Abstract
While many clinical laboratory tests are now highly automated, body fluid cell counting, particularly in low-cellularity samples such as cerebral spinal fluid (CSF), is often performed manually. Here, we report a simple, cost-effective method to obtain white and red blood cell counts from human body fluids such as CSF. The method consists of a compact, automated, and low-cost fluorescence microscope system, coupled to a sample chamber containing all of the necessary reagents in dry form to stain and prepare the sample. Sample focus and scanning are handled automatically, and the acquired multimodal images are automatically analyzed to extract cell counts. Comparison with manual counting on over 200 clinical samples shows excellent agreement. As the system counts a substantially larger image region than a standard manual cell count, we find our sensitivity to extremely low cellularity samples to potentially be higher than the manual gold standard, evidenced by our system recording images of cells in samples whose cell count was registered as “0” by a trained user. Thus, our system holds promise for routine, automated, and sensitive analysis of body fluids whose cellularity extends across a wide dynamic range.
Graphical abstract
Similar content being viewed by others
References
CLSI. Body fluid analysis for cellular composition; approved guidelines. CLSI document H56-A.Wayne. Clinical and Laboratory Standard Institute: PA; 2006.
Sandhaus LM. Is the hemocytometer obsolete for body fluid cell counting? Am J Clin Path. 2016;145(3):294–5.
Martín MJA, Queral LA, Frías LS, Amado LV, Merino A, de Guadiana-Romualdo LG. Automated cell count in body fluids: a review. Adv Lab Med. 2021;20210011.
Buoro S, Peruzzi B, Fanelli A, Seghezzi M, Manenti B, Lorubbio M, et al. Two-site evaluation of the diagnostic performance of the Sysmex XN Body Fluid (BF) module for cell count and differential in cerebrospinal fluid. Int J Lab Hematol. 2018;40(1):26–33.
Buoro S, Seghezzi M, Dominoni P, Moioli V, Manenti B, Previtali G, et al. Lack of harmonization in high fluorescent cell automated counts with body fluids mode in ascitic, pleural, synovial, and cerebrospinal fluids. Int J Lab Hematol. 2019;41(2):277–86.
Seo JY, Lee S-T, Kim S-H. Performance evaluation of the new hematology analyzer Sysmex XN-series. Int J Lab Hematol. 2015;37(2):155–64.
Huang W-H, Lu L-P, Wu K, Guo F-Y, Guo J, Yu J-L, et al. Extent of agreement between the body fluid model of Sysmex XN-20 and the manual microscopy method. J Clin Lab Anal. 2017;31(5):e22101.
Zhu H, Sencan I, Wong J, Dimitrov S, Tseng D, Nagashima K, et al. Cost-effective and rapid blood analysis on a cell-phone. Lab Chip. 2013;13(7):1282–8.
Smith ZJ, Gao T, Chu K, Lane SM, Matthews DL, Dwyre DM, et al. Single-step preparation and image-based counting of minute volumes of human blood. Lab Chip. 2014;14(16):3029–36.
Forcucci A, Pawlowski ME, Majors C, Richards-Kortum R, Tkaczyk TS. All-plastic, miniature, digital fluorescence microscope for three part white blood cell differential measurements at the point of care. Biomed Opt Express. 2015;6(11):4433–46.
Powless A, Feekin L, Hutcheson J, Alapat D, Muldoon T. Low-cost computing and network communication for a point-of-care device to perform a 3-part leukocyte differential. Proc. SPIE 9715; 2016.
Xie D, Xie Y, Liu P, Tong L, Hu C, Shao P, et al. Performance of a cost-effective and automated blood counting system for resource-limited settings operated by trained and untrained users. J Biophotonics. 2018;11(2):e201700030.
Li X, Deng Q, Liu H, Lei Y, Fan P, Wang B, et al. A smart preparation strategy for point-of-care cellular counting of trace volumes of human blood. Anal Bioanal Chem. 2019;411(13):2767–80.
Lv M, Zhao X, Chen F, Yu M, Li C, Sun J. A rapid white blood cell classification system based on multimode imaging technology. J Biophotonics. 2020;13(11):e202000197.
Chen Y, Chen X, Li M, Fan P, Wang B, Zhao S, et al. A new analytical platform for potential point-of-care testing of circulating tumor cells. Biosens Bioelectron. 2021;171:112718.
Bachar N, Benbassat D, Brailovsky D, Eshel Y, Glück D, Levner D, et al. An artificial intelligence-assisted diagnostic platform for rapid near-patient hematology. medRxiv; 2021.
Chen X, Luo P, Hu C, Yan S, Lu D, Li Y, et al. Nanometer precise red blood cell sizing using a cost-effective quantitative dark field imaging system. Biomed Opt Express. 2020;11(10):5950–66.
Li Y, Zheng R, Wu Y, Chu K, Xu Q, Sun M, et al. A low-cost, automated parasite diagnostic system via a portable, robotic microscope and deep learning. J Biophotonics. 2019;12(9):e201800410.
Gao T, Smith ZJ, Lin T-Y, Carrade Holt D, Lane SM, Matthews DL, et al. Smart and fast blood counting of trace volumes of body fluids from various mammalian species using a compact, custom-built microscope cytometer. Anal Chem. 2015;87(23):11854–62.
Powless AJ, Prieto SP, Gramling MR, Conley RJ, Holley GG, Muldoon TJ. Evaluation of acridine orange staining for a semi-automated urinalysis microscopic examination at the point-of-care. Diagnostics. 2019;9(3).
Campbell RA, Eifert RW, Turner GC. OpenStage: a low-cost motorized microscope stage with sub-micron positioning accuracy. PLoS One. 2014;9(2):e88977.
Lu Q, Liu G, Xiao C, Hu C, Zhang S, Xu RX, et al. A modular, open-source, slide-scanning microscope for diagnostic applications in resource-constrained settings. PLoS One. 2018;13(3):e0194063.
Yan Z, Chen G, Xu W, Yang C, Lu Y. Study of an image autofocus method based on power threshold function wavelet reconstruction and a quality evaluation algorithm. Appl Opt. 2018;57(33):9714–21.
Kim YR, Ornstein L. Isovolumetric sphering of erythrocytes for more accurate and precise cell volume measurement by flow cytometry. Cytometry. 1983;3(6):419–27.
Traganos F, Darzynkiewicz Z, Sharpless T, Melamed MR. Simultaneous staining of ribonucleic and deoxyribonucleic acids in unfixed cells using acridine orange in a flow cytofluorometric system. J Histochem Cytochem. 1977;25(1):46–56.
Chen Z, Wang Y, Zeng A, Chen L, Wu R, Chen B, et al. The clinical diagnostic significance of cerebrospinal fluid d-lactate for bacterial meningitis. Clin Chim Acta. 2012;413(19):1512–5.
Veerman A, Huismans L, Zantwijk I. Storage of cerebrospinal fluid samples at room temperature. Acta Cytol. 1985;29:188–9.
Powless A, Conley R, Freeman K, Muldoon T. Considerations for point-of-care diagnostics: evaluation of acridine orange staining and postprocessing methods for a three-part leukocyte differential test. J Biomed Opt. 2017;22(3):035001.
de Jonge R, Brouwer R, de Graaf MT, Luitwieler RL, Fleming C, de Frankrijker-Merkestijn M, et al. Evaluation of the new body fluid mode on the Sysmex XE-5000 for counting leukocytes and erythrocytes in cerebrospinal fluid and other body fluids. Clin Chem Lab Med. 2010;48(5):665–75.
Funding
This research was funded by the Ministry of Science and Technology of China’s National Key Research and Development Program, Grant number 2016YFA0201300 (ZJS) and the Chongqing Municipal Science and Technology Commission, grant number cstc2017shmsA130083 (HD, ZJS), whose support is gratefully acknowledged.
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
Ethics approval
This study was approved by the Ethics Committee of the Children’s Hospital of Chongqing Medical University, approval number:(2016)年伦审(研)第(67)号 (Translation: 2016 IRB Research approval number 67).
Source of biological material
The samples utilized in this study were anonymized discarded patient samples from the Clinical Laboratory of the Children’s Hospital of Chongqing Medical University, which were not collected for the purposes of this study.
Conflict of interest
The authors declare no competing interests.
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
ESM 1
(PDF 668 kb)
Rights and permissions
About this article
Cite this article
Lu, Q., Chu, K., Dou, H. et al. A sample-preparation-free, automated, sample-to-answer system for cell counting in human body fluids. Anal Bioanal Chem 413, 5025–5035 (2021). https://doi.org/10.1007/s00216-021-03466-6
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00216-021-03466-6