration was clustered at lower values for the CTR group, while in HZN it was more variable but more frequent at higher values C. At lamRegarding the enzymes, the GGT activity level was equal to 50 ± 13 UI/L in the CTR group and 42 ± 16 UI/L in the HZN one, while the LDH activity level was 1077 ± 166 UI/L and 1132 ± 182 UI/L in CTR and HZN ewes, respectively. In the colostrum, GGT concentration was around 10,533 ± 1103 UI/L in the CTR group and 10,291 ± 1275 UI/L in the HZN one, while LDH was 973 ± 213 UI/L and 1024 ± 195 UI/L in CTR and HZN group, respectively. In the newborns, the GGT activity level was highly variable with 755 ± 970 UI/L and 1076 ± 1517 UI/L in the CTR and HZN group. Finally, mean ± SD lamb’s LDH activity level was 1503 ± 412 UI/L and 1403 ± 362 UI/L in CTR and HZN newborns.p < 0.05). A strong positive correlation was detected between IgG concentration and GGT activity level in lambs , while a minimal correlation was found between IgG and LDH . GGT and LDH also had a moderate correlation in newborns . A significant correlation was also detected between IgG concentration and GGT activity level in the colostrum , whereas the GGT-LDH and IgG-LDH correlation was moderate   .To the best of our knowledge, the present study assessed for the first time the effect of hazelnut skin integration in sheep diet on the immunological quality of colostrum and passive immunity transfer to newborns. The results showed that the inclusion of hazelnut skin in the ewes’ diet during the peri-partum period significantly increased the IgG concentration of the colostrum and lamb’s serum. The IgG concentration was significantly correlated with GGT and LDH activity levels.c9) and PUFA, such as C18:2n6 (one of the precursors of conjugated linoleic acid-CLA). In a previous study [Thanks to the FA composition of hazelnut skin, the HZN ewes ingested higher amounts of MUFA . Considering that the two groups were balanced, and the experimental design has been carried out to minimize other confounding factors , the difference in the colostral IgG concentration could suggest an effect of the inclusion of hazelnut skin in the diet, considering its tocopherols and phenolic compounds.Vela  and AlveIn the current study, IgG concentration > 15 g/L was recorded in all the lambs; this value is considered an indicator of efficient passive immune transfer ,15. In bThe evaluation of GGT and LDH enzyme activity both in sheep’s serum and colostrum showed interesting results. GGT activity levels were found to agree with the literature . On the Our study provided evidence of the correlation between IgG concentration and enzyme activity levels in sheep’s blood and colostrum. The greatest IgG-GGT correlation was found in lamb’s serum and colostrum. Increased GGT expression is related to the high levels of colostral IgG and milk protein ,27. The Concerning LDH analysis, the results showed that GGT-LDH correlation was always moderate both in lamb’s serum and in the colostrum. Although few papers described a correlation between IgG and LDH in the colostrum, our study showed a highly significant moderate correlation. As previously suggested, this outcome can be explained as an indicator of udder health, and a suitable index for the IgG concentration in milk ,86. GGT Our findings considered a small sample size, as witnessed by the great variability of the data. The experimental trial was carried out in pens adapted for the breeding of only 20 animals, in accordance with the sheep welfare guidelines . Beyond Our work demonstrated that hazelnut skin affects the IgG concentration in ewe’s colostrum, with an increased antibody coverage in lambs and better proactive immune defense against neonatal infectious diseases. The inclusion of hazelnut skin in ruminant feeding represents an interesting source of bioactive compounds with antioxidant activity that affords a paramount opportunity to benefit the health of farmed animals. The study suggests that the high content of MUFA, PUFA, and the oleic acid of hazelnut skin could ameliorate the IgG concentration in the colostrum, thus improving passive immunity transfer to newborns. Our findings confirm that IgG levels may be indirectly measured by GGT in both the blood and colostrum, and LDH evaluation could predict IgG concentrations in the colostrum. Moreover, our findings provide useful information to the feeding industry for new nutritional strategies by promoting a circular economy in the livestock system.


Difficulty in interpreting white blood cell (WBC) counts in cerebrospinal fluid (CSF) complicates the diagnosis of neonatal meningitis in traumatic lumbar punctures (LP). The aim of our study was to determine the correction factor for WBC counts in traumatic LP that offers the greatest diagnostic efficacy in meningitis.3 CSF and pleocytosis as WBCs ≥ 20 cells/mm3 CSF. The CSF RBC:WBC ratio was analyzed by linear regression to determine a new correction factor. Cell count adjustments were also studied using the 500:1, the 1,000:1 ratio method, and the peripheral blood RBC:WBC ratio, using ROC curves and studies of accuracy (sensitivity and specificity).We conducted a retrospective observational study of LP in neonates between January 2014 and December 2020. Traumatic LP was defined as a red blood cell (RBC) count ≥ 1,000 cells/mmOverall, 41.0% of the 1,053 LPs included in the study were traumatic. The best results for effective WBC correction were the method based on the peripheral blood ratio  and the 400:1 ratio  obtained from linear regression .Both the peripheral blood correction and the 400:1 correction reduce the number of neonates classified with pleocytosis who were not eventually diagnosed with meningitis. Both methods might be a useful tool to clarify the neonatal meningitis diagnosis, offering neonatologists the possibility to assess the WBC count in traumatic LP. Meningitis occurs most often in the first month of life and is associated with high rates of morbidity and mortality –3. The sTraumatic LP occurs when the needle used to perform the procedure causes accidental bleeding in the subarachnoid space. It is estimated that about 30%-50% of neonatal CSF samples are contaminated with blood. The introduction of peripheral blood into the CSF complicates the interpretation of the results by increasing the WBC count, due to the higher concentration of WBC in the plasma compared to CSF –3, 5–7. One possible way of obtaining a correct interpretation of CSF WBC values in case of traumatic LP is to apply correction factors using the CSF RBC count . CorrectWe performed a retrospective observational study of lumbar punctures performed on neonates (≤ 28 days) admitted to the neonatology unit of the Hospital Universitario Nuestra Señora de Candelaria between January 1, 2014 and December 31, 2020 who underwent LP with subsequent cell count, PCR for viruses and bacteria, and CSF culture to rule out bacterial or viral meningitis in the context of neonatal sepsis. The study was approved by the ethical committee of the Complejo Hospitalario Universitario de Canarias (CHUNSC_2020_104).CSFs for which cell counts were unavailable or that had coagulated were excluded. In cases in which several CSF samples were collected from the neonate during admission, the first was selected for the study.General patient data  and diagnosis at discharge  were collected from the DragoAE Hospital Information System.3), WBC (cells/mm3), glucose (mmol/L), and proteins (g/L)], and microbiological results in CSF (cultures with identified microorganism and PCR for bacteria and viruses) and peripheral blood (cultures with identified microorganism) were obtained from the OpenLab Laboratory Computer System (LCS). The gold standard visual method, Neubauer’s chamber, was used for cell counting. CSF biochemical parameters were analyzed using the Dimension RxL Max  automated system. Blood parameters were obtained using the Sysmex XN automatic analyzer . Samples for blood culture were received in BACT/ALERT® Culture Media . As these are pediatric blood cultures, only a single sample (1–5 mL) was required, which was incubated in the BACT/ALERT® 3D for a standardized period of 5 days. In case of tested positive, subcultures were carried out in blood, chocolate and Schaedler agar, by seeding by discharge or isolation. Samples for CSF culture were received in polypropylene sterile tubes and were sown with a calibrated loop for isolation in blood, chocolate and McConkey agar and in fluid thioglycollate medium. Those were incubated at 37ºC for 24–48 h in aerobiosis except chocolate agar which was incubated in chamber at 5–10% CO2. The FilmArray meningitis panel  that analyzes the most common pathogens causing meningitis, including the following viruses, bacteria and yeasts, was used to identify the causative pathogen: Escherichia coli K1, Haemophilus influenzae, Listeria monocytogenes, Neisseria meningitidis, Streptococcus agalactiae, Streptococcus pneumoniae, Cytomegalovirus (CMV), Enterovirus, Herpes simplex virus 1 (HSV-1), Herpes simplex virus 2 (HSV-2), Human herpesvirus 6 (HHV-6), Human parechovirus, Varicella zoster virus (VZV) and Cryptococcus neoformans/gatti.Blood parameters , CSF laboratory parameters  cells/mm3 for neonates with non-traumatic LPs. The median CSF WBC count was 30.0 [12.0–82.8] cells/mm3 for neonates with traumatic LPs and 7.0 [2.0–13.0] cells/mm3 for neonates with non-traumatic LPs .The median [interquartile range (IQR)] CSF RBC count was 12,093.5  cells/mmA CSF RBC:WBC correction ratio of 400:1 was estimated by linear regression  , and the proportion of peripheral blood RBCs to WBCs in determining meningitis  are shown in Table When compared with the uncorrected WBC count, different correction factors showed lower sensitivity but higher specificity and area under the curve for the detection of meningitis. With regards to bacterial meningitis, sensitivity did not differ from the uncorrected WBC count (100%), although in regards to viral meningitis, correction factors showed lower sensitivity (75%).The peripheral blood correction and 400:1 correction showed the highest specificity values for bacterial and viral meningitis  in comparison with the uncorrected WBC count . With these two methods, the peripheral blood correction and 400:1 correction, the number of neonates classified with CSF pleocytosis is substantially reduced: zero cases of bacterial meningitis and one single case of viral meningitis were misclassified.Diagnostic efficacy was also evaluated in CSF with non-traumatic LPs. The results are summarized in Table 3  to more than 10,000 RBC/mm3. However, the most widely used cut-off point used is 1,000 RBC/mm3 [The high rate of traumatic LPs in neonates complicates clinical decisions surrounding the risk of both bacterial and viral meningitis . Further RBC/mm3 .3. These data are consistent with studies reporting rates of up to 46% [In our retrospective study of neonates undergoing LP over a period of 7 years, we observed a considerable number of traumatic LPs (41.0%), defined as ≥ 1,000 RBC/mmp to 46% , 9, althBaziomo et al.  suggest The differential diagnosis between bacterial and viral meningitis is often complex, especially in neonates where clinical examination is of limited value . Most neWhen we compare the AUC of the different correction methods for WBC counts in CSF, we obtain higher figures with the corrections 400:1, 500:1, 1000:1, and with the correction based on peripheral blood than that obtained with uncorrected WBC. Moreover, specificity values for uncorrected WBCs were 38% and 37% in bacterial and viral meningitis, respectively, while these figures increased significantly with all correction factors applied. If we focus on corrections that are not based on ratios in peripheral blood, we see that, although the factors of 500:1 and 1000:1 are the most widely used in the literature, the factor 400:1 obtained the best results in terms of specificity . However, the peripheral blood correction obtained even better results .Although the peripheral blood correction factor obtained the greatest specificity, it is more difficult to apply because recent blood data are required and more complex mathematical calculations must be employed. On the other hand, although correction by the 400:1 factor yields a slightly lower specificity, this method only requires CSF data and the mathematical calculation is easier. The specificity of the 500:1 correction is slightly lower than that of the 400:1 correction and may also be recommended because neonatologists are more familiar with this method.3, conclude that WBC correction in traumatic LP is not useful because it leads to an underestimation of the real number of WBC present in CSF. It may certainly result in missed cases of meningitis that can entail serious clinical consequences. However, they did not take into account the possible morbidity resulting from unnecessary use of antibiotics in patients, which we believe should be a consideration. Lyons et al. [The two largest studies to date evaluating WBC correction in traumatic LP reach conflicting conclusions. Since all these authors, including us, used different traumatic LP cut-off points, comparisons between studies are more difficult to interpret. For example, Greenberg et al. , who defs et al.  obtainedWhen comparing our results in corrected traumatic LPs, we observed that sensitivity and specificity results barely differed from those obtained from non-traumatic LPs that represent optimal CSF conditions and maximum reachable sensitivity and specificity. This substantial reduction in false positives, in relation with uncorrected WBC in traumatic LPs, with no increase in false negatives (missed diagnosis of meningitis), regarding the optimal reachable CSF conditions (non-traumatic LPs), demonstrates that correcting the WBC in traumatic LPs is a valuable procedure that is feasible for use in daily practice.Our study has some limitations. Firstly, it is a single-center study with a limited number of cases of bacterial and viral meningitis. Large-scale multicenter studies are required to evaluate the interpretation of WBC count in traumatic LPs. It should, however, be noted that this is the first study of these characteristics to be carried out in the Spanish population. Secondly, we were unable to determine whether the study neonates received antibiotics prior to the LP procedure, which would overestimate WBC correction. However, this is an increasingly unusual clinical practice. In most cases, empirical therapy is initiated after LP is performed, except in cases where LP is not possible in the first instance due to clinical instability of the patient. Despite this, our study also included the cohort of neonates with non-traumatic LP in order to make a more reliable comparison. Thirdly, we did not select an upper limit for CSF RBC count in our definition of traumatic LP, so we cannot ensure that samples are exclusively peripheral blood. Fourthly, clinical data for the study neonates were not considered in this study. Clinicians treating neonates in the context of bacterial or viral meningitis should take into account not only CSF WBC counts, but also other clinical and laboratory variables.Although CSF correction factors should be used with caution in neonates, we show that correcting WBC values improved diagnostic yield in both bacterial and viral meningitis. Specifically, we recommend the use of both peripheral blood data and the 400:1 correction, the latter being readily available and easier to use. With both methods, it is possible to substantially reduce the number of neonates classified with CSF pleocytosis in whom bacterial or viral meningitis are finally not diagnosed. This approach offers neonatologists the possibility to assess WBC in CSF from traumatic LPs and to avoid the systematic administration of antibiotics in all these neonates.