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The effects of incubation media on the assessment of the shape of human erythrocytes by flow cytometry: a contribution to mathematical data interpretation to enable wider application of the method

Authorized Users Only
2021
Authors
Drvenica, Ivana
Mojsilović, Slavko
Stančić, Ana
Marković, Dragana
Kovačić, Marijana
Maslovarić, Irina
Rapajić, Ivana
Vučetić, Dušan
Ilić, Vesna
Article (Published version)
Metadata
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Abstract
Flow cytometry (FC) analysis of erythrocyte shape and related biomechanical properties, such as osmotic fragility, have not moved from a research tool to regular clinical testing. The main reason is existing evidence that various pre-analytical factors influence the mathematical interpretation of the data obtained. With an aim to contribute to the standardization and broaden the use of FC for human erythrocyte shape assessment, freshly prepared peripheral blood erythrocytes isolated from healthy donors were incubated in iso and hypo-osmotic solutions (pure saline, saline with potassium and calcium, and phosphate buffered saline) and examined by FC using values of forward scatter (FSC) and side scatter (SSC). Kurtosis, skewness, Pearson's second skewness coefficient of dissymmetry (PCD), and spherical index, calculated from FSC distributions, were used for the erythrocyte shape evaluation. In all isotonic media FSC distribution and FSC-based morphology parameters showed huge inter-indiv...idual and inter-medium variation. With decreasing osmolality, in all media and samples, the size of the erythrocytes increased, and swelling index and kurtosis decreased. However, changes in skewness and PCD were influenced by the medium used and the sample tested. Compared to FSC, SSC signal in isotonic and its change in hypotonic media showed lower inter-individual variation and was not influenced by the type of medium. We propose a spherical index and kurtosis as FSC-based indicators of erythrocyte shape. As more resistant to the influence of the preanalytical treatment, SSC data appeared to be unfairly neglected for the assessment of erythrocyte shape, in comparison to the usually employed FSC data.

Keywords:
Human erythrocytes / Flow cytometry / Osmotic swelling / Morphology / Sphericity / Multimodal distribution
Source:
European Biophysics Journal, 2021, 50, 6, 829-846
Publisher:
  • Springer Nature
Funding / projects:
  • Ministry of Education, Science and Technological Development, Republic of Serbia, Grant no. 200015 (University of Belgrade, Institute for Medical Research) (RS-200015)
  • HEMMAGINERO - Hemoglobin-Based Spectroscopy and Nonlinear Imaging of Erythrocytes and Their Membranes As Emerging Diagnostic Tool (RS-6066079)

DOI: 10.1007/s00249-021-01527-3

ISSN: 0175-7571; 0175-7571

[ Google Scholar ]
URI
http://rimi.imi.bg.ac.rs/handle/123456789/1176
Collections
  • Radovi istraživača / Researchers' publications
Institution/Community
Institut za medicinska istraživanja
TY  - JOUR
AU  - Drvenica, Ivana
AU  - Mojsilović, Slavko
AU  - Stančić, Ana
AU  - Marković, Dragana
AU  - Kovačić, Marijana
AU  - Maslovarić, Irina
AU  - Rapajić, Ivana
AU  - Vučetić, Dušan
AU  - Ilić, Vesna
PY  - 2021
UR  - http://rimi.imi.bg.ac.rs/handle/123456789/1176
AB  - Flow cytometry (FC) analysis of erythrocyte shape and related biomechanical properties, such as osmotic fragility, have not moved from a research tool to regular clinical testing. The main reason is existing evidence that various pre-analytical factors influence the mathematical interpretation of the data obtained. With an aim to contribute to the standardization and broaden the use of FC for human erythrocyte shape assessment, freshly prepared peripheral blood erythrocytes isolated from healthy donors were incubated in iso and hypo-osmotic solutions (pure saline, saline with potassium and calcium, and phosphate buffered saline) and examined by FC using values of forward scatter (FSC) and side scatter (SSC). Kurtosis, skewness, Pearson's second skewness coefficient of dissymmetry (PCD), and spherical index, calculated from FSC distributions, were used for the erythrocyte shape evaluation. In all isotonic media FSC distribution and FSC-based morphology parameters showed huge inter-individual and inter-medium variation. With decreasing osmolality, in all media and samples, the size of the erythrocytes increased, and swelling index and kurtosis decreased. However, changes in skewness and PCD were influenced by the medium used and the sample tested. Compared to FSC, SSC signal in isotonic and its change in hypotonic media showed lower inter-individual variation and was not influenced by the type of medium. We propose a spherical index and kurtosis as FSC-based indicators of erythrocyte shape. As more resistant to the influence of the preanalytical treatment, SSC data appeared to be unfairly neglected for the assessment of erythrocyte shape, in comparison to the usually employed FSC data.
PB  - Springer Nature
T2  - European Biophysics Journal
T1  - The effects of incubation media on the assessment of the shape of human erythrocytes by flow cytometry: a contribution to mathematical data interpretation to enable wider application of the method
EP  - 846
IS  - 6
SP  - 829
VL  - 50
DO  - 10.1007/s00249-021-01527-3
ER  - 
@article{
author = "Drvenica, Ivana and Mojsilović, Slavko and Stančić, Ana and Marković, Dragana and Kovačić, Marijana and Maslovarić, Irina and Rapajić, Ivana and Vučetić, Dušan and Ilić, Vesna",
year = "2021",
abstract = "Flow cytometry (FC) analysis of erythrocyte shape and related biomechanical properties, such as osmotic fragility, have not moved from a research tool to regular clinical testing. The main reason is existing evidence that various pre-analytical factors influence the mathematical interpretation of the data obtained. With an aim to contribute to the standardization and broaden the use of FC for human erythrocyte shape assessment, freshly prepared peripheral blood erythrocytes isolated from healthy donors were incubated in iso and hypo-osmotic solutions (pure saline, saline with potassium and calcium, and phosphate buffered saline) and examined by FC using values of forward scatter (FSC) and side scatter (SSC). Kurtosis, skewness, Pearson's second skewness coefficient of dissymmetry (PCD), and spherical index, calculated from FSC distributions, were used for the erythrocyte shape evaluation. In all isotonic media FSC distribution and FSC-based morphology parameters showed huge inter-individual and inter-medium variation. With decreasing osmolality, in all media and samples, the size of the erythrocytes increased, and swelling index and kurtosis decreased. However, changes in skewness and PCD were influenced by the medium used and the sample tested. Compared to FSC, SSC signal in isotonic and its change in hypotonic media showed lower inter-individual variation and was not influenced by the type of medium. We propose a spherical index and kurtosis as FSC-based indicators of erythrocyte shape. As more resistant to the influence of the preanalytical treatment, SSC data appeared to be unfairly neglected for the assessment of erythrocyte shape, in comparison to the usually employed FSC data.",
publisher = "Springer Nature",
journal = "European Biophysics Journal",
title = "The effects of incubation media on the assessment of the shape of human erythrocytes by flow cytometry: a contribution to mathematical data interpretation to enable wider application of the method",
pages = "846-829",
number = "6",
volume = "50",
doi = "10.1007/s00249-021-01527-3"
}
Drvenica, I., Mojsilović, S., Stančić, A., Marković, D., Kovačić, M., Maslovarić, I., Rapajić, I., Vučetić, D.,& Ilić, V.. (2021). The effects of incubation media on the assessment of the shape of human erythrocytes by flow cytometry: a contribution to mathematical data interpretation to enable wider application of the method. in European Biophysics Journal
Springer Nature., 50(6), 829-846.
https://doi.org/10.1007/s00249-021-01527-3
Drvenica I, Mojsilović S, Stančić A, Marković D, Kovačić M, Maslovarić I, Rapajić I, Vučetić D, Ilić V. The effects of incubation media on the assessment of the shape of human erythrocytes by flow cytometry: a contribution to mathematical data interpretation to enable wider application of the method. in European Biophysics Journal. 2021;50(6):829-846.
doi:10.1007/s00249-021-01527-3 .
Drvenica, Ivana, Mojsilović, Slavko, Stančić, Ana, Marković, Dragana, Kovačić, Marijana, Maslovarić, Irina, Rapajić, Ivana, Vučetić, Dušan, Ilić, Vesna, "The effects of incubation media on the assessment of the shape of human erythrocytes by flow cytometry: a contribution to mathematical data interpretation to enable wider application of the method" in European Biophysics Journal, 50, no. 6 (2021):829-846,
https://doi.org/10.1007/s00249-021-01527-3 . .

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