Increased Oxidative Stress in Acute Myeloid Leukemia Patients

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Increased Oxidative Stress in Acute Myeloid Leukemia Patients

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Increased Oxidative Stress in Acute Myeloid Leukemia Patients after Red Blood Cell Transfusion, but Not Platelet Transfusion, Results Mainly from the Oxidative/Nitrative Protein Damage: An Exploratory Study

Kamila Czubak-Prowizor 1,2,* , Jacek Trelinski 3, Paulina Stelmach 4, Piotr Stelmach 4, Agnieszka Madon 5 and Halina Malgorzata Zbikowska 1

Citation: Czubak-Prowizor, K.; Trelinski, J.; Stelmach, P.; Stelmach, P.; Madon, A.; Zbikowska, H.M. Increased Oxidative Stress in Acute Myeloid Leukemia Patients after Red Blood Cell Transfusion, but Not Platelet Transfusion, Results Mainly from the Oxidative/Nitrative Protein Damage: An Exploratory Study. J. Clin. Med. 2021, 10, 1349. https:// doi.org/10.3390/jcm10071349
Academic Editor: Farhad Ravandi-Kashani
Received: 3 March 2021 Accepted: 23 March 2021 Published: 25 March 2021
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

1 Department of General Biochemistry, Faculty of Biology and Environmental Protection, University of Lodz, Pomorska 141/143, 90-236 Lodz, Poland; [email protected]uni.lodz.pl
2 Department of Cytobiology and Proteomics, Medical University of Lodz, Mazowiecka 6/8, 92-215 Lodz, Poland
3 Department of Coagulation Disorders, Medical University of Lodz, Ciolkowskiego 2, 93-510 Lodz, Poland; [email protected]
4 Department of Haematology, Medical University of Lodz, Copernicus Memorial Hospital, Ciolkowskiego 2, 93-510 Lodz, Poland; [email protected] (P.S.); [email protected] (P.S.)
5 Laboratory of Transfusion Serology and Blood Bank, Copernicus Memorial Hospital, Pabianicka 62, 93-513 Lodz, Poland; [email protected]
* Correspondence: [email protected] or [email protected]
Abstract: Chronic oxidative stress (OS) can be an important factor of acute myeloid leukemia (AML) progression; however, there are no data on the extent/consequence of OS after transfusion of packed red blood cells (pRBCs) and platelet concentrates (PCs), which are commonly used in the treatment of leukemia-associated anemia and thrombocytopenia. We aimed to investigate the effects of pRBC/PC transfusion on the OS markers, i.e., thiol and carbonyl (CO) groups, 3-nitrotyrosine (3-NT), thiobarbituric acid reactive substances (TBARS), advanced glycation end products (AGE), total antioxidant capacity (TAC), SOD, GST, and LDH, in the blood plasma of AML patients, before and 24 h post-transfusion. In this exploratory study, 52 patients were examined, of which 27 were transfused with pRBCs and 25 with PCs. Age-matched healthy subjects were also enrolled as controls. Our results showed the oxidation of thiols, increased 3-NT, AGE levels, and decreased TAC in AML groups versus controls. After pRBC transfusion, CO groups, AGE, and 3-NT significantly increased (by approximately 30, 23, and 35%; p < 0.05, p < 0.05, and p < 0.01, respectively) while thiols reduced (by 18%; p < 0.05). The PC transfusion resulted in the raise of TBARS and AGE (by 45%; p < 0.01 and 31%; p < 0.001), respectively). Other variables showed no significant post-transfusion changes. In conclusion, transfusion of both pRBCs and PCs was associated with an increased OS; however, transfusing the former may have more severe consequences, since it is associated with the irreversible oxidative/nitrative modifications of plasma proteins.
Keywords: acute myeloid leukemia; blood platelet; red blood cell; transfusion; oxidative stress markers

Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

1. Introduction
Acute myeloid leukemia (AML) is a hematologic malignancy distinguished by a rapid, uncontrolled clonal growth of myeloid lineage cells in the bone marrow. In AML patients, severe anemia and a bleeding risk remain significant clinical problems, resulting from the infiltration by leukemic cells in the bone marrow and the suppressive effect of a cytotoxic drug therapy [1]. In the treatment of deep, leukemia-associated anemia (hemoglobin (Hgb) level below 7 g/dL) or profound thrombocytopenia (platelet (PLT) count below 20,000/µL) transfusion of packed red blood cells (pRBCs) and platelet concentrates (PCs) are regularly used [2].

J. Clin. Med. 2021, 10, 1349. https://doi.org/10.3390/jcm10071349

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Blood transfusion is often associated with some risk factors, due to a genetic difference between the transfused blood cells and the tissues of the recipient’s system, as well as the possibility of transferring a plethora of biologically active molecules along with the blood components [3,4]. During their blood bank storage, in the presence of the additive solutions (up to 42 days, at 4 ± 2 ◦C), red blood cells (RBCs) undergo progressive aging-associated changes and oxidative damage, which are collectively referred to as the storage lesion [3]. Oxidative stress (OS) is an undesirable phenomenon that is well documented to occur in the stored pRBCs, in the majority due to the Hgb release and breakdown [3,5]. Platelets are stored for up to 5–7 days, at 22 ◦C, the PLT storage lesion is mainly coordinated by platelet-activating signals, which ultimately lead to platelet aggregation and release of granular contents and expression of sequestered membrane proteins (selectin-P, CD40L) on the outer surface [4]. Procedures such as leukocyte filtration and irradiation before storage also lead to a different degree of the lesion to RBCs/PLTs [6].
Evidence for chronic OS, caused by the increased production of reactive oxygen/ reactive nitrogen species (ROS/RNS) and/or depletion of the antioxidant defense systems, has been found in several hematopoietic malignancies including AML [7,8]. Some reports indicate that relapse in this disease is associated with increased OS markers within the leukemic blasts, suggesting that ROS production may be an important factor of AML progression [8]. Transfusion dependency at diagnosis and transfusion intensity during initial chemotherapy were associated with poorer outcomes in AML adult patients [1], but the underlying molecular mechanisms are not well understood. The current data on the post-transfusion OS in pRBC recipients are controversial and limited to studies on few selected patient groups (neonatal, critically ill, and receiving chronic transfusion therapy) [9–12], while the impact of PC transfusion on the redox imbalance has not been investigated yet.
This exploratory study aim was to investigate the effects of pRBC or PC transfusions on the OS level in plasma of AML patients. A wide panel of the OS markers, such as the concentrations of total thiols, carbonyl (CO) groups, 3-nitrotyrosine (3-NT), thiobarbituric acid reactive substances (TBARS) and advanced glycation end products (AGE), total antioxidant capacity (TAC), activities of superoxide dismutase (SOD) and glutathione transferase (GST), and lactic dehydrogenase (LDH), before and 24 h post-transfusion of the blood component, were assessed. The hypothesis for this study was that OS increases following blood transfusion. To our knowledge, this is the first study to examine the relationship between blood transfusion and OS in AML patients.
2. Materials and Methods 2.1. Chemicals
2,2 -azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 5,5 -dithio-bis-(2-nitrobenzoic acid) (DTNB), 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), hydrogen peroxide, nicotinamide adenine dinucleotide (NADH), reduced glutathione (GSH), 1-chloro2,4-dinitrobenzene (CDNB), sodium pyruvate, thiobarbituric acid (TBA), trichloroacetic acid (TCA), 2,4-dinitrophenylhydrazine (DNPH), anti-DNP antibody produced in rabbit, antirabbit IgG (whole molecule)–peroxidase antibody produced in goat, SOD Determination Kit, and Sigma Fast™ OPD Tablet Sets were purchased from Sigma-Aldrich Chemical Co., Warsaw, Poland. Pierce™ BCA Protein Assay Kit and rabbit anti-goat IgG secondary antibody biotin were from Thermo Fisher Scientific, Waltham, MA, USA. OxiSelect™ Advanced Glycation End Product (AGE) Competitive ELISA Kit was obtained from Cell Biolabs, San Diego, US. Anti-nitro tyrosine antibody and streptavidin (HRP) were purchased from Abcam, Cambridge, Great Britain. Other chemicals, all of the analytical grade, were obtained from POCh, Gliwice, Poland.
2.2. Study Design and Patient Selection
The study group was recruited between March 2017 and December 2018, among adult patients with AML (general condition 0–3 in the ECOG (Eastern Cooperative Oncology

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Group) scale) during a hospitalization at the Department of Hematology of the Copernicus Memorial Hospital in Lodz (Poland). In this exploratory study, 52 patients, diagnosed with AML on the basis of the diagnostic criteria of the WHO classification from 2008, participated in the study. Of these, 27 patients were transfused with the irradiated leukocyte-reduced pRBCs, and 25 patients were treated with the irradiated leukocyte-reduced PCs. Each patient could be included only one time.
Criteria for exclusion from the study were as follows: renal failure (GFR (glomerular filtration rate) <30 mL/min), liver failure (the activity of alanine aminotransferase (ALT) and/or aspartate aminotransferase (AST) three-fold upper limit of normal), autoimmune diseases, endocrine disorders, and other malignancies. During hospitalization, patients were not administered substances with antioxidant properties.
The control group comprised 43 healthy volunteers. The group of AML patients was comparable (homogeneous) with the control group in terms of age and sex. The mean age of the control group was 54.5 ± 9.5 years (ranged from 22 to 71 years), while the median was 55 years; women constituted about 45% of this group, men the remaining 55%. The volunteers were not diagnosed with any chronic disease or other diseases that could affect the analyzed parameters of oxidative stress.
2.3. Blood Collection and Isolation of Plasma
Venous blood from AML patients was drawn twice, before (approximately 30 min) and after (24 h) transfusion of the blood component. Blood samples were collected into the S-Monovette® blood collection tubes containing 3.8% citrate (Sarstedt, Nümbrecht, Germany) (blood to citrate ratio was 9:1). The collected blood was immediately (within 15 min after blood collection) centrifuged (10 min, 1800× g, 20 ◦C) to obtain citrated plasma. Each plasma sample was aliquoted (to avoid freeze-thaw cycles), frozen at −32 ◦C, and stored at this temperature until assayed, but no longer than a week after blood collection.
2.4. Measurement of the Oxidative Stress-Related Markers
All analyzes were performed in 96-well microplates and absorbances were measured in the plate reader (SPECTROstar® Nano BMG LABTECH GmbH; Offenburg, Germany).
2.4.1. Protein Oxidative/Nitrative Modifications
The total plasma thiols were determined with Ellman’s reagent (DTNB) [13,14]. Briefly, 10 mM phosphate buffer (pH 8.0) containing 10% SDS (20 µL) was mixed with plasma (20 µL). Then, 160 µL of the same buffer was added and the absorbance (A0) was measured at 412 nm. Next, 0.04% DTNB in 10 mM phosphate buffer (pH 8.0) was added (16.6 µL). After incubation (1 h, 37 ◦C), the absorbance at 412 nm (A1) was recorded. The concentration of thiols was calculated based on the absorbance difference (A1–A0) using the molar extinction coefficient (ε = 13,600 M−1 cm−1).
The protein CO groups were detected by the enzyme-linked immunosorbent assay (ELISA) according to Buss et al. [15] with modifications by Alamdari et al. [16]. Wells of a 96-well microplate were coated with plasma (10 µg protein/mL; 100 µL) and incubated overnight at 4 ◦C. Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific; Waltham, MA, USA) was used to measure the total plasma protein concentration. Plasma samples adsorbed to wells were reacted with 0.0024% DNPH solution (200 µL, pH 6.2) and probed with the rabbit anti-DNP primary antibody (200 µL; 60 min) followed by a second antirabbit antibody conjugated with horseradish peroxidase (200 µL, 60 min). More details on the method were previously described [17].
Detection of 3-NT in the plasma proteins was carried out by a competitive ELISA, according to Khan et al. [18], as described previously [19] with some modifications. Wells of a 96-well microplate were coated with nitrated human fibrinogen (3-NT-Fg; standard antigen, 100 µL/well) at a concentration of 1 µg/mL and incubated overnight at 4 ◦C. Wells were washed, blocked with 1% milk, and washed again. Blood plasma (free antigen) was pre-incubated (30 min, ambient temperature) with the diluted (1:40,000) anti-3-NT antibody

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(plasma:antibody was 1:1), and was then added to the wells (incubation: overnight at 4 ◦C). Next, wells (after washing) were probed with the biotinylated secondary antibody (100 µL; 60 min; 1: 2000) followed by the streptavidin complex with biotinylated horseradish peroxidase (100 µL; 60 min, 1:10,000). OPD substrate solution (Sigma Fast™ OPD Tablet Sets) was applied, and after incubation (10 min), the reaction was stopped by the addition of 40% sulfuric acid (50 µL). The absorbance was measured at 490 nm. The calibration curve (0.0018–2.0 µg/mL) was prepared by serial dilutions of nitrated Fg. The nitration was performed by treating the solution of human Fg (2 mg/mL) with 1 mM peroxynitrite. The 3-NT concentration in Fg was determined spectrophotometrically by measuring the absorbance at 430 nm, after which it was calculated using the molar extinction coefficient (ε = 4400 M−1 cm−1).
2.4.2. Lipid Peroxidation Lipid peroxidation was quantified by measuring the concentration of TBARS [14,20].
Briefly, equal volumes of plasma and 15% trichloroacetic acid (TCA) containing 0.25 M HCl were mixed and incubated in an ice bath (30 min). After centrifugation (7000× g, 10 min), the supernatant (200 µL) was collected into clean tubes and 200 µL 0.37% TBA containing 0.25 M HCl was added. Samples were incubated (10 min, 100 ◦C), and after cooling, equal volumes of clear solutions were transferred to wells on a 96-well microplate. The absorbance was measured at 535 nm, the TBARS concentration was calculated using the molar extinction coefficient for MDA (ε = 156,000 M−1 cm−1).
2.4.3. Advanced Glycation End Products The content of AGE was evaluated using OxiSelect™ Advanced Glycation End Prod-
uct (AGE) Competitive ELISA Kit (Cell Biolabs; San Diego, CA, USA). The assay was performed according to the manufacturer protocol. An aliquot of 50 µL of plasma (or AGE-BSA standard) was applied. A standard curve for AGE-BSA was prepared in the concentration range (0–100 µg/mL).
2.4.4. Total Antioxidant Capacity TAC was assessed by the method of Erel [21]. The details of the method were previ-
ously described [22]. A calibration curve in the concentration range of 0.008–1.0 µmol/mL was prepared using Trolox, a reference antioxidant. The TAC of plasma was calculated on the basis of differences in absorbance A1–A0 and the standard curve.
2.4.5. SOD Activity SOD activity was determined using SOD Determination Kit (Sigma-Aldrich; St. Louis,
MO, USA). The assay was performed according to the manufacturer protocol. The SOD activity was expressed as the inhibition rate (%).
2.4.6. GST Activity GST activity was estimated as described by Habig et al. [23], using 1-chloro-2,
4-dinitrobenzene (CDNB) and GSH as substrates. The GST activity was calculated using the millimolar extinction coefficient for 2,4-dinitrophenyl-S-glutathione (ε = 9.6 mM−1 cm−1).
2.4.7. LDH Activity LDH activity was estimated by the method of Wroblewski and La Due [24] and it was
calculated in international units per liter (IU/L).
2.4.8. Statistical Analysis All results are presented as mean values ± standard error (SE). Each plasma sample
was performed in triplicate. The normal distribution of the results was analyzed using a Shapiro–Wilk test. Then, homogeneity of variance was tested using the Brown-Forsythe test. In the next step, a two-way analysis of variance (ANOVA) followed by Tukey’s

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post-hoc test was performed to assess the significance of differences between values. All data were analyzed using statistical software StatSoft Inc. “Statistica” v. 13.1. p < 0.05 was accepted as statistically significant. All presented figures were prepared using GraphPad Prism 5 Software.
3. Results
Fifty-two patients were included, of which 27 were transfused with pRBCs (24 patients received two pRBC units, 3 patients received one pRBC unit) and 25 were transfused with PCs (all received one unit). Detailed characteristics of the patients enrolled in the study are presented in Table 1. The storage time of the pRBCs was in majority between 2 to 10 days; only four of the transfused units had been stored longer (up to 11–26 days). Therefore, it was not possible to assess the impact of the RBC storage duration on the OS level. Likewise, only five patients received “older” PCs (stored more than 3 days).

Table 1. Characteristics of acute myeloid leukemia (AML) patients before and 24 h after transfusion of red blood cells or platelets.

Transfused Blood Component

pRBCs n = 27

PCs n = 25

Age (years) [median (range)]

59 (26–74)

58 (37–89)

Gender (female/male)

10/17

13/12

Blood component “age” [median (range)]

6.00 (2.00–26.00)

3.00 (1.00–5.00)

PLT [×103/µL] (150.00–400.00) *
Hgb [g/dL] (11.00–15.20) * RBC [×106/µL] (3.50–5.00) * WBC [×103/µL] (4.40–11.30) *
INR (0.80–1.20) *
APTT [s] (26.00–40.00) * Fibrinogen [mg/dL] (200.00–393.00) * Ferritin [ng/mL] (13.00–400.00) * CRP [mg/L] (0.00–5.00) *

before after before after before after before after before after before after before after before after before after

30.00 (4.00–256.00) 29.00 (3.00–226.00)
7.50 (6.20–8.90) 8.60 (6.90–10.40) 2.39 (1.85–2.95) 2.85 (2.15–3.43) 0.78 (0.03–135.48) 0.66 (0.03–86.27) 1.23 (0.89–1.41) 1.23 (0.90–1.38) 31.90 (22.70–49.70) 31.15 (22.80–51.30) 375.00 (190.00–850.00) 324.00 (176.00–894.00) 1774.50 (688.70–7666.00) 1720.00 (671.00–4957.00) 31.40 (1.00–386.90) 32.61 (1.00–483.33)

12.00 (3.00–35.00) 36.00 (2.00–88.00) 8.40 (7.20–10.90)
8.30 (6.5–10.5) 2.86 (2.24–3.49) 2.82 (2.12–3.56) 0.49 (0.07–27.16) 0.64 (0.08–19.20) 1.20 (0.92–1.91) 1.19 (0.88–1.54) 32.90 (24.30–54.00) 32.30 (24.50–53.50) 351.00 (128.00–804.00) 326.00 (156.00–778.00) 1170.00 (199.60–1329.00) 1185.00 (173.50–2260.00) 39.32 (1.97–306.90) 33.80 (2.08–313.20)

The values of the determined parameters are presented as medians (range of values). APTT—the activated partial thromboplastin time; CRP—C-reactive protein; Hgb—hemoglobin; INR—normalized prothrombin time; PCs—platelet concentrates; PLT—platelets; pRBCs—packed red blood cells; RBC—red blood cells; WBC—white blood cells; *—normal ranges.

As shown in Figure 1, the concentration of thiols in all AML patients (before transfusion) was found to be reduced by approximately 30% (p < 0.001) compared to the control group. After the pRBC transfusion, a statistically significant decrease (by 18%; p < 0.05) in the plasma thiols was noted, compared to before transfusion (Figure 1).
Significant post-transfusion increases (by approximately 30 and 35%; p < 0.05 and p < 0.01, respectively) in the amount of CO groups and 3-NT level were seen only in the case of pRBC transfusion, compared to pre-transfusion values (Figures 2 and 3).

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As shown in Figure 1, the concentration of thiols in all AML patients (before transfusion) was found to be reduced by approximately 30% (p < 0.001) compared to the control group. After the pRBC transfusion, a statistically significant decrease (by 18%;6pof<16 0.05) in the plasma thiols was noted, compared to before transfusion (Figure 1).

Figure 1. The concentration of total thiols in the plasma of healthy volunteers (control group) and FiAguMreL 1p.aTtiheenctsonbceefonrteraatinodn aofftetor ttarlatnhsifoulssioinntohfetphleasbmloaodofchoemalpthoynevnotl.uSnctaetetresr(pcolonttsroslhgorwouapll) oafntdhe J. Clin. Med. 2021, 10, x FOR PEER REAVIMdEaWLtapvaatileunetssobbetfaoirneeadn. dDaafttaearrterapnrsefsuesnitoendoafsthmeebalnoo±dScEo.m**p*opn Significant post-transfusion increases (by approximately 30 and 35%; p < 0.05 and p < 0.01, respectively) in the amount of CO groups and 3-NT level were seen only in the case of pRBC transfusion, compared to pre-transfusion values (Figures 2 and 3).
Pre-transfusion (baseline) levels of 3-NT and AGE were found to be significantly higher (above 60% and 54–78%, respectively; p < 0.01 or p < 0.001), whereas the TAC of plasma and SOD activity were significantly less than in healthy volunteers. However, the baseline TAC differed significantly between the patient groups (p < 0.05), whereas the reduced SOD activity, compared to control, was noted only in the group receiving PLTs. Total antioxidant capacity considers the cumulative effect of all plasma antioxidants. Similarly, LDH activity (a marker of cell damage) was significantly higher (by 65%; p < 0.01) in AML patients prior to PC transfusion versus the volunteers. There were no significant differences in the levels of other markers between patients with AML and the control group.
Transfusion of the PCs, but not pRBCs, resulted in enhanced lipid peroxidation, measured by the concentration of TBARS (Figure 4). The post-transfusion increase in TBARS level (by 45%; p < 0.01) compared to the value before transfusion was shown. Interestingly, an increase in plasma AGE concentration, compared to that of pretransfusion, was noted both in patients receiving pRBCs (by approximately 23%; p < 0.05) and those transfused with the PCs (approximately 31%; p < 0.001) (Figure 5).
Figure 2. The carbonyl group content in plasma proteins in healthy volunteers (control group) Figure 2. The carbonyl group content in plasma proteins in healthy volunteers (control group) and AManLdpAatMieLntspbateifeonrtesabnedfoarfetearntrdanasfftuersitornanosfftuhseiobnlooofdtchoembploonodenct.oSmcaptotenrepnlto. tsScshatotwer aplllootfsthsheodwataall vaoluf eths eobdtaatianevda.luDeastaoabrteaipnreeds.enDtaedtaaasrme pearens±enStEe.d#aps
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FigFiugruere33.. TThhee lleevveellofo3f-N3-TNiTn pinlaspmlaaspmroateipnrsoitneihnesaltihny hveoalulnthteyervs o(clounntrtoelergsro(ucpo)natnrdolAgMroLup) and paptiaetinentsts bbeeffoorreeanadnadftearfttrearnstfruasinosnfuofstihoenbloofodthcoembploonoednt.cSocmattperopnloetnsts.hSowcaatltleorf tphleodtsatashvaolwuesall of th vaolubteasinoebd.taDianteada.reDparetaseantreedparsemseeannte±dSaEs. *m* pe versus PCs; statistically significant according to the Tukey test. pRBCs—packed red blood cells;
pRPBCCs—s pvleartesluetscPonCcse;ntsrtaatetiss. tically significant according to the Tukey test. pRBCs—packed red cells; PCs—platelet concentrates.
Pre-transfusion (baseline) levels of 3-NT and AGE were found to be significantly higher (above 60% and 54–78%, respectively; p < 0.01 or p < 0.001), whereas the TAC of plasma and SOD activity were significantly less than in healthy volunteers. However, the baseline TAC differed significantly between the patient groups (p < 0.05), whereas the reduced SOD activity, compared to control, was noted only in the group receiving PLTs. Total antioxidant capacity considers the cumulative effect of all plasma antioxidants. Similarly, LDH activity (a marker of cell damage) was significantly higher (by 65%; p < 0.01) in AML patients prior to PC transfusion versus the volunteers. There were no significant differences in the levels of other markers between patients with AML and the control group.
Transfusion of the PCs, but not pRBCs, resulted in enhanced lipid peroxidation, measured by the concentration of TBARS (Figure 4). The post-transfusion increase in TBARS level (by 45%; p < 0.01) compared to the value before transfusion was shown. Interestingly, an increase in plasma AGE concentration, compared to that of pre-transfusion, was noted both in patients receiving pRBCs (by approximately 23%; p < 0.05) and those transfused with the PCs (approximately 31%; p < 0.001) (Figure 5).

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FigFuigruer4e. T4.heThcoenccoennctreanttiroantiofnthoifotbhairobbiaturbrictuarciicdarceiadctrievaectsiuvbestsaunbcsetsan(TceBsA(RTSB)AinRSth)einpltahsempalaosfma of J. Clin. Med. 2021, 10, x FOR PEER REhVeIhaEleWtahlythvyolvuonlutenetrese(rcso(nctoronltrgorlogurpo)uapn)daAndMAL MpaLtiepnattsiebnetfsorbeeafonrde aafntedr atrfatenrsfturasinosnfuosfiothneobflot1oh0deobflo19od
cocmopmopnoennet.nStc. aStctearttperloptslosthsoswhoawll oaflltohfetdhaetadavtaaluveasluoebstaoibnteadi.nDeda.taDaarteaparreesepnretesdenatsedmaesanm±eaSnE.±##SE. p <##0.0p1 PCs—platelet concentrates.
Figure 5. The concentration of advanced glycation end products (AGE) in the plasma of healthy Fivgoulruen5t.eTerhse(ccoonnctreonltrgartoiuonp)oafnaddvAaMncLedpagtliyecnattsiobnefeonred apnrdodauftcetrs t(rAaGnsEfu) isniotnheofptlhasembalooofdhceoamlthpyonent. volunteers (control group) and AML patients before and after transfusion of the blood component. ScSacttaetrteprloptlsotsshoshwowallaolfl othfethdeatdaavtaalvuaelsuoebstoabintaeidn.eDda. tDa aatrae aprreespernetseedntaesdmaesamn e±aSnE±. **S*Ep. *<*0* .p00<10.001 cocmopmapreadretdo ttohethceonctornotlrgorlogurpo;u#pp; #< 0p.0<5,0#.0#5#,p#<##0.p00<1 0c.o0m01pacoremdppaorsetd- vpeorsstu-svperres-utsrapnrsef-utsriaonns;fu^^si^on; p <ˆˆ0ˆ .p00<10c.o00m1pcaormedpaproesdt-ptroasnts-ftruasniosnfupsiRoBnCpsRvBeCrssuvsePrsCuss; sPtCatsi;stsitcaatlilsyticsiagllnyifsiicgannitfiaccacnotrdacincogrtdointhgeto the TuTkuekyeytetset.stp. RpBRCBsC—s—papcakcekdedrerdedblboloododceclelsl;lsP; CPsC—s—plpaltaetleltectocnocnecnetnrtartaetse.s.

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When comparing antioxidant defense markers (TAC and activities of SOD, GST), we
foundWthhatenthceosme vpaarriianbgleasnstihooxwideadntndoesfiegnnsieficmaanrtkcehrasn(gTeAaCftaenrdtraacntsivfuitsiieosno(fnSeOitDhe,rGpSRTB),Cwse
noforuPnCds)thcaotmthpeasreedvatroiatbhleessesphroiworedtontorasnigsfnuisfiicoannt(Fcihgaunrgese 6a–ft8e)r. tMraonrsefouvseiorn, n(onesittahteisrtpicRaBllCy s singonrifPicCans)t cpoomstp- avreerdsutsopthree-sterapnrsifourstioontrcahnasnfugseisoinn(LFDigHuraecst6iv–i8t)y. wMeorreefoovuenr,dn(oFisgtautriest9ic).ally
significant post- versus pre-transfusion changes in LDH activity were found (Figure 9).

J. Clin. Med. 2021, 10, x FOR PEER REFViIFgEiuWgruere6.6T. hTehTeATCACofopflapslmasamian ihnehaletahlythvyovluonlutenetrese(rcso(nctornotlrgorlogurpo)uapn)danAdMALMpLatpieanttisenbtesfobre1ef2oaronefda1n9 d afateftretrratrnasnfusfsuiosinonofotfhtehbelobolodocdocmopmopnoennet.nSt.caStctaetrteprloptlsosths oswhoawllaolfl tohfethdeatdaavtaalvuaelsuoebstoabintaeidn.eDda. tDa ata araereprperseesnetnedtedasams emanea±nS±E. S* Ep.<*0p.0<5, 0**.0*5p, <**0*.0p01< c0o.0m0p1acroemd tpoatrhede ctoontthroelcgornoturpol; #grpo pRpBRCBCs—s—papcakcekdedrerdedblbolododceclelsll;sP; CPCs—s—plpaltaetleeltect ocnocnecnentrtartaetse.s.

Figure 7. The activity of plasma SOD in healthy volunteers (control group) and AML patients before Figure 7. The activity of plasma SOD in healthy volunteers (control group) and AML patients beafnordeaafntedratfrtaenrstfruasnisofnusoifonthoefbthloeobdlocoodmcpoomnpenotn.eSnct.aSttcearttpelroptslosthsoswhoawll aolfl othfethdeadtaatvaavluaeluseosbtained. obDtaaitnaeadr.eDparteaseanretepdreassemnteeadna±s mSEea. n* p± concentrates.

J. Clin. Med. 2021, 10, x FOR PEER REVIEW J. Clin. Med. 2021, 10, 1349

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FiFgiugruere8.8.TThheeaaccttiivviittyy ooffppllaassmmaaGGSTSTinihnehaletahlythvyolvunotleuenrste(ecrosnt(rcoolngtrrooulpg)raonudpA) ManLdpAatMienLtspbaetfioernets

beafnodreafatnerdtraafntesfrutsriaonnsoffutshieobnlooofdthcoembplooondencto. Smcaptotenrepnlot.tsSschaottwerapllloofttshsehdoawta vaallluoefstohbetadinatead.vDalautaes

obatraeinpereds.eDntaetda aasrempearens±enSteEd. ˆaps
PCsisg;nsitfiactaisntticaaclcloyrdsiinggnitfoictahne tTauckceoyrtdeisnt.gptRoBtChse—Tpuakcekyedteresdt. bplRooBdCcse—llsp; aPcCkse—dprleadtelbeltocoodncceenltlrsa;tPesC. s—

platelet concentrates.

FigFuigruer9e.9T. hTeheacatcitvivitiytyooff eexxttrraacceelllluulalarrLLDDHHininhehaeltahlythvyolvuonltueenrtse(ecrosn(tcroolngtrrooul pg)roanudp)AaMnLd pAaMtieLnts pabtieefnotrse baenfdoraeftaenr dtraanftsefrustrioannsoffutshioenbloofotdhecobmlopoodnecnotm. pSocantetenrt.pSloctasttsehropwloatlsl sohfotwheadllatoaf vthaleudesata valoubetasionbedta. iDnaetda.aDreatpareasreenpterdesaesnmteedana±s mSEe.a*n* ±p 4. Discussion
This study has evaluated the impact of pRBC transfusion on the OS level in AML
MedClinTransfusionVolunteersAml Patients