Analytical and Bioanalytical Chemistry

, Volume 411, Issue 13, pp 2767–2780 | Cite as

A smart preparation strategy for point-of-care cellular counting of trace volumes of human blood

  • Xinyi Li
  • Qiong Deng
  • Hongping Liu
  • Youwang Lei
  • Pengwei Fan
  • Bin Wang
  • Yangfei Chen
  • Zachary J. Smith
  • Yuchen TangEmail author
  • Tingjuan GaoEmail author
Paper in Forefront


Blood counting is one of the most commonly ordered clinical assays, and is often part of the basis for initial diagnosis and screening for disease. While substantial prior research has shown the ability of portable instruments to accurately produce blood counts through image- or flow-based cytometry, these methods require complex sample preparation using either costly commercial imaging chambers or complicated reagents. To address these issues, in this paper we developed a method to prepare trace volumes of whole blood aimed at portable blood counting. The strategy is based on pre-storing dry-form reagents and fabricating a specifically designed cell counter. In order to obtain total cell counts for red blood cells, platelets, and 3-part differentials of white blood cells, two parallel counting chambers with different depths are made from cost- and environmentally friendly materials using soft lithography. As little as 1 μl of whole blood is prepared with pre-stored reagents in centrifuge vials, whereas red blood cells are sphered and white blood cells are stained at the same time. Driven by the capillary force, prepared blood samples enter the hydrophilic chambers automatically. Monolayers of cells are formed when the blood dilution factors and the chamber depths are co-optimized. Combined with our previous custom-built instrument and automated analysis algorithm, the sample preparation strategy allows producing counting results with excellent agreement to a gold-standard clinical hematology instrument. The success of this preparation method may further advance applications of our technology for global use in low-resource settings where central hematology laboratories are not accessible.

Graphical abstract

Graphical Abstract


Blood counting POCT Image-based cytometry Sample preparation 


Funding information

This work is supported by the National Natural Science Foundation of China (21605054), Central China Normal University (CCNU) new PI start-up fund (210-31102), and self-determined research funds of CCNU (234-20205016002, 234-20205170355) from the colleges’ basic research and operation of Ministry of Education (MOE). TG acknowledges support from the Program of Introducing Talents of Discipline to University of China (111 program, B17019), and the CCNU Program of Innovation and Entrepreneurship for college students (220-20205180419). ZJS acknowledges support from the 1000 Young Talents Global Recruitment Plan, and from the Ministry of Science and Technology of the People’s Republic of China (2016YFA020130).

Compliance with ethical standards

The authors declare that they have no conflict of interest.

Supplementary material

216_2019_1738_MOESM1_ESM.pdf (850 kb)
ESM 1 (PDF 849 kb)


  1. 1.
    Tefferi A, Hanson CA, Inwards DJ. How to interpret and pursue an abnormal complete blood cell count in adults. Mayo Clin Proc. 2005;80(7):923–36.CrossRefGoogle Scholar
  2. 2.
    Verbrugge SE, Huisman A. Verification and standardization of blood cell counters for routine clinical laboratory tests. Clin Lab Med. 2015;35(1):183–96.CrossRefGoogle Scholar
  3. 3.
    Chabot-Richards DS, George TI. White blood cell counts reference methodology. Clin Lab Med. 2014;35(1):11–24.CrossRefGoogle Scholar
  4. 4.
    Pai NP, Vadnais C, Denkinger C, Engel N, Pai M. Point-of-care testing for infectious diseases: diversity, complexity, and barriers in low- and middle-income countries. PLoS Med. 2012;9(9):e1001306.CrossRefGoogle Scholar
  5. 5.
    Peeling RW, Mabey D. Point-of-care tests for diagnosing infections in the developing world. Clin Microbiol Infect. 2010;16(8):1062–9.CrossRefGoogle Scholar
  6. 6.
    Peters DH, Garg A, Bloom G, Walker DG, Brieger WR, Rahman MH. Poverty and access to health care in developing countries. Ann N Y Acad Sci. 2008;1136(1):161–70.CrossRefGoogle Scholar
  7. 7.
    Boppart SA, Richards-Kortum R. Point-of-care and point-of-procedure optical imaging technologies for primary care and global health. Sci Transl Med. 2014;6(253):253rv2.CrossRefGoogle Scholar
  8. 8.
    Browne AW, Ramasamy L, Cripe TP, Ahn CH. A lab-on-a-chip for rapid blood separation and quantification of hematocrit and serum analytes. Lab Chip. 2011;11(14):2440–6.CrossRefGoogle Scholar
  9. 9.
    Vercruysse D, Dusa A, Stahl R, Vanmeerbeeck G, Wijs KD, Liu C, et al. Three-part differential of unlabeled leukocytes with a compact lens-free imaging flow cytometer. Lab Chip. 2015;15(4):1123–32.CrossRefGoogle Scholar
  10. 10.
    Shi W, Guo L, Kasdan H, Tai YC. Four-part leukocyte differential count based on sheathless microflow cytometer and fluorescent dye assay. Lab Chip. 2013;13(7):1257–65.CrossRefGoogle Scholar
  11. 11.
    Berkel CV, Gwyer JD, Deane S, Green N, Holloway J, Hollis V, et al. Integrated systems for rapid point of care (PoC) blood cell analysis. Lab Chip. 2011;11(7):1249–55.CrossRefGoogle Scholar
  12. 12.
    Zhu H, Mavandadi S, Coskun AF, Yaglidere O, Ozcan A. Optofluidic fluorescent imaging cytometry on a cell phone. Anal Chem. 2011;83(17):6641–7.CrossRefGoogle Scholar
  13. 13.
    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.CrossRefGoogle Scholar
  14. 14.
    Ben-Yosef Y, Marom B, Hirshberg G, D'Souza C, Larsson A, Bransky A. The HemoScreen, a novel haematology analyser for the point of care. J Clin Pathol. 2016;69(8):720–5.CrossRefGoogle Scholar
  15. 15.
    Leshansky AM, Bransky A, Korin N, Dinnar U. Tunable nonlinear viscoelastic “focusing” in a microfluidic device. Phys Rev Lett. 2007;98(23):234501.CrossRefGoogle Scholar
  16. 16.
    Del Giudice F, Romeo G, D’Avino G, Greco F, Netti P, Maffettone PL. Particle alignment in a viscoelastic liquid flowing in a square-shaped microchannel. Lab Chip. 2013;13(21):4263–71.CrossRefGoogle Scholar
  17. 17.
    D’Avino G, Romeo G, Villone MM, Greco F, Netti PA, Maffettone PL. Single line particle focusing induced by viscoelasticity of the suspending liquid: theory, experiments and simulations to design a micropipe flow-focuser. Lab Chip. 2012;12(9):1638–45.CrossRefGoogle Scholar
  18. 18.
    Song Y, Huang Y, Liu X, Zhang X, Ferrari M, Qin L. Point-of-care technologies for molecular diagnostics using a drop of blood. Trends Biotechnol. 2014;32(3):132–9.CrossRefGoogle Scholar
  19. 19.
    Pacaud D, Lemay J, Guay P, Buithieu M, Yale J. Assessment of blood volumes obtained by capillary punctures in older children and adolescents with diabetes. Pediatr Res. 1996;39:95.CrossRefGoogle Scholar
  20. 20.
    Wong ECC. Hematology analyzers: special considerations for pediatric patients. Clin Lab Med. 2015;35:165–81.CrossRefGoogle Scholar
  21. 21.
    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.CrossRefGoogle Scholar
  22. 22.
    Smith ZJ, Gao T, Lane SM, Wachmann-Hogiu S, Dwyre DM, Heifetz L, et al. Portable Blood Count Monitor, U.S. Patent No. 20,140,273,064. Washington, DC: U.S. Patent and Trademark Office; 2014.Google Scholar
  23. 23.
    Gao T, Smith ZJ, Lin T, Holt DC, 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.CrossRefGoogle Scholar
  24. 24.
    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.CrossRefGoogle Scholar
  25. 25.
    Kim YR, Ornstein L. Isovolurnetric sphering of erythrocytes for more accurate and precise cell volume measurement by flow cytometry. Cytometry. 1983;3(6):419–27.CrossRefGoogle Scholar
  26. 26.
    Ban T, Kasatani K, Kawasaki M, Sato H. Fluorescence decay of the acridine orange-sodium dodecyl sulfate system: formation of dye-rich induced micelles in the premicellar region. Photochem Photobiol. 1983;37(2):131–9.CrossRefGoogle Scholar
  27. 27.
    Moulik SP, Ghosh S, Das AR. Interaction of acridine orange monohydrochloride dye with sodiumdodeeylsulfate (SDS), cetyltrimethylammoniumbromide (CTAB) and p-tert-octylphenoxypolyoxy ethanol (Triton X 100) surfactants. Colloid Polym Sci. 1979;257(6):645–55.CrossRefGoogle Scholar
  28. 28.
    Gerola AP, Costa PF, Quina FH, Fiedler HD, Nome F. Zwitterionic surfactants in ion binding and catalysis. Curr Opin Colloid Interface Sci. 2017;32:39–47.CrossRefGoogle Scholar
  29. 29.
    Nabity MB, Harr KE, Camus MS, Flatland B, Vap LM. ASVCP guidelines: allowable total error hematology. Vet Clin Pathol. 2018;47(1):9–21.CrossRefGoogle Scholar
  30. 30.
    Buttarello M, Plebani M. Automated blood cell counts. Am J Clin Pathol. 2008;130(1):104–15.CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Xinyi Li
    • 1
  • Qiong Deng
    • 1
  • Hongping Liu
    • 2
  • Youwang Lei
    • 3
  • Pengwei Fan
    • 4
  • Bin Wang
    • 4
  • Yangfei Chen
    • 1
  • Zachary J. Smith
    • 5
  • Yuchen Tang
    • 1
    Email author
  • Tingjuan Gao
    • 1
    Email author
  1. 1.Key Laboratory of Pesticide and Chemical Biology of Ministry of Education, and Chemical Biology Center, College of ChemistryCentral China Normal UniversityWuhanChina
  2. 2.State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Institute of Physics and MathematicsChinese Academy of SciencesWuhanChina
  3. 3.Prenatal Diagnostics CenterJieyang Women and Children’s Health Care HospitalJieyangChina
  4. 4.Clinical Laboratory CenterWuhanChina
  5. 5.Department of Precision Machinery and Precision InstrumentationUniversity of Science and Technology of ChinaHefeiChina

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