Comparative Analysis of the Osmotic Fragility of Erythrocytes Across Various Taxa of Vertebrates

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Abstract

The osmotic fragility of erythrocytes serves as a crucial parameter indicating the cells' ability to endure variations in the osmotic environment. Disorders in this attribute are often correlated with a spectrum of pathologies, encompassing hemolytic anemias, malignant tumors, and cardiovascular dysfunctions. Notably, osmotic fragility exhibits variability across different animal species and closely intertwines with their respective ecosystems. A methodology for assessing osmotic fragility has been devised utilizing a laser particle analyzer, facilitating the real-time monitoring of cell concentration changes under controlled temperature conditions. The species examined include Homo sapiens, Rattus norvegicus domestica, Coturnix japonica domestica, Rana ridibunda, Carassius carassius, and Lampetra fluviatilis. The methodology is presented in two variants: (1) manual water additions and (2) automated medium dilution. Key parameters characterizing osmotic fragility include H50 (the osmolality causing lysis in half of the susceptible cells), H90 (lysis in 90% of the cells), and W (heterogeneity in lysis fragility within the cell population). The findings obtained through the developed method did not show statistically significant deviations from the results obtained using spectrophotometry and flow cytometry concerning parameters such as H50 and W. Moreover, no noteworthy disparities were observed between the outcomes of the automatic and manual methodologies. Erythrocytes of aquatic and semi-aquatic animals exhibit significantly higher resistance to hypotonic lysis. Among all species examined, amphibian (Rana ridibunda) and lamprey (Lampetra fluviatilis) erythrocytes demonstrated the lowest osmotic fragility. The most pronounced variability in resistance levels was detected among amphibians, with differences nearly doubling in comparison to other taxa examined. While mammalian erythrocytes (including those of humans and rats) exhibited similar fragility levels, they displayed less uniformity in their resistance profiles. Bird erythrocytes, on the other hand, demonstrated a half-lysis occurrence at higher osmolality levels compared to mammalian erythrocytes. Nonetheless, bird erythrocytes (Coturnix japonica domestica) lysed over a considerably wider osmotic range and contained a subset of cells resilient to hypotonic lysis. These findings indicate that erythrocytes of lower vertebrates possess lower osmotic fragility compared to those of higher vertebrates, a phenomenon likely attributable to embryonic characteristics, ecto-/endothermy, and habitat considerations.

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About the authors

B. A. Gerda

Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences

Author for correspondence.
Email: bgergda2525@gmail.com
Russian Federation, Saint Petersburg

E. A. Skverchinskaya

Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences

Email: bgergda2525@gmail.com
Russian Federation, Saint Petersburg

A. Yu. Andreeva

Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences; A.O. Kovalevsky Institute of Biology of the Southern Seas of the Russian Academy of Sciences

Email: bgergda2525@gmail.com
Russian Federation, Saint Petersburg; Sevastopol

A. A. Volkova

Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences

Email: bgergda2525@gmail.com
Russian Federation, Saint Petersburg

S. P. Gambaryan

Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences

Email: bgergda2525@gmail.com
Russian Federation, Saint Petersburg

I. V. Mindukshev

Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences

Email: bgergda2525@gmail.com
Russian Federation, Saint Petersburg

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2. Fig. 1. Examples of primary data obtained by spectrophotometry and flow cytometry. (a) - spectrophotometry. Different colors show plots of optical density (OD) versus wavelength (λ), the wavelength used (540 nm) is marked. OD increases as osmolality decreases. The point before the onset of lysis (Rmax) is 150 mOsm. (b) - flow cytometry. A decrease in osmolality leads to a decrease in the number of recorded events in the region with cells (RBC) and an increase in the number of microparticles (MP). The point before the onset of lysis (Rmax) is 200 mOsm. The above data were obtained on human erythrocytes. Red highlighting - area with erythrocytes, blue highlighting - area with microparticles.

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3. Fig. 2. Example of primary data obtained by manual laser diffraction method and visualization of key parameters on the lysis curve. (a) - laser diffraction method (manual). Light scattering intensity increases due to the increase in cell volume at osmolalities of 200 and 150 mOsm. It then decreases as osmolality decreases further as a result of hemolysis. The point before the onset of lysis (Rmax ) is 150 mOsm. (b) is the lysis curve plotted using the described algorithm. Data were obtained on avian erythrocytes to demonstrate incomplete cell lysis and the Lysmax parameter. Key parameters characterizing erythrocyte stability (H50, H90, H10, W and, Lysmax) are displayed.

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4. Fig. 3. Correction of light scattering intensity. (a) - theoretical and experimental dependences of intensity on the concentration of dispersed particles for latexes with diameters of 6, 10 and 16 μm. Theoretical and experimental dependences coincide at kd = 6.3, V = 8.33. (b) - light scattering intensities before and after correction. The data were obtained on human erythrocytes. The corrected dependence shows an increase in intensity due to an increase in erythrocyte volume.

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5. Fig. 4. Osmotic resistance of human erythrocytes at 25 and 37 C. (a) - comparison by H90. (b) - comparison by H50. (c) - comparison by W. Whiskers - minimum and maximum, n = 12 for 37 C, n = 6 for 25 C, + - mean value, ns - p > 0.05, ** - p < 0.01, ** - p < 0.001, Welch's t test. (d) - dependences of the percentage of lysis (Lys) at experimental points on the osmolality of the medium (Osm). Semitransparent cloud - standard deviation (sd).

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6. Fig. 5. Comparison of manual laser diffraction method with flow cytometry and spectrophotometry in the assessment of osmotic resistance of human erythrocytes. (a) - comparison by H90. (b) - comparison by H50. (c) - comparison by W. FC - flow cytometry (n = 6), SPh - spectrophotometry (n = 5), LDM - manual laser diffraction method (n = 6), whiskers - minimum and maximum, + - mean value, ns - p > 0.05, * - p < 0.05, Tukey's multiple comparisons test. (d) - QQ-plot for H90. (e) - QQ-plot for H50. (f) - QQ-plot for W. Predicted - quantiles of the standard normal distribution. (g) - dependences of the percentage of lysis (Lys) at experimental points on the osmolality of the medium (Osm); translucent cloud - standard deviation (sd).

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7. Fig. 6. Comparison of results of automatic and manual laser diffraction methods in analyzing osmotic resistance of human erythrocytes. (a) - comparison of results for H90. (b) - comparison by H50. (c) - comparison by W. Whiskers - minimum and maximum, + - mean value, n = 15, ns - p > 0.05, Paired t test. (d) - QQ-plot for H90. (e) - QQ-plot for H50. (f) - QQ-plot for W. Predicted - quantiles of the standard normal distribution. (g) - correlations between manual and automatic methods for H90 parameters. (h) - correlation by H50. (i) - correlation by W. upper left corner - Pearson r and p-value. LDM - manual laser diffraction method, LDA - automatic laser diffraction method.

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8. Fig. 7. Comparison of the results of automatic and manual laser diffraction methods in the analysis of osmotic resistance of rat erythrocytes. (a) - comparison of results for H90. (b) - comparison by H50. (c) - comparison by W. Whiskers - minimum and maximum, + - mean value, n = 7, ns - p > 0.05, Paired t test. (d) - QQ-plot for H90. (e) - QQ-plot for H50. (f) - QQ-plot for W. Predicted - quantiles of the standard normal distribution.

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9. Fig. 8. Comparison of osmotic resistance of erythrocytes of different vertebrate species. (a) - comparison of results for H90. (b) - comparison by H50. (c) - comparison by W. Temperatures: 15C for Lampetra fluviatilis (n = 30), 16C for Carassius carassius (n = 12), 18C for Rana ridibunda (n = 15), 41C for Coturnix japonica domestica (n = 6) and 37C for mammals (n = 15 for Homo sapiens, n = 7 for Rattus norvegicus). Translucent cloud is standard deviation (sd). ns - p > 0.05, * - p < 0.05, ** - p < 0.01, *** - p < 0.001, Tukey's multiple comparisons test. (d) - dependencies of the percentage of lysis (Lys) on the osmolality of the medium (Osm). The translucent cloud is the standard deviation (sd). The manual laser diffraction method was used.

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10. Fig. 9. Comparison of osmotic resistance of erythrocytes of different vertebrate species in %-x of initial osmolality. (a) - comparison of results for H90. (b) - comparison by H50. (c) - comparison by W. Temperatures and physiologic osmolalities: 15C and 260 mOsm for Lampetra fluviatilis (n = 30), 16C and 260 mOsm for Carassius carassius (n = 12), 18C and 220 mOsm for Rana ridibunda (n = 15), 41C and 320 mOsm for Coturnix japonica (n = 6), 37C and 283 mOsm for Homo sapiens (n = 15), 37C and 294 mOsm for Rattus norvegicus (n = 7). Translucent cloud is standard deviation (sd). ns - p > 0.05, * - p < 0.05, ** - p < 0.01, *** - p < 0.001, Tukey's multiple comparisons test. (d) - dependencies of the percentage of lysis (Lys) on the osmolality of the medium (Osm). The translucent cloud is the standard deviation (sd). The manual laser diffraction method was used.

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11. Fig. 10. Comparison of heterogeneity of higher vertebrate erythrocyte populations in terms of resistance to hypoosmotic lysis. (a) - Comparison in absolute values (mOsm). (b) - Comparison in relative values (percent of physiologic plasma osmolality). Temperatures and physiologic osmolalities: 41C and 320 mOsm for Coturnix japonica (n = 6), 37C and 283 mOsm for Homo sapiens (n = 15), 37C and 294 mOsm for Rattus norvegicus (n = 7). The manual laser diffraction method was used. ns - p > 0.05, ** - p < 0.01, *** - p < 0.001, Tukey's multiple comparisons test.

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