Dielectric Response of Cytoplasmic Water and Its Connection to the

Article pubs.acs.org/JPCB

Dielectric Response of Cytoplasmic Water and Its Connection to the Vitality of Human Red Blood Cells. II. The Influence of Storage Evgeniya Levy,† Marcelo David,† Gregory Barshtein,‡ Saul Yedgar,‡ Leonid Livshits,‡ Paul Ben Ishai,§ and Yuri Feldman*,† †

Department of Applied Physics, The Rachel and Selim Benin School of Engineering and Computer Science, The Hebrew University of Jerusalem, Edmond J. Safra Campus, Jerusalem, 91904, Israel ‡ Department of Biochemistry & Molecular Biology, IMRIC, Faculty of Medicine, The Hebrew University of Jerusalem, Ein Kerem, Jerusalem, 91120, Israel § Department of Physics, Ariel University, P.O.B. 3, Ariel 40700, Israel S Supporting Information *

ABSTRACT: Maintaining an appropriate inventory of packaged blood products is a critical part of modern medicine. Consequently, the assessment of red blood cell (RBC) functionality is instrumental for the monitoring of the quality of stored RBC (sRBC) in the blood bank. We present a comprehensive study of sRBC lesion kinetics in SAGM (saline, adenine, glucose, mannitol) solution, using microwave dielectric spectroscopy (0.5−50 GHz) and cell deformability. As part of the research, we have isolated the microwave dielectric response of cytoplasmic water in sRBC. The extracted dielectric parameters are sensitive to the age of the cells and, in particular, to the critical moment of transition from discocyte to echinocyte. From the analysis of the dielectric relaxation as a function of storage-duration, we postulate that the behavior is rooted in the delicate interplay between bound and bulk water in the cellular interior. In particular, the microwave dielectric response reflects the moment when the continuous diffusion of oxygen to the cell and the oxygenation of hemoglobin affects the role played by water in the maintenance of cell integrity. These results open a possible new avenue for the noninvasive inspection of stored red blood cells, permitting a true inventory system for the modern blood bank.

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INTRODUCTION Donated human red blood cells (RBCs) are routinely stored for a period of 35−42 days. This time limit has been determined mainly on the basis of their life span in the circulatory system and on changes in their biochemical parameters.1 Contrastingly, a growing number of studies have shown that the transfusion of some bank-stored RBCs may be a source of injury rather than benefit to the recipients.2−6 Increased hospital mortality, intubation within 72 h, renal failure, and sepsis or septicaemia are some examples of conditions associated with the use of nonfunctioning transfused blood.7,8 Furthermore, modern medicine requires an appropriate inventory of packaged blood products and this is estimated to represent 1% of overall healthcare costs in highly developed countries.9 It would seem to be that there is a pressing need for a more precise assessment of stored red blood cell (sRBC) functionality as a consequence of their shelf life. During cold storage, the contents of sRBC units undergo slow detrimental changes that are collectively termed storage lesion. Storage- and aging-related processes lead to significant metabolic and structural changes of erythrocytes, such as a decrease in ATP, a loss of volume, a change in morphology, and a massive vesicle generation.10,11 Specifically, the convex © XXXX American Chemical Society

rounded protrusions in RBCs (characterizing echinocytes) and the further released vesicles have a very specific membrane organization and internal content with elevated concentrations of hemoglobin-derived molecules and clusters of band 3 proteins.12−14 These are collectively a number of biochemical alterations in RBC properties, including, for example, an increase in membrane-bound globin,15 an oxidation of skeleton proteins, and a strong elevation of cell rigidity (decreasing deformability).16 This situation is further complicated by a strong donor variability of sRBC properties.17,18 There exist a number of biochemical methods to gauge sRBC changes and RBC viability. However, they involve invasive sampling and a 24 h time frame, greatly limiting their application in real time blood bank inventory. As a result, usually no testing of the sRBC functionality is performed and a “FIFO” (first-in-firstout) policy is enforced.19 There are exceptions for certain special recipients, e.g., neonates, thalassemia, and transplant patients.20 Recently, a number of works have hinted that Received: March 21, 2017 Revised: April 23, 2017 Published: April 28, 2017 A

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standard conditions (2−6 °C). The concentration of RBCs (Hematocrit about 60%) in suspension was controlled by a complete blood counter (Automated Hematology Analyzer, XP-300, Symex America, Inc., USA). Dielectric Spectroscopy. Dielectric measurements were carried out in the frequency range from 500 MHz to 50 GHz using the Microwave Network Analyzer (Keysight N5245A PNA-X), together with a Flexible Cable and Slim-Form Probe (Keysight N1501A Dielectric Probe Kit). The calibration of the system was performed with the aid of three references: air, the Keysight standard short circuit, and pure water at 25 °C. A special stand for the Slim-Form Probe was designed and combined with a sample cell holder for liquids (total volume ∼7.8 mL). The holder was enveloped by a thermal jacket and attached to a Julabo CF 41 oil-based heat circulatory system. The cell was held at 25 °C with temperature fluctuations less than 0.1 °C. The whole measuring system was placed in an airconditioned room maintained at 25 ± 1 °C (see part I of this series29). Each sample was measured at least four times in succession. The real and imaginary parts ε′(ω) and ε″(ω) were evaluated using the Keysight N1500A Materials Measurement Software with an accuracy of Δε′/ε′ = 0.05, Δε″/ε″ = 0.05.37 Note, three of the samples were measured starting from the fourth day of aging and the other three starting from the 11th day, respectively. All sample bags were stored at 4 °C, and the assays from them were heated to 25 °C before the measurement. Concurrently, pure water and buffer solution were measured under the same conditions. Determination of Single Cell Spectra. The actual measurement is a convolution of the dielectric response of both RBCs and the external buffer. The cytosolic water spectra can be isolated from the bulk response of the suspension, once the dc conductivity has been removed, using the Kraszewski mixture formula as in part I:29,38

dielectric sampling could provide a convenient, safe, and noninvasive method for the assessment of sRBC viability.21,22 Fresh and healthy RBCs or erythrocytes are nonspherical biconcave discs and consist of a cellular membrane and a cytoplasm that is rich in hemoglobin, while aged and/or lesioned RBCs are typically transformed into echinocytes; i.e., the cells become spheroidal with large external outgrowths.21,23,24 The plasma membrane of erythrocytes is nonconductive compared to their cytoplasm, which results in charge accumulation and interfacial polarization. This leads to the well-known β-dispersion in the RBC dielectric spectrum at 0.l−10 MHz.21,22 Since the change in membrane structure is one of the markers of RBC aging and sensitivity of dielectric spectroscopy to RBC membrane properties alteration has been previously shown,25,26 there have been attempts to solve the problem of noninvasive blood quality monitoring by monitoring the β dispersion.21,27,28 These have not been met with much success. There are a number of practical reasons why this frequency range is not the most practical for applications. Principal among them is the high salinityand consequently high conductivityof the storage buffer of sRBCs. This leads to two negative effects. First, the conductivity is so high that it masks the true bulk dielectric response. Second, the accumulation of a polarization layer at the interface of the plastic bag and the solution shields the contents from the probing electromagnetic field. However, we have shown in part I29 that variations in the membrane cannot be divorced from accompanying changes to the cell interior. One would expect that the complicated cascade of biochemical reactions leading to changes in membrane structure would have a knock-on effect in the cytoplasm of the cell interior. As this is a concentrated aqueous solution of macromolecules, organic molecules, and ions, it is not unreasonable to assume that this would lead to changes in the dielectric response of cellular water,30−34 commonly known as the γ dispersion, situated in the frequency range 0.5−75 GHz.35 An added advantage is that at these frequencies interfacial polarizations, contributions from the cell membrane, and the tumbling of any polar entities in the cytoplasm or membrane will not play a noticeable role in the dielectric response. In the first part of this series, we successfully applied the logic above to investigate the effect of buffer glucose concentration on the dielectric response of the cytoplasm of RBCs.29 In this paperthe second part of our workwe will follow the same general routine to study the cytoplasm of erythrocytes during cold storage. These results will be supplemented by direct measurements of cell deformability.16,36

⎛ (ε* )1/2 − (ε* )1/2 (1 − φ) ⎞2 buff ⎟⎟ ε*cell = ⎜⎜ mix φ ⎝ ⎠

(1)

Here, ε*mix, ε*cell, and ε*buff are the dielectric permittivity of the mixture, the interior of the cell, and the buffer, respectively. The volume fraction of the RBCs (hematocrit) is represented by φ. Determination of RBC Deformability. The present research employed the computerized Cell Flow-Properties Analyzer (CFA), designed and constructed in house.16 The CFA enables the monitoring of RBC hemodynamic characteristics as a function of shear stress, under conditions resembling those in microvessels, by a direct visualization of their dynamic organization in a narrow-gap flow-chamber that has been placed under a microscope.16,39,40 RBC deformability is determined by monitoring the elongation of RBCs, while they are stuck to a polystyrene slide, under flow-induced sheer stress.16 In brief, 50 μL of RBC suspension (1% hematocrit, in PBS supplemented by 0.5% of albumin) is inserted into the flow-chamber (adjusted to 200 μm gap) containing an uncoated slide (purchased from Electron Microscopy Science (Washington, PA)). The RBCs that adhere to the slide surface are then subjected to controllable flow-induced sheer stress (3.0 Pa), and their deformability is determined by the change in cell shape. This change is expressed by the elongation ratio, ER = a/b, where a is the major cellular axis and b is the minor cellular axis. ER = 1 reflects a round RBC, undeformed by the applied sheer stress. The CFA contains an image analysis program capable of automatically measuring the ER for individual cells.

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MATERIALS AND METHODS Cell Preparation. Blood was drawn from six healthy donors in the Hadassah Hospital Blood Bank, following informed consent according to the Helsinki Committee Regulations Permit (98290, Hadassah Hospital, Jerusalem, Israel), and collected into standard sterile bags, containing citrate phosphate dextrose (CPD). Immediately following collection, RBCs were isolated by centrifugation (Roto Silenta 630RS, Tuttlingen, Germany) for 6 min (2367 rpm, 24 °C) followed by removal of the plasma and resuspended in SAGM. RBCs were filtrated for leukoreduction. For monitoring of alteration in RBC properties during cell storage, 15 mL of RBCs from the standard unit has been sterilely transferred to special small (120 mL) storage bags. The samples were then stored under the B

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Figure 1. (a) Dielectric loss spectra at 25 °C of fresh RBC suspension in SAGM (purple squares) and the derived losses of cellular cytoplasm for the same sample (red circles) compared to SAGM (green triangles) and water (black stars); dc conductivity has been removed. (b) The derived losses of cellular cytoplasm for fresh RBC (red circles) and stored ones (blue triangles). The lines are the fitting curves.

Figure 2. Average of cell’s elongation ratio during days of cold storage for two different samples.

of H-bond network rearrangements in the vicinity of the different solute molecules.29,31−34 Both the real and imaginary parts of the recalculated spectra of the single cell response were fitted using the in-house software, Datama.43 The fitting function used was based on the CC function (eq 2), and the solid lines of Figure 1 show the quality of the obtained fit. The fitting parameters are presented in Table 1 (Supporting Information). The accepted biophysical marker for the storage-induced aging of RBC is their deformability.16,17,44 In the present research, we described deformability of sRBC by average of cell’s elongation ratio (AER).45,46 As stated above, this parameter was measured concurrently with the dielectric spectra. Figure 2 shows the dielectric relaxation time as a function of storage duration, superimposed on the graph of AER for the same samples. It is clear that a significant change in relaxation times, τ, coincides with a similar variation in AER. The abrupt change in the derived relaxation times is a clear indication of a dramatic change in blood viability. The deformability is a macroscopic physical property of the cell, whereas the relaxation time represents a direct access to the microscopic situation of the cell interior. Its related size of the apparent dipole moment of interior water structures and its red shift at the critical day, compared to the relaxation times of fresh cell cytoplasm, indicates a reorganization of the bulk water

The deformability distribution of a large RBC population (at least 2500 ± 300 cells) is then provided for shear stress of 3.0 Pa,16 and an average value of ER (AER) has been calculated (see part I of this series29).

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RESULTS AND DISCUSSION

The imaginary parts of typical recalculated complex dielectric permittivity spectra are presented in Figure 1. Note, the dc conductivity has been removed before recalculation. Typically, water’s relaxation peak can be described by the phenomenological Cole−Cole (CC) function:41 ε*(f ) = ε′(ω) − iε″(ω) = εh +

Δε 1 + (iωτ )α′

(2)

Here, ε′ and ε″ are the real and imaginary parts of the complex permittivity, ω = 2πf is the cyclic frequency, and i2 = −1. The parameter εh denotes the extrapolated high-frequency permittivity, and Δε = εl − εh is the relaxation amplitude (with the low frequency permittivity limit denoted by εl). The exponent α (0 < α ≤ 1) is a measure of the symmetrical broadening. In the case of pure water, for frequencies up to 40 GHz, α can be set to 1, resulting in a Debye relaxation.42 However, whenever water interacts with another dipolar or charged entity, a symmetrical broadening of its dispersion peak and a change in the attendant relaxation time is induced. The origin of the alteration of the dielectric loss peak is defined by the dynamics C

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storage time.50,51 These trends would affect the balance between bound and bulk water. This leads to an intriguing hypothesis for the behavior of the dielectric parameters and for the demise of the cell. As storage time continues, the dielectric strength, Δε, and the relaxation times, τ, remain constant, indicating that the bulk water structures in the cytoplasm remain relatively stable, both in number density and in apparent dipole moment. However, the behavior of α suggests that the rate of exchange between these same structures and water bound around proteins such as hemoglobin is constantly decreasing. This is happening despite an ever-changing internal environment. It is known that there is a constant diffusion of O2 into the blood portion through the PVC wall of the bag.52 After the first week of RBC storage, pO2 increased,53 and ultimately, Hb O2 saturation is reached ≈99% by the end of a six-week storage period53 due to irreversible Hb oxygenation through increased oxygen affinity.54 As it is allosteric protein and as the oxygenation induces subsequent conformational transitions,55 the ratio of bound to bulk water volumes must be steadily changing, due to binding of additional water molecules to Hb.56,57 The implication is that less and less bound/bulk water interfaces are available. However, the same bulk water interior maintains cell integrity by regulating osmotic pressure, a problem as cellular biochemistry progresses in the interior. As a consequence, the drop in pH that accompanies the production of lactic acid50 makes the maintenance of cell topology an impossible task, failing eventually around day 25 catastrophically. At this point, α returns to its initial values and the relaxation time rises to a constant value, close to that of bulk water for the measurement temperature.35 This is intriguing, as the dielectric strength continues to drop. Physically, the cell becomes an echinocyte.23 Coupled with the cascade of various biochemical reactions that accompany the demise of the cell, it could be that water effectively “detaches” from its role of the membrane. This would account for the constant behavior of both α and τ.

structures into larger formations. The other parameters of the fitting, α and Δε, further elucidate this process. The relaxation times, τ, the broadening parameter, α, and the relaxation amplitude, Δε, of the cytoplasm are presented in Figure 3. For all samples, the dominant feature is the sharp

Figure 3. Experimental relaxation times τ, the broadening parameter α, and the relaxation amplitude Δε for one cell during days of cold storage for two different samples.

increase of the relaxation time value around 25−35 days of aging. A strong correlation between α and the storage time is observed, with α becoming a good indicator of aging. It has been shown that the broadening parameter α reflects the rate of interactions of dipole relaxation units with their surroundings and that it can be expressed as α=

ln(Nτ /N0τ ) ln(τ /τc)

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CONCLUSIONS

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ASSOCIATED CONTENT

We have succeeded in isolating the microwave dielectric response of cytoplasmic water in stored red blood cells. The extracted dielectric parameters are sensitive to the age of the cells and, in particular, to the critical moment of transition from discocyte to echinocyte. From the analysis of the dielectric relaxation as a function of age, we postulate that the behavior is rooted in the delicate interplay between bound and bulk water in the cellular interior. In particular, the microwave dielectric response reflects the moment when the continuous diffusion of oxygen to the cell and the oxygenation of hemoglobin affects the role played by water in the maintenance of cell integrity. These results open a possible new avenue for the noninvasive inspection of stored red blood cells, permitting a true inventory system for the modern blood bank.

(3) 29,30

This is a recursive fractal model that connects the average number density of interactions, Nτ, to the time frame of τ, within which they occur. The time scale, τc, is a normalization factor linked to the characteristic number density, N0τ, that defines the interaction volume. In our case, the dielectric response is related to the apparent dipole moment of bulk water structures, and consequently, τ is proportional to their mesoscopic dimension.47,48 In this case, the rate of interaction, represented by α, is really the ever-constant exchange between bulk and bound water around cytoplasmic entities. An example of such would be proteins like hemoglobin. The dielectric strength of the relaxation is related to the number density, n, of relaxing units and their apparent dipole

S Supporting Information *

n·⟨M2⟩

moment,49 ⟨M⟩, Δε ∼ kT . From Figure 3, one notes that, while both τ and Δε remain constant until the critical period (shaded gray in the figure), from day 10, α monotonically decreases up to the same critical moment. From NMR and Raman studies of sRBC, the oxygenation of hemoglobin and the buildup of lactate acid also show a linear dependence on the

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.7b02662. The table of the dielectric parameters for RBC cytoplasm as a function of days in cold storage for two different samples (PDF) D

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Evgeniya Levy: 0000-0001-6954-5576 Yuri Feldman: 0000-0002-8742-090X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We would like to thank Keysight Technologies Israel Ltd. for the loan of Vector Network Analyzer Agilent N5245A PNA-X. We are grateful to Dr. Ivan Popov for helpful discussion and to Ms. Olga Friedman for her technical assistance.

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REFERENCES

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DOI: 10.1021/acs.jpcb.7b02662 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.jpcb.7b02662 J. Phys. Chem. B XXXX, XXX, XXX−XXX