{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,19]],"date-time":"2026-03-19T21:15:21Z","timestamp":1773954921888,"version":"3.50.1"},"reference-count":53,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2024,1,19]],"date-time":"2024-01-19T00:00:00Z","timestamp":1705622400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"INESC TEC SEED project","award":["CEEIND\/017801\/2018"],"award-info":[{"award-number":["CEEIND\/017801\/2018"]}]},{"name":"INESC TEC SEED project","award":["SFRH\/BD\/09136\/2020"],"award-info":[{"award-number":["SFRH\/BD\/09136\/2020"]}]},{"name":"Funda\u00e7\u00e3o para a Ci\u00eancia e Tecnologia (FCT)","award":["CEEIND\/017801\/2018"],"award-info":[{"award-number":["CEEIND\/017801\/2018"]}]},{"name":"Funda\u00e7\u00e3o para a Ci\u00eancia e Tecnologia (FCT)","award":["SFRH\/BD\/09136\/2020"],"award-info":[{"award-number":["SFRH\/BD\/09136\/2020"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biosensors"],"abstract":"<jats:p>Spectral point-of-care technology is reagentless with minimal sampling (&lt;10 \u03bcL) and can be performed in real-time. White blood cells are non-dominant in blood and in spectral information, suffering significant interferences from dominant constituents such as red blood cells, hemoglobin and billirubin. White blood cells of a bigger size can account for 0.5% to 22.5% of blood spectra information. Knowledge expansion was performed using data augmentation through the hybridization of 94 real-world blood samples into 300 synthetic data samples. Synthetic data samples are representative of real-world data, expanding the detailed spectral information through sample hybridization, allowing us to unscramble the spectral white blood cell information from spectra, with correlations of 0.7975 to 0.8397 and a mean absolute error of 32.25% to 34.13%; furthermore, we achieved a diagnostic efficiency between 83% and 100% inside the reference interval (5.5 to 19.5 \u00d7 109 cell\/L), and 85.11% for cases with extreme high white blood cell counts. At the covariance mode level, white blood cells are quantified using orthogonal information on red blood cells, maximizing sensitivity and specificity towards white blood cells, and avoiding the use of non-specific natural correlations present in the dataset; thus, the specifity of white blood cells spectral information is increased. The presented research is a step towards high-specificity, reagentless, miniaturized spectral point-of-care hematology technology for Veterinary Medicine.<\/jats:p>","DOI":"10.3390\/bios14010053","type":"journal-article","created":{"date-parts":[[2024,1,19]],"date-time":"2024-01-19T07:00:32Z","timestamp":1705647632000},"page":"53","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Reagentless Vis-NIR Spectroscopy Point-of-Care for Feline Total White Blood Cell Counts"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8400-0248","authenticated-orcid":false,"given":"Teresa Guerra","family":"Barroso","sequence":"first","affiliation":[{"name":"1H-TOXRUN\u2014One Health Toxicology Research Unit, University Institute of Health Sciences (IUCS), CESPU, CRL, 4585-116 Gandra, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0485-8023","authenticated-orcid":false,"given":"Carla","family":"Queir\u00f3s","sequence":"additional","affiliation":[{"name":"LAQV-REQUIMTE, Faculty of Sciences, University of Porto, R. Campo Alegre, 4169-007 Porto, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1923-2218","authenticated-orcid":false,"given":"Filipe","family":"Monteiro-Silva","sequence":"additional","affiliation":[{"name":"INESC TEC\u2014Institute for Systems and Computer Engineering, Technology and Science\u2014Campus da FEUP, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8486-6113","authenticated-orcid":false,"given":"Filipe","family":"Santos","sequence":"additional","affiliation":[{"name":"INESC TEC\u2014Institute for Systems and Computer Engineering, Technology and Science\u2014Campus da FEUP, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4346-2259","authenticated-orcid":false,"given":"Ant\u00f3nio Hugo","family":"Greg\u00f3rio","sequence":"additional","affiliation":[{"name":"Anicura CHV\u2014Veterinary Hospital Center, R. Manuel Pinto de Azevedo 118, 4100-320 Porto, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9058-3147","authenticated-orcid":false,"given":"Rui Costa","family":"Martins","sequence":"additional","affiliation":[{"name":"INESC TEC\u2014Institute for Systems and Computer Engineering, Technology and Science\u2014Campus da FEUP, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2024,1,19]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"130138","DOI":"10.1016\/j.snb.2021.130138","article-title":"Point-of-care Vis-SWNIR spectroscopy towards reagent-less hemogram analysis","volume":"343","author":"Barroso","year":"2021","journal-title":"Sens. Actuators B Chem."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Barroso, T.G., Ribeiro, L., Greg\u00f3rio, H., Monteiro-Silva, F., Neves dos Santos, F., and Martins, R.C. (2022). Point-of-Care Using Vis-NIR Spectroscopy for White Blood Cell Count Analysis. Chemosensors, 10.","DOI":"10.3390\/chemosensors10110460"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"8","DOI":"10.1016\/j.aca.2018.04.004","article-title":"Near infrared spectroscopy: A mature analytical technique with new perspectives\u2014A review","volume":"1026","author":"Pasquini","year":"2018","journal-title":"Anal. Chim. Acta"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"2427","DOI":"10.1021\/ac00172a022","article-title":"Quantitative effects of an absorbing matrix on near-infrared diffuse reflectance spectra","volume":"60","author":"Olinger","year":"1988","journal-title":"Anal. Chem."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"580","DOI":"10.1366\/14-07645","article-title":"Matrix Effects in Quantitative Assessment of Pharmaceutical Tablets Using Transmission Raman and Near-Infrared (NIR) Spectroscopy","volume":"69","author":"Hartman","year":"2015","journal-title":"Appl. Spectrosc."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"129681","DOI":"10.1016\/j.snb.2021.129681","article-title":"Paper-based microfluidics: Simplified fabrication and assay methods","volume":"336","author":"Nishat","year":"2021","journal-title":"Sens. Actuators B Chem."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"129762","DOI":"10.1016\/j.snb.2021.129762","article-title":"Plasmonic colorimetric PCR for Rapid molecular diagnostic assays","volume":"337","author":"Jiang","year":"2021","journal-title":"Sens. Actuators B Chem."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"129915","DOI":"10.1016\/j.snb.2021.129915","article-title":"Colorimetric point-of-care paper-based sensors for urinary creatinine with smartphone readout","volume":"340","author":"Speichert","year":"2021","journal-title":"Sens. Actuators B Chem."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Zhou, L., Vestri, A., Marchesano, V., Rippa, M., Sagnelli, D., Picazio, G., Fusco, G., Han, J., Zhou, J., and Petti, L. (2023). The Label-Free Detection and Identification of SARS-CoV-2 Using Surface-Enhanced Raman Spectroscopy and Principal Component Analysis. Biosensors, 13.","DOI":"10.3390\/bios13121014"},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Popov, E., Polishchuk, A., Kovalev, A., and Vitkin, V. (2023). Raman Spectroscopy for Urea Breath Test. Biosensors, 13.","DOI":"10.3390\/bios13060609"},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Monteiro-Silva, F., Jorge, P.A.S., and Martins, R.C. (2019). Optical Sensing of Nitrogen, Phosphorus and Potassium: A Spectrophotometrical Approach toward Smart Nutrient Deployment. Chemosensors, 7.","DOI":"10.3390\/chemosensors7040051"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"106710","DOI":"10.1016\/j.compag.2022.106710","article-title":"Unscrambling spectral interference and matrix effects in Vitis vinifera Vis-NIR spectroscopy: Towards analytical grade \u2018in vivo\u2019 sugars and acids quantification","volume":"194","author":"Martins","year":"2022","journal-title":"Comput. Electron. Agric."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"213","DOI":"10.1016\/j.bios.2012.02.050","article-title":"Optical diagnosis of laryngeal cancer using high wavenumber Raman spectroscopy","volume":"35","author":"Lin","year":"2012","journal-title":"Biosens. Bioelectron."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Coelho, B.F.O., Nunes, S.L.P., de Fran\u00e7a, C.A., dos Santos Costa, D., do Carmo, R.F., Prates, R.M., Filho, E.F.S., and Ramos, R.P. (2024). On the feasibility of Vis\u2013NIR spectroscopy and machine learning for real time SARS-CoV-2 detection. Spectrochim. Acta Part A Mol. Biomol. Spectrosc., 308.","DOI":"10.1016\/j.saa.2023.123735"},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Burns, D., and Ciurczak, E. (2001). Handbook of Near Infrared Analysis, Marcel Dekker, Inc.. [2nd ed.].","DOI":"10.1201\/9781003042204"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"259","DOI":"10.1016\/j.bios.2017.09.004","article-title":"Detection of BCG bacteria using a magnetoresistive biosensor: A step towards a fully electronic platform for tuberculosis point-of-care detection","volume":"100","author":"Barroso","year":"2018","journal-title":"Biosens. Bioelectron."},{"key":"ref_17","unstructured":"Willard, M.D., and Tvedten, H. (2012). Small Animal Clinical Diagnosis by Laboratory Methods, W.B. Saunders. [5th ed.]."},{"key":"ref_18","unstructured":"Burton, A.G., and Jandrey, K.E. (2018). Textbook of Small Animal Emergency Medicine, John Wiley & Sons, Ltd.. Chapter 64."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"7917","DOI":"10.1016\/S0021-9258(18)43366-2","article-title":"Association-dependent absorption spectra of oxyhemoglobin A and its subunits","volume":"256","author":"Philo","year":"1981","journal-title":"J. Biol. Chem."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Tan, H., Liao, S., Pan, T., Zhang, J., and Chen, J. (2020). Rapid and simultaneous analysis of direct and indirect bilirubin indicators in serum through reagent-free visible-near-infrared spectroscopy combined with chemometrics. Spectrochim. Acta Part A Mol. Biomol. Spectrosc., 233.","DOI":"10.1016\/j.saa.2020.118215"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"11","DOI":"10.1016\/j.cll.2014.10.007","article-title":"White Blood Cell Counts: Reference Methodology","volume":"35","author":"George","year":"2015","journal-title":"Clin. Lab. Med."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"604","DOI":"10.1111\/vcp.12398","article-title":"Evaluation of performance of veterinary in-clinic hematology analyzers","volume":"45","author":"Rishniw","year":"2016","journal-title":"Vet. Clin. Pathol."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Riond, B., Wa\u00dfmuth, A.K., Hartnack, S., Hofmann-Lehmann, R., and Lutz, H. (2015). Study on the kinetics and influence of feline platelet aggregation and deaggregation. BMC Vet. Res., 11.","DOI":"10.1186\/s12917-015-0590-7"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"515","DOI":"10.3343\/alm.2022.42.5.515","article-title":"Unreliable Automated Complete Blood Count Results: Causes, Recognition, and Resolution","volume":"42","author":"Gulati","year":"2022","journal-title":"Ann. Lab. Med."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1111\/vcp.12583","article-title":"ASVCP guidelines: Allowable total error hematology","volume":"47","author":"Nabity","year":"2018","journal-title":"Vet. Clin. Pathol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"747","DOI":"10.1177\/1098612X17706471","article-title":"The feline blood film: 2. Leukocyte and platelet morphology","volume":"19","author":"Harvey","year":"2017","journal-title":"J. Feline Med. Surg."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"128","DOI":"10.1111\/vcp.12682","article-title":"What is your diagnosis? A feline blood smear","volume":"48","author":"Meredith","year":"2019","journal-title":"Vet. Clin. Pathol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"044010","DOI":"10.1088\/2050-6120\/4\/4\/044010","article-title":"The manifestation of optical centers in UV\u2013Vis absorption and luminescence spectra of white blood human cells","volume":"4","author":"Yashchuk","year":"2016","journal-title":"Methods Appl. Fluoresc."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"251","DOI":"10.1016\/S0165-022X(02)00004-0","article-title":"Novel methodology for the follow-up of acute lymphoblastic leukemia using FTIR microspectroscopy","volume":"51","author":"Ramesh","year":"2002","journal-title":"J. Biochem. Biophys. Methods"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"385","DOI":"10.1016\/S0022-2143(03)00025-8","article-title":"Preliminary results of evaluation of progress in chemotherapy for childhood leukemia patients employing Fourier-transform infrared microspectroscopy and cluster analysis","volume":"141","author":"Ramesh","year":"2003","journal-title":"J. Lab. Clin. Med."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1274","DOI":"10.1021\/ja076069p","article-title":"Compact biocompatible quantum dots functionalized for cellular imaging","volume":"130","author":"Liu","year":"2007","journal-title":"J. Am. Chem. Soc."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"24821","DOI":"10.1038\/srep24821","article-title":"A reliable Raman-spectroscopy-based approach for diagnosis, classification and follow-up of B-cell acute lymphoblastic leukemia","volume":"6","author":"Valente","year":"2016","journal-title":"Sci. Rep."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"e67101421657","DOI":"10.33448\/rsd-v10i14.21657","article-title":"Diagnosis, classification and monitoring of leukemia based on Raman spectroscopy","volume":"10","author":"Lima","year":"2021","journal-title":"Res. Soc. Dev."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"687","DOI":"10.1016\/j.leukres.2005.10.011","article-title":"Continuous monitoring of WBC (biochemistry) in an adult leukemia patient using advanced FTIR-spectroscopy","volume":"30","author":"Sahu","year":"2006","journal-title":"Leuk. Res."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"827","DOI":"10.1016\/j.bbagen.2011.06.010","article-title":"Pre-screening and follow-up of childhood acute leukemia using biochemical infrared analysis of peripheral blood mononuclear cells","volume":"1810","author":"Zelig","year":"2011","journal-title":"Biochim. Biophys. Acta (BBA)-Gen. Subj."},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Chaber, R., Kowal, A., Jakubczyk, P., Arthur, C., \u0141ach, K., Wojnarowska-Nowak, R., Kusz, K., Zawlik, I., Paszek, S., and Cebulski, J. (2021). A Preliminary Study of FTIR Spectroscopy as a Potential Non-Invasive Screening Tool for Pediatric Precursor B Lymphoblastic Leukemia. Molecules, 26.","DOI":"10.3390\/molecules26041174"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"611","DOI":"10.1177\/0003702820985856","article-title":"Infrared Spectroscopy of Blood","volume":"75","author":"Kochan","year":"2021","journal-title":"Appl. Spectrosc."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"6955","DOI":"10.1039\/D0AN00752H","article-title":"Diagnosis of inaccessible infections using infrared microscopy of white blood cells and machine learning algorithms","volume":"145","author":"Agbaria","year":"2020","journal-title":"Analyst"},{"key":"ref_39","unstructured":"Little, S.E. (2012). The Cat, W.B. Saunders."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"134442","DOI":"10.1016\/j.snb.2023.134442","article-title":"Reagent-less spectroscopy towards NPK sensing for hydroponics nutrient solutions","volume":"395","author":"Silva","year":"2023","journal-title":"Sens. Actuators B Chem."},{"key":"ref_41","doi-asserted-by":"crossref","unstructured":"Nalepa, J., Marcinkiewicz, M., and Kawulok, M. (2019). Data Augmentation for Brain-Tumor Segmentation: A Review. Front. Comput. Neurosci., 13.","DOI":"10.3389\/fncom.2019.00083"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"60","DOI":"10.1186\/s40537-019-0197-0","article-title":"A survey on Image Data Augmentation for Deep Learning","volume":"6","author":"Shorten","year":"2019","journal-title":"J. Big Data"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"1221","DOI":"10.1093\/clinchem\/46.8.1221","article-title":"Flow Cytometry: Principles and Clinical Applications in Hematology","volume":"46","author":"Brown","year":"2000","journal-title":"Clin. Chem."},{"key":"ref_44","unstructured":"INESCTEC (2020). AgIoT\u2014Modular Solution and Open Source IoT Solution for Agrofood Domain, INESCTEC."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"625","DOI":"10.1016\/0731-7085(91)80188-F","article-title":"Extended multiplicative signal correction and spectral interference subtraction: New preprocessing methods for near infrared spectroscopy","volume":"9","author":"Martens","year":"1991","journal-title":"J. Pharm. Biomed. Anal."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/0003-2670(86)80028-9","article-title":"Partial least squares regression: A tuturial","volume":"185","author":"Geladi","year":"1988","journal-title":"Anal. Acta"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"72","DOI":"10.1002\/cem.1180","article-title":"Re-interpretation of NIPALS results solves PLSR inconsistency problem","volume":"23","author":"Ergon","year":"2009","journal-title":"J. Chemom."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"311","DOI":"10.1002\/(SICI)1099-128X(199707)11:4<311::AID-CEM478>3.0.CO;2-4","article-title":"The geometry of partial least squares","volume":"11","author":"Phatak","year":"1997","journal-title":"J. Chemom."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"e3117","DOI":"10.1002\/cem.3117","article-title":"Local partial least squares based on global PLS scores","volume":"33","author":"Shen","year":"2019","journal-title":"J. Chemom."},{"key":"ref_50","unstructured":"R-Core-Team (2020). R: A Language and Environment for Statistical Computing, R Foundation for Statistical Computing. Available online: http:\/\/www.r-project.org\/."},{"key":"ref_51","unstructured":"Harr, K.E., Flatland, B., Nabity, M.B., and Freeman, K.P. (2013). ASVCP Guidelines\u2014Allowable Total Error: Biochemistry, American Society for Veterinary Clinical Pathology."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"11","DOI":"10.1016\/j.ab.2017.01.002","article-title":"Determination of extinction coefficients of human hemoglobin in various redox states","volume":"521","author":"Meng","year":"2017","journal-title":"Anal. Biochem."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"466","DOI":"10.1111\/vcp.12383","article-title":"Quality requirements for veterinary hematology analyzers in small animals\u2014A survey about veterinary experts\u2019 requirements and objective evaluation of analyzer performance based on a meta-analysis of method validation studies: Bench top hematology analyzer","volume":"45","author":"Cook","year":"2016","journal-title":"Vet. Clin. Pathol."}],"container-title":["Biosensors"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2079-6374\/14\/1\/53\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T13:46:01Z","timestamp":1760103961000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2079-6374\/14\/1\/53"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,1,19]]},"references-count":53,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2024,1]]}},"alternative-id":["bios14010053"],"URL":"https:\/\/doi.org\/10.3390\/bios14010053","relation":{},"ISSN":["2079-6374"],"issn-type":[{"value":"2079-6374","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,1,19]]}}}