Non-irradiated Red Blood Cells Research Articles

The Effects of Medical Diagnostic Low Dose X-rays after in vitro Exposure of Human Red Blood Cells: Hemolysis and Osmotic Fragility

Although low dose X-rays are widely used in medical radiology, the harmful effects of exposure to low dose X-rays during diagnostic radiological examination is still a commonly discussed topic. In this current study, we investigated the effects of medical diagnostic low dose X-rays after an in vitro exposure of human red blood cells. We gave red blood cells an in vitro exposure to a total dose of 0.03, 0.05, and 0.1 mGy of medical diagnostic X-rays. Non-irradiated cells served as the control group. Hemolysis and osmotic fragility tests were used to determine the effects of medical diagnostic low dose X-rays at 10, 30, and 60 min post-irradiation. The results indicated no changes in the percentage of hemolysis and osmotic fragility parameters in red blood cells exposed to these low dose X-rays, when compared to the corresponding non-irradiated red blood cells at all harvest time-points. These finding suggested that there were no harmful effects of the diagnostic low dose X-rays in an in vitro exposure of human red blood cells.

Laser-induced changes of in vitro erythrocyte sedimentation rate.

The study of the effects of low-level laser (LLL) radiation on blood is important for elucidating the mechanisms behind the interaction of LLL radiation and biologic tissues. Different therapy methods that involve blood irradiation have been developed and used for clinical purposes with beneficial effects. The aim of this study was to compare the effects of different irradiation protocols using a diode-pumped solid-state LLL (λ=405nm) on samples of human blood by measuring the erythrocyte sedimentation rate (ESR). Human blood samples were obtained through venipuncture into tubes containing EDTA as an anticoagulant. Every sample was divided into two equal aliquots to be used as an irradiated sample and a non-irradiated control sample. The irradiated aliquot was subjected to a laser beam with a wavelength of 405nm and an energy density of 72J/cm2. The radiation source had a fixed irradiance of 30mW/cm2. The ESR change was observed for three different experimental protocols: irradiated whole blood, irradiated red blood cells (RBCs) samples re-suspended in non-irradiated blood plasma, and non-irradiated RBCs re-suspended in irradiated blood plasma. The ESR values were measured after laser irradiation and compared with the non-irradiated control samples. Irradiated blood plasma in which non-radiated RBCs were re-suspended was found to result in the largest ESR decrease for healthy human RBCs, 51%, when compared with RBCs re-suspended in non-irradiated blood plasma. The decrease in ESR induced by LLL irradiation of the plasma alone was likely related to changes in the plasma composition and an increase in the erythrocyte zeta potential upon re-suspension of the RBCs in the irradiated blood plasma.

Transfusion of irradiated red blood cell units with a potassium adsorption filter: A randomized controlled trial.

The irradiation of red blood cells (RBCs) causes damage of the RBC membrane with increased potassium (K) leak during storage compared with nonirradiated RBC units of similar age. A previous in vitro study showed a mean reduction of K of 94 ± 5% with a potassium adsorption filter (PAF). A prospective, single-center, nonblinded, randomized controlled trial (RCT) was designed to evaluate the safety and efficacy of transfusing irradiated RBC units with the PAF. Patients 18 years of age or older who received irradiated RBC units due to chemotherapy-induced anemia were randomly assigned to receive irradiated RBC units with the PAF (PAF group) or with the standard blood infusion set (control group). Primary outcome measures were safety and efficacy of the PAF (absolute change in hemoglobin [Hb] and K, respectively, in patient's blood values after transfusing the irradiated RBC units with or without the PAF). A total of 63 irradiated RBC units were transfused to 17 patients in the control group, and a total of 56 irradiated RBC units were transfused to 13 patients in the PAF group. The absolute change of Hb (9.3 ± 6.3 g/L vs. 8.1 ± 5.8 g/L; p = 0.3) and the absolute change of K (-0.01 ± 0.4 mmol/L vs. -0.01 ± 0.3 mmol/L; p = 0.2) were comparable between the two groups of the trial. The transfusion of 1 irradiated RBC unit with the PAF was as safe and efficacious as the transfusion of 1 irradiated RBC unit with the standard blood infusion set in patients with chemotherapy-induced anemia.

A moderate protective effect of quercetin against γ-irradiation- and storage-induced oxidative damage in red blood cells for transfusion

Purpose: To investigate the extent of γ-irradiation-induced oxidative membrane damage and antioxidant activity of quercetin in long-term, cold stored (4°C) acid-citrate-dextrose- preserved human red blood cells (RBC).Materials and methods: The extracellular activity of lactate dehydrogenase (LDH) was measured to assess RBC membrane integrity. Lipid peroxidation and reduced glutathione (GSH) levels were quantified by thiobarbituric acid-reactive substances (TBARS) and Ellman's reagent, respectively.Results: During storage of non-irradiated RBC (up 21 days) the LDH activity in the supernatant increased with time. In contrast to a low dose of ionizing radiation (30 Gy), irradiation at higher, but still clinically relevant doses, of 40–50 Gy resulted in elevation of the post-storage extracellular LDH activity. Quercetin (2–50 μM) dissolved in dimethyl sulfoxide (DMSO) significantly increased the LDH release in the irradiated and non-irradiated RBC, reflecting an increase of RBC membrane permeability. In the presence of ethanol as a solvent quercetin protected RBC against storage-induced oxidative damage – it inhibited the LDH release, GSH depletion, and lipid peroxidation.Conclusion: The level of protection offered by quercetin against the radiation- and storage-induced oxidative damage to RBC does not seem to be sufficient to warrant its application as an additive for conservation purposes. The findings indicate that the solvent can modulate a response of RBC to water-insoluble antioxidants changing their properties from anti-oxidative to pro-oxidative.

Does quercetin protect human red blood cell membranes against γ-irradiation?

ObjectivesRadioprotective potential of quercetin, a powerful free radical scavenger, was investigated in human red blood cells (RBCs) and in isolated RBC membranes exposed to γ-irradiation-induced oxidative stress.MethodsRBCs and RBC membrane suspensions were irradiated (50 Gy) in the presence of quercetin (2–50 µM). Oxidative damage of the membranes was analysed by protein carbonyl measurement (enzyme-linked immunosorbent assay). In RBCs, the concentration of glutathione (GSH) was determined. Lipid peroxidation in RBCs, and for comparison in plasma and peripheral lymphocytes, was quantified by the amount of thiobarbituric acid-reactive substances (TBARS). Radiation-induced damage of the RBC membrane integrity was evaluated by the degree of haemolysis.ResultsQuercetin (50 µM) brought back the level of carbonyls to normal in γ-irradiated RBC membrane proteins and inhibited radiation-induced lipid peroxidation in plasma and lymphocytes, by 75 and 96%, respectively. However, it moderately decreased reduced/oxidized glutathione (GSH/GSSG) ratio and significantly increased TBARS concentrations, by 60 and 28% in irradiated and non-irradiated RBCs, respectively. Haemolysis rate was much higher in RBCs irradiated in the presence of quercetin vs. non antioxidant.DiscussionIn non-cellular systems (RBC membranes or plasma) and in lymphocytes, quercetin shows antioxidative/radioprotective activity but in whole RBCs it acts as a pro-oxidant and a cytotoxic substance. The possible mechanisms of such action are discussed.

Genetic Diagnosis in Recently Transfused Patients

Analysis of recently transfused patients is usually postponed to avoid spurious results because of contamination with donor's cells. However, little is known about the extent of this influence in routine molecular diagnostic tests. To elucidate this question, we tested a mix of blood samples from 2 α-1-antitrypsin-deficient patients diagnosed as Pi*Z homozygous with 1 normal donor at 1:1, 1:10, 1:20, and 1:30 proportions. Human identification panel and Pi*Z allele detection were used to establish the detection limit of a blood mixture. Mixtures of 1:1 and 1:10 were easily detected with both techniques, whereas for 1:30, it was necessary to change the equipment settings to identify the mixture. Moreover, the heterozygous pattern observed for the mixtures on Pi*Z genotyping was weaker at this level of mixture. We further evaluated the degree of mixture detectable in 20 transfused patients who received 1 blood unit (concentrate of irradiated or nonirradiated red blood cells) using the human identification panel. Two days after the transfusion, the presence of the donor's markers was not detected, suggesting that after this time point the levels of admixture are below 1:30. The methods applied in the present study showed adequate sensitivity to identify alleles of the so-called "smaller population" of cells up to 3%, approximately. The same result was obtained in a "diagnostic situation," in which the blood mixture was submitted to a PCR-RFLP protocol to detect a mutation.

Transfusion‐associated graft‐versus‐host disease in a liver transplant recipient: an unusual presentation and review of the literature

Transfusion-associated graft-versus-host disease (TA-GVHD) is a rare, nearly universally fatal complication from transfusion of nonirradiated cellular blood components, occurring when a recipient's immune system is unable to recognize and destroy transfused T lymphocytes. Irradiation of cellular components eliminates this risk. We present an unusual case of a liver transplant recipient developing TA-GVHD 13 weeks after transfusion of a random unit of nonirradiated red blood cells (RBCs) that happened to be from a donor homozygous for an HLA haplotype shared by the patient. This study was a single case review of a liver transplant recipient who developed skin GVHD and marrow aplasia. Clinical course and the chimerism studies involving the patient, the liver donor, and the blood donor are detailed. The patient presented 3 months posttransplant with GVHD of his skin and marrow aplasia. In addition to standard antigraft immunosuppression, this patient had started the interleukin-1 receptor antagonist anakinra on Posttransplant Day 13 for an acute gout flare. Chimerism studies on the patient's peripheral blood identified a population of CD3 cells that did not originate with either the patient or his liver donor. HLA studies and microsatellite profiling of the unknown CD3 population identified the source of the patient's TA-GVHD, a unit of nonirradiated, nonleukoreduced apheresis RBCs. Use of an immunomodulating agent may have contributed to the development of TA-GVHD in a liver transplant patient who received a random unit of nonirradiated RBCs by chance from an unrelated haploidentical donor.

Influence of late irradiation on the in vitro RBC storage variables of leucoreduced RBCs in SAGM additive solution

There exists only few data on in vitro and in vivo effects of gamma irradiation of leucoreduced red blood cells (RBCs). Reported studies reflect the effects of early irradiation and subsequent storage. The effects of irradiation on RBCs shortly before the end of their shelf-life have not been examined. We studied 160 RBC units that were stored in the additive solution saline-adenine-glucose-mannitol and leucoreduced on the collection day. Forty components were irradiated on day +14 with 30 Gy, 40 on day +28, 40 on day +35, and 40 served as nonirradiated controls. In vitro evaluation of all units was performed on days +3, +7, +14, +21, +28, +35, and +42 from the collection day. Gamma irradiation induced leakage of potassium ions and lactate dehydrogenase and enhanced in vitro haemolysis rate in the irradiated components, which started to increase faster than that of nonirradiated RBCs from the day of irradiation, i.e. from day +14 in units that were irradiated on day +14, from day +28 in units that were irradiated on day +28, and from day +35 in units that were irradiated on day +35. This study presents data on the in vitro quality of leucoreduced RBCs that have been irradiated on days +14, +28, or +35 after collection. Our findings support the proposal that the current limitation of the age of RBCs on the day of gamma irradiation may be replaced by staged limitations depending on the time of irradiation.

Age of blood as a limitation for transfusion: potential impact on blood inventory and availability

Evolving concerns about storage lesions for red blood cells (RBCs) have led to ongoing trials evaluating the benefits of transfusing fresher blood to acutely ill patients. We evaluated several RBC maximum shelf lives (MSLs) and their impact on RBC availability and outdate rate. First, we determined the mean age of the RBC units in our inventory by analyzing the data set of 18,987 nonirradiated RBC units transfused at our institution from April 2008 to March 2009. Second, we determined the feasibility of issuing RBC units of a designated age to patients using the same data set. We defined six scenarios where RBC units have different MSLs: Scenarios 1, 2, 3, 4, and 5 used a MSL of 7, 14, 21, 28, and 35 days, respectively. Scenario 6 used a combination of different MSLs depending on the category of patients. RBC units spent on average 8.6 days on the shelf with a mean age of 10.2 days at delivery and 18.8 days at issue for transfusion. Using the original 18,987 data points, we observed a shortfall in the availability of RBC units, decreased by 51, 20, 10, 4, 1, and 0% and an increase in the outdate rate to 3.2, 2.2, 1.0, 0.6, 0.4, and 4.5% for Scenarios 1, 2, 3, 4, 5, and 6, respectively, compared to baseline. Changing the MSL for RBC units would require novel approaches to RBC inventory management to meet hospital demands with acceptable outdate rates.

Susceptibility of oxidative stress on red blood cells exposed to gamma rays: Hemorheological evaluation

Irradiation has been shown to induce biochemical changes in stored red blood cells (RBCs) and to generate reactive oxygen species (ROS). This study evaluated the hemorheological properties, the degree of lipid peroxidation and the oxidative susceptibility of irradiated RBCs. Furthermore, we investigated the radioprotective role of N-t-butyl hydroxylamine (NtBHA) against gamma-ray exposure of RBCs. RBC concentrates were irradiated with a minimum dose of 25 Gy, and were exposed to FeSO4 to examine the oxidative susceptibility. RBC deformability was evaluated by the use of a microfluidic ektacytometer, in relation to the hematological and biochemical properties. The deformability of the irradiated RBCs was significantly lower than that of control. Exposure to gamma rays significantly increased the mean corpuscular volume (MCV) and lipid peroxidation. Changes in RBC deformability were more prominent in irradiated RBCs than in non-irradiated RBCs also under conditions of oxidative stress. The deformability of NtBHA treated RBCs prior to irradiation was not altered as compared with irradiated RBCs not treated with NtBHA. In conclusion, irradiation reduces RBC deformability during storage and the irradiated RBCs seem susceptible to oxidative stress. NtBHA may not have a protective role against the effects of gamma-ray exposure in RBCs but further evaluation of NtBHA or another radioprotective compound is required.

Extracellular potassium concentrations in red blood cell suspensions after irradiation and washing

Extracellular potassium concentration [K(+)e] increases with duration of red blood cell storage. Sometimes red blood cells (RBC) are washed before transfusion to infants to reduce [K(+)e] of these components. AABB standards permit storage of washed RBCs at 4 degrees C for 24 hours. The [K(+)e] of washed RBCs during storage is not known. Experiments were performed to provide those data. One day after outdating, 26 RBC units were washed without irradiation or before or after irradiation (25 Gy), and [K(+)e] was measured for 24 hours. [K(+)e] was measured also immediately before transfusion of 29 nonoutdated irradiated and washed RBC units. After washing, [K(+)e] increased in a time-dependent fashion. [K(+)e] increased more rapidly in preparations of irradiated than nonirradiated RBCs. [K(+)e] was less after washing after irradiation (1.6 +/- 0.3, 2.4 +/- 0.3, 3.0 +/- 0.3, 3.6 +/- 0.3. 4.2 +/- 0.4, 5.3 +/- 0.5, 8.6 +/- 1.0, and 14.3 +/- 1.3 mEq/L at 0, 1, 2, 3, 4, 6, 12, and 24 hr; mean +/- SD) than washing before irradiation (p < 0.001). The increase in [K(+)e] during the first 6 hours after washing after irradiation was linear (0.61 +/- 0.08 mEq K(+)/L/hr). The probability of a unit of RBCs having a [K(+)e] greater than 5 mEq per L is 0.0 to 0.2 percent 3 hours after washing irradiated RBCs and 0.0 to 1.1 percent 6 hours after washing nonirradiated RBCs. [K(+)e] increases after washing irradiated and nonirradiated packed RBCs. After irradiation and washing, the [K(+)e] for the initial 6 hours can be predicted from the [K(+)e] immediately after washing. There is a low probability that a unit of RBCs would have a [K(+)e] greater than 5 mEq per L during 6 hours of storage at 4 degrees C after washing if the cells are not irradiated and for 3 hours if the cells are irradiated.

Leakage of Potassium from Red Blood Cells following Gamma Ray Irradiation in the Presence of Dipyridamole, Trolox, Human Plasma or Mannitol

Transfusion-associated graft-versus-host disease (TA-GVHD) is a fatal complication of blood transfusion resulting from the contamination of blood products by leukocytes. In order to prevent this disease, gamma or X-ray irradiation of blood components,which can inactivate leukocytes, is currently used. However, the minimal doses needed to destroy lymphocytes promote the leakage of potassium from red blood cells (RBCs), which can induce other side effects, such as hyperpotassemia and cardiac arrest. The reactive oxygen species (ROS) generated by the irradiation of aqueous solutions may accelerate the leakage through oxidation of the RBC membrane. Here we studied the effect of dipyridamole, Trolox, human plasma or mannitol on the leakage of potassium from RBCs following irradiation. RBC preparations (hematocrit; 30%) containing antioxidants were irradiated at 30 Gy and stored at 4 degrees C for 7 d. The leakage of potassium from the RBCs caused by the irradiation was significantly suppressed by dipyridamole (more than 50 microM), Trolox (more than 5 mM) or human plasma (50%). Mannitol (80 mM) is used to inhibit hemolysis as a constituent of MAP solution, which is a solution used for the storage of RBC products in Japan. Here it was clarified that the leakage of potassium from not only irradiated but also non-irradiated RBCs was unexpectedly promoted by mannitol. The amount of mannitol in MAP solution may have to be reconsidered. The osmotic pressure of the RBC preparation increased in a manner dependent on the concentration of mannitol. The elevated osmotic pressure may promote the leakage. In conclusion, although antioxidants have the potential to suppress the leakage of potassium ascribed to the irradiation, the extent of the suppression (10-20%) by dipyridamole (DPM), Trolox or human plasma seems insufficient for the clinical use of these agents as an additive for MAP solution.