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RESEARCH ARTICLE
Polyvinylalcohol-carbazate (PVAC) reduces red
blood cell hemolysis
Felix SellbergID1, Fanny Fredriksson2, Thomas Engstrand3, Tim Melander Bowden4,
Bo Nilsson1, Jaan Hong1, Folke Knutson1, David Berglund1*
1 Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden, 2 Department
of Women’s and Children’s Health, Section of Pediatric Surgery, Uppsala University, Uppsala, Sweden,
3 Department of Surgical Sciences, Uppsala University, Uppsala, Sweden, 4 Department of Chemistry—
Ångstrom Laboratory, Uppsala University, Uppsala, Sweden
Erythrocyte concentrates were split into 5-mL portions. PVAC was dissolved in NaCl (250 μL)
and added to the erythrocytes over a broad range of concentrations. As control, NaCl alone
was added at a volume equivalent to that of the PVAC solution. Erythrocyte concentrates were
stored in cold (4˚C) or room temperature (20˚C) for 2 weeks in polypropylene test tubes. For
erythrocytes stored at room temperature more PVAC was added after one week because the
hemolysis was expected to be greater in warm compared to cold storage. During contemporary
storage of RBC the temperature is lowered to stop the metabolism and increase pH during
storage. [14] Experiments in cold storage were done with six donors in duplicates and experi-
ments in warm storage were done with samples from two donors in duplicates. Supernatant
samples were regularly collected from the erythrocyte concentrates after centrifugation (2000 x
g for 10 m). Free Hb was measured with the plasma-low Hb method (Hemocue, Angelholm,
Sweden). The percentage of hemolysis was calculated by dividing the supernatant concentra-
tion by the total hemoglobin concentration for each sample.
Storage of RBC in 96-well plates
Erythrocyte concentrates were diluted in of NaCl (v/v 100/100 μL) with or without additional
PVAC, PVA (Sigma) and ethyl-carbazate (EC) (Sigma). The final volume of 200 μl was added
to a round bottom 96-well cell culture plate with a lid, allowing oxygen exchange, and stored
for one month at 4˚C. Plates were assayed for hemolysis at day 0, 4, 7, 14, 21 and 28. At analysis
the plate was spun down (2000 g x 10 min) and supernatant (100 μL) was analyzed for absor-
bance (540 nm) using a Synergy HTX plate reader (BioTek). The 540-nm wavelength was used
because it represents the absorbance peak for the oxygenated state of hemoglobin, the most
common state during storage.
Assessment of hemoglobin levels
To exclude any direct interference of PVAC with the detection of hemolysis, whole blood was
centrifuged (2000 x g for 10 min) and the erythrocyte portion was lysed using ethanol and
recentrifuged. Free Hb was obtained and dissolved in PBS to a concentration of 10 g/L. PVAC
was added to the free Hb and incubated for 2 h before centrifugation (2000 x g for 10 min).
Free Hb was measured with the plasma-low Hb method (Hemocue). The spectrum was also
Fig 1. The chemical structure of PVAC. Polyvinylalcohol-carbazate (PVAC) condensation reaction with aldehyde at neutral conditions leads to the formation of a stable
carbazone adduct and a water molecule. Unmodified repeat units of PVA are denoted with n and carbazate groups conjugated to repeat units are denoted by m. The level
of substitution of PVA with carbazate groups is about 10% (n = 0.9; m = 0.1).
https://doi.org/10.1371/journal.pone.0225777.g001
PVAC reduces red blood cell hemolysis
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isotonic NaCl or PBS. However, in a hypotonic solution an increase in binding was observed.
MFI for cells in hypotonic solution was increased, 1021 vs 335 (mean difference 686, 389–983,
p =<0.001). (Fig 3B) Altered concentrations of NaCl in the hypotonic solution did not differ.
Aldehyde resistance after storage
Aldehyde levels 60 min after addition of acetaldehyde showed that RBC from four donors
stored in PVAC were more efficient to neutralize acetaldehyde. The effect was dose dependent.
Addition of 2.5 mg/mL PVAC reduced the amount of free aldehydes by 55% (p =<0.001).
(Fig 4)
Blood compatibility
Blood compatibility was studied in a whole blood model where whole blood from three circu-
lated in chambers for 2 h with adding either PVAC, a stimulating material (thrombogenic
metal) or NaCl and subsequently sampled for analyses. PVAC did not activate the coagulation
system as levels of TPC and TAT were comparable to the control. The complement cascade,
assessed by TCC and C3a, was similarly unaffected compared to the control. (Fig 5)
Mass spectrometry
PVAC conjugated to biotin associated with beads showed no difference in the gel pattern or in
the final MS analysis between the proteins isolated with or without additional PVAC. For
detailed results please see the supporting information. (S1 File and S3 and S4 Figs)
Fig 2. RBC stored in test tubes with PVAC added to the solution. (a) RBC stored in test tubes for two weeks at 4˚C, a dose-dependent reduction of hemolysis was
seen with increasing doses of PVAC. 2.5 (0.25%, 0.11–0.35, p = 0.0048) and 0.5 (0.17%, 0.01–0.33, p = 0.0478) mg/mL of PVAC compared to the control. (b) RBC
stored in test tubes for two weeks at room temperature (20˚C), where hemolysis was measured three times a week. A second dose of PVAC or NaCl was added after
one week. Mean hemolysis in the control after two weeks was 3.1% and the addition of PVAC lowered hemolysis in all groups: 2.5 (1.75%, p< 0.001), 0.5 (1.86%,
RBC from four donors stored in the presence of a low-molecular-weight carbazate (ethyl-car-
bazate, EC), PVA backbone and, PVAC displayed different patterns of hemolysis over time.
During the first few days of storage the groups did not differ, but after 14 days all but the lowest
concentration of EC (2.5 mg/mL, 500 and, 100 μg/mL) had a higher level of hemolysis com-
pared to the control. (Fig 6A) After one month storage of RBC (from eight different blood
donors), all concentrations of EC (2.5 mg/mL, 500, 100 and, 20 μg/mL, 288.5, 257.8, 120.5 and,
24.9% mean increase, respectively) and the lowest concentration of PVA (20 μg/mL, 34.1%
mean increase) had a higher level of hemolysis compared to the control. All concentrations
but 20 μg/mL of PVAC lowered the level of hemolysis (2.5 mg/mL, 500 and, 100 μg/mL, 39.6,
22,1 and, 19.6% mean decrease, respectively). (Fig 6B)
Fig 3. Osmotic stability of RBC stored with PVAC and association studied with flow cytometry. (a) RBC were exposed to a hypotonic solution and sampled
for free hemoglobin. Hemolysis was measured as absorbance at 540 nm, with values normalized to the control. Addition of PVAC resulted in a dose-dependent
reduction of hemolysis, with a mean reduction of 63% for PVAC 2.5 mg/mL compared to the control (p = 0.0026). (b) RBC incubated with FITC-conjugated
PVAC in a hypotonic or isotonic solution. The MFI was higher when the RBC were incubated in a hypotonic solution compared to an isotonic solution, mean
difference 686 (389–983, p =<0.001).
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PVAC reduces red blood cell hemolysis
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room temperature and that PVAC can act as an inhibitor when additional stress is put on the
cells.
One of the factors contributing to the storage lesion is oxidative injury, [17] with previous
studies linking increasing concentrations of malondialdehyde (MDA) to the progress of the
storage lesion. [12] RBC stored in PVAC exhibited an increased aldehyde binding capacity,
which could help blood storage by preventing oxidative by-products from injuring the RBC.
In addition, when red blood cells were tested for osmotic fragility after exposure to PVAC, the
level of hemolysis decreased substantially. PVAC thus seems to also provide a membrane stabi-
lizing effect, making erythrocytes more resistant to lysis with hypotonic solution. When ana-
lyzed with flow cytometry the signal for RBC increased more than threefold when the cells
were in hypotonic solution. This along with the membrane stabilizing effect indicate that
PVAC associates directly with RBC during stress. When separately assessing the different com-
ponents of PVAC, a low-molecular-weight carbazate (ethyl-carbazate) negatively affected stor-
age resulting in an increase of hemolysis after storage for one month. The backbone used in
PVAC synthesis, PVA, also resulted in an increase in hemolysis after one month of storage at
certain concentrations. Still, this effect was not as pronounced as that of ethyl-carbazate.
Indeed, PVA seemed rather inert in comparison, which is not unexpected considering its
widespread use in materials commonly used in medicine and elsewhere. It is therefore not the
individual chemical properties of either the carbazate moiety or the PVA backbone alone that
are responsible for the observed effects but rather their combination that results in properties
that are able to reduce hemolysis.
Based on the present study, PVAC seems to be a promising agent worthy of further investi-
gation for its ability to reduce hemolysis. So far, no in vivo toxicity has been observed with
PVAC, even in very high concentrations administered intravenously in rodents (unpublished
data). Future studies will be aimed at assessing the biodistribution and clearance after in vivoadministration of PVAC to approach clinical applications. Whereas the best potential clinical
use for PVAC remains to be determined, the broader concept of neutralizing aldehydes and
other reactive components through activated polymers serves as a proof-of-concept for a novel
group of biologically active substances.
Supporting information
S1 Fig. Solubility of PVAC with varying degrees of conjugation.
(TIF)
S2 Fig. Ligand binding capabilities with varying degrees of conjugation.
(TIF)
S3 Fig. Association of biotin-PVAC with magnetic anti-biotin beads.
(TIF)
S4 Fig. Gel picture of bands isolated via IP.
(TIF)
S5 Fig. Abosorbance spectra of hemoglobin in the presence of PVAC.
(TIF)
S6 Fig. Raw image file for gel picture.
(TIF)
S1 File. Detailed description of experiments, in addition results from MS and IP.
(DOCX)
PVAC reduces red blood cell hemolysis
PLOS ONE | https://doi.org/10.1371/journal.pone.0225777 December 6, 2019 11 / 13