Bìol. Tvarin. 2021; 23 (2): 32–36.
Received 19.05.2021 ▪ Accepted 25.06.2021 ▪ Published online 01.07.2021

The role of the dehydration stage in the post-hypertonic hemolysis of mammalian erythrocytes

O. E. Nipot, O. O. Shapkina, P. M. Zubov, N. V. Orlova, N. M. Shpakova

This email address is being protected from spambots. You need JavaScript enabled to view it.

Institute of Problems of Cryobiology and Cryomedicine NAS of Ukraine,
23 Pereyaslavska str., Kharkiv, 61016, Ukraine, This email address is being protected from spambots. You need JavaScript enabled to view it.

The aim of this study was to assess the level of damage to mammalian erythrocytes under post-hypertonic shock depending on the concentration of NaCl in the dehydration medium and to determine the effect of hypertonic NaCl solutions on the condition of mammalian erythrocytes by flow cytometry. To achieve this goal, spectrophotometric and cytometry research methods were used. The data obtained showed that post-hypertonic lysis of mammalian erythrocytes depends on the concentration of NaCl in the dehydration medium. The most sensitive to the effects of post-hypertonic shock are rat erythrocytes, the least sensitive are rabbit cells. Cytometry studies revealed significant changes in the histograms of the distribution of erythrocytes of all mammalian species with increasing salt concentration in the dehydration medium. These changes are species-specific and are probably related to changes in cell volume and morphology. The data revealed a relationship between the level of post-hypertonic hemolysis and the values of such indicators as the median distribution and the coefficient of variation. Thus, an increase in the sensitivity of mammalian erythrocytes to post-hypertonic shock with increasing salt concentration in dehydration medium was usually accompanied by a decrease in the median cell division, and higher values of the coefficient of variation are characteristic of mammalian erythrocytes resistant to post-hypertonic shock.

Key words: mammalian erythrocytes, dehydration, post-hypertonic shock, cytometry, median, coefficient of variation, distribution

  1. Adan A, Alizada G, Kiraz Y, Baran Y, Nalbant A Flow cytometry: basic principles and applications. Critic. Rev. Biotechnol. 2017; 37 (2): 163–176. DOI: 10.3109/07388551.2015.1128876.
  2. Ameri M, Schnaars HA, Sibley JR, Honor DJ. Stability of hematologic analytes in monkey, rabbit, rat, and mouse blood stored at 4°C in EDTA using the ADVIA 120 hematology analyzer. Vet. Clin. Pathol. 2011; 40 (2): 188–193. DOI: 10.1111/j.1939-165X.2011.00304.x.
  3. Betticher DC, Geiser J. Resistance of mammalian red blood cells of different size to hypertonic milieu. Comp. Biochem. Physiol. A Comp. Physiol. 1989; 93 (2): 429–432. DOI: 10.1016/0300-9629(89)90061-3.
  4. Bojic S, Murray A, Bentley BL, Spindler R, Pawlik P, Cordeiro JL, Bauer R, de Magalhães JP. Winter is coming: the future of cryopreservation. BMC Biol. 2021;19 (1): 56. DOI: 10.1186/s12915-021-00976-8.
  5. García JM, Ardila AM. Cell volume variation under different concentrations of saline solution (NaCL).  Rev. Colomb. Anestesiol. 2009; 37 (2): 101–105. DOI: 10.1016/S0120-3347(09)72002-7.
  6. Ghosh S, Chakraborty I, Chakraborty M, Mukhopadhyay A, Mishra R, Sarkar D. Evaluating the morphology of erythrocyte population: An approach based on atomic force microscopy and flow cytometry. Biochim. Biophys. Acta. 2016; 1858 (4): 671–681. DOI: 10.1016/j.bbamem.2016.01.021.
  7. Gienger J, Gross H, Ost V, Bär M, Neukammer J. Assessment of deformation of human red blood cells in flow cytometry: measurement and simulation of bimodal forward scatter distributions. Biomed. Opt. Exp. 2019; 10 (9): 4531–4550. DOI: 10.1364/BOE.10.004531.
  8. Gorey A, Biswas D, Kumari A, Gupta S, Sharma N, Chen GCK, Vasudevan S. Application of continuous-wave photoacoustic sensing to red blood cell morphology. Laser. Med. Sci. 2019; 34: 487–494. DOI: 10.1007/s10103-018-2621-7.
  9. Lahmann JM, Benson JD, Higgins AZ. Concentration dependence of the cell membrane permeability to cryoprotectant and water and implications for design of methods for post-thaw washing of human erythrocytes. Cryobiol. 2018; 80: 1–11. DOI: 10.1016/j.cryobiol.2017.12.003.
  10. Mishra Ragh, Sarkar D, Bhattacharya S, Mallick S, Chakraborty M, Mukherjee D, Kar M, Mishra Rosh. Quantifying morphological alteration of RBC population from light scattering data. Clin. Hemorheol. Microcirc. 2015; 59 (4): 287–300. DOI: 10.3233/CH-131726.
  11. Muldrew K. The salting-in hypothesis of post-hypertonic lysis. Cryobiol. 2008; 57 (3): 251–256. DOI: 10.1016/j.cryobiol.2008.09.007.
  12. Nemeth N, Sogor V, Kiss F, Ulker P. Interspecies diversity of erythrocyte mechanical stability at various combinations in magnitude and duration of shear stress, and osmolality. Clin. Hemorheol. Microcirc. 2016; 63 (4): 381–398. DOI: 10.3233/CH-152031.
  13. Piagnerelli M, Zouaoui Boudjeltia K, Brohee D, Vereerstraeten A, Piro P, Vincent JL, Vanhaeverbeek M. Assessment of erythrocyte shape by flow cytometry techniques. J. Clin. Pathol. 2007; 60 (5): 549–554. DOI: 10.1136/jcp.2006.037523.
  14. Semionova KA, Nipot OE, Yershova NA, Shapkina OO, Shpakova NM. Temperature, osmolality, and glucose determine the erythrocyte resistance to post-hypertonic stress. Rep. Nat. Acad. Sci. Ukr. 2020; 4: 99–106. DOI: 10.15407/dopovidi2020.04.099. (in Ukrainian)
  15. Semionova KA, Yershova NA, Yershov SS, Orlova NV, Shpakova NM. Peculiarities of posthypertonic lysis in erythrocytes of several mammals. Probl. Cryobiol. Cryomed. 2016; 26 (1): 73–83. DOI: 10.15407/cryo26.01.073. (in Russian)
  16. Yamamoto A, Saito N, Yamauchi Y, Takeda M, Ueki S, Itoga M, Kojima K, Kayaba H. Flow cytometric analysis of red blood cell osmotic fragility. J. Lab. Automat. 2014; 19 (5): 483–487. DOI: 10.1177/2211068214532254.
  17. Zemlyanskykh NG, Kovalenko IF, Babijchuk LA. Peculiarities of modifications in geometric parameters and changes in osmotic fragility of human erythrocytes following their exposure in sucrose and PEG-1500 solutions. Probl. Cryobiol. Cryomed. 2017; 27 (4): 296–310. DOI: 10.15407/cryo27.04.296. (in Russian)