Influence of prestorage leukoreduction and subsequent irradiation on in vitro red blood cell (RBC) storage variables of RBCs in additive solution saline-adenine-glucose-mannitol
Robert Zimmermann, MD, Transfusionsmedizinische und Haemostaseologische Abteilung, Universitätsklinikum Erlangen, Krankenhausstrasse 12, D-91054 Erlangen, Germany; e-mail: email@example.com.
BACKGROUND: There exists only very few data on in vitro and in vivo effects of gamma irradiation of red blood cells (RBCs) that have been leukoreduced by filtration before a subsequent irradiation. Reported studies reflect neither the current Food and Drug Administration (FDA) nor the European recommendations on timing of irradiation and subsequent storage.
STUDY DESIGN AND METHODS: We studied 40 RBC units that were prepared from inline filtered whole blood and 40 RBC units that were filtered after component separation. All RBCs were stored in the additive solution saline-adenine-glucose-mannitol and leukoreduced on the collection day. In both groups, 20 components were irradiated on Day +14 with 30 Gy, and 20 served as nonirradiated controls. In vitro evaluation of both irradiated and nonirradiated RBC units was performed before and after irradiation on Days +1, +7, +14, +21, +28, +35, and +42 from the collection day.
RESULTS: Gamma irradiation induced enhanced leakage of potassium ions and lactate dehydrogenase and an enhanced in vitro hemolysis rate in the irradiated components. However, in vitro hemolysis rate of both nonirradiated and irradiated components was remarkably lower than 0.8 percent, and the preservation of adenosine triphosphate over 42 days was satisfying.
CONCLUSIONS: This study reflects the current FDA and European recommendations on timing of irradiation and subsequent storage. Our findings together with recent results of other investigations on the effect of gamma irradiation on leukoreduced RBCs allow the proposal that a storage time up to 28 days after irradiation is allowable.
- LR of RBCs
leukoreduction of red blood cells
- LR of WB
leukoreduction of whole blood
transfusion-associated graft-versus-host disease.
Irradiation of blood components with gamma rays is the strategy of choice to prevent transfusion-associated graft-versus-host disease (TA-GVHD) in severely immunocompromised recipients.1-3 Although gamma irradiation primarily targets nucleic acids of lymphocytes, which contaminate cellular blood components, the irradiation may also produce effects on nonlymphoid blood cells. These effects are small and probably without clinical significance when leukoreduced platelet (PLT) components are irradiated.4 Gamma irradiation of plasma results in a significant but very weak activation of the coagulation and fibrinolytic system.5 However, when red blood cells (RBCs) are irradiated, a remarkable alteration of the components' properties is noted.3 Irradiation of RBCs results in lipid peroxidation by reactive oxygen species, affects membrane integrity, decreases cell deformability and elasticity, accelerates the leakage of potassium ions, and alters intracellular purine nucleotides.6-11 Consequently, in vitro hemolysis of RBCs stored after irradiation is accelerated and in vivo recovery of transfused irradiated RBCs is decreased.1-3,12
Due to these observations, authorities limited the maximal storage time of irradiated RBCs. The US Food and Drug Administration (FDA) has recommended that irradiated RBCs should be stored until the end of shelf life, but no longer than 28 days from the time of irradiation.1 The Council of Europe has recommended that irradiation of RBCs should not be performed later than 14 days after collection and that irradiated RBCs should not be stored longer than 14 days after irradiation.13 These recommendations are based on data from irradiated RBCs that were not leukoreduced. However, it might be that the irradiation damage of RBCs is not only caused by the procedure itself but also by the degradation of white blood cells (WBCs), the contents of which might secondarily damage RBCs. Meanwhile, universal leukoreduction of cellular blood components has become standard in most European countries. In Germany universal leukoreduction was required after January 2001.14 The most relevant benefits that convinced the German authority Paul-Ehrlich Institute to mandate the universal leukoreduction of cellular blood components were the prevention of both transfusion-mediated immunosuppression15 and transmission of cell-associated pathogens.16 British hemovigilance data show that the introduction of leukoreduction reduced the frequency of TA-GVHD.17 Nevertheless, TA-GVHD may rarely be induced by leukoreduced components. Therefore, patients at risk for TA-GVHD get irradiated leukoreduced RBCs.
However, there exists only very few data on in vitro and in vivo effects of gamma irradiation on RBCs that have been leukoreduced by filtration before a subsequent irradiation. Davey and colleagues18 reported an improved in vivo 24-hour survival of irradiated and leukoreduced RBCs in the additive solution (AS) AS-3 in comparison to irradiated but not leukoreduced units. Wagner and Myrup19 compared in vitro variables of leukoreduced and irradiated, of not leukoreduced but irradiated, and of leukoreduced but not irradiated RBC units in the two different ASs AS-1 and AS-3. Leukoreduction significantly reduced the deleterious effect of irradiation on adenosine triphosphate (ATP), glucose levels, hemolysis, and mean corpuscular volume (MCV) for AS-3 units and significantly decreased the deleterious effect of irradiation on glucose levels, hemolysis, and MCV for AS-1 units. In both studies, the irradiation dose was 25 Gy, and irradiation took place on the collection day with a subsequent storage period of 42 days before component analysis. Therefore, both studies reflect neither the current FDA recommendations nor the European recommendations, which restrict the expiration dating period for RBC products to no more than 28 and 14 days, respectively, from the date of irradiation.
Other information on the irradiation effect on leukoreduced RBCs is missing. Therefore, we conducted the present in vitro study. We studied 40 RBC units that were prepared from inline filtered whole blood and 40 RBC units that were filtered after component separation. All RBCs were stored in the AS saline-adenine-glucose-mannitol (SAG-M) and leukoreduced on the collection day. In both groups, 20 components were irradiated on Day +14 with 30 Gy, and 20 served as nonirradiated controls.
MATERIALS AND METHODS
Eighty leukoreduced, SAG-M–preserved RBC units were obtained from the Institute for Transfusion Medicine Suhl (limited liability company). Forty units had been manufactured by component separation from inline filtered whole blood, and 40 units had been inline filtered as RBCs after component separation of unfiltered whole blood. The filtration was done on Day 0 and the units were cooled to 2 to 6°C thereafter. After overnight storage, that is, on Day +1 after collection, the units were shipped to our institution. The required storage and shipping temperatures of 2 to 6 and 2 to 8°C, respectively, were guaranteed by the manufacturer. Twenty of each group of 40 units were gamma-irradiated on Day +14 after blood collection with a dose of 30 Gy using a blood irradiator containing a 137Cs source (OB 29/4 Type 882, STS Steuerungstechnik und Strahlenschutz GmbH, Braunschweig, Germany). The other 20 of each group of 40 units served as nonirradiated controls. The irradiated RBC units were irradiated in such a manner that the internal midplane of the container received the 30-Gy dose with a minimum of at least 25 Gy and a maximum of 40 Gy at any other part of the suspension as recommended.13 The irradiation instrument canister was loaded with one to three components at one time. An irradiation indicator (International Specialty Products, Wayne, NJ) was applied to each RBC unit and irradiated to document a dose delivery of at least 25 Gy. No additional indicator was placed at the same spot on the container in each irradiation procedure. In vitro evaluation of the irradiated and the nonirradiated RBC units was performed before and after irradiation on Days +1, +7, +14, +21, +28, +35, and +42 from the collection day.
After careful mixing of the RBCs, 20-mL samples were aseptically drawn via a sampling site coupler. After centrifugation of the RBC suspension, the supernatant was used for analysis of potassium, phosphate, glucose, free hemoglobin (Hb), and lactate dehydrogenase (LDH). Blood cell counts, Hb, hematocrit (Hct), and pH were analyzed from the RBC suspension. One-milliliter volumes of RBCs were mixed under vigorous shaking with trichloroacetic acid (final concentration. 0.37 mol/L) and put on ice for 5 minutes. After centrifugation at 2000 × g for 10 minutes, the supernatant was stored frozen at −80°C until analysis of ATP and 2,3-diphosphoglycerate acid (2,3-DPG).
Measurements in vitro
The volumes were calculated by dividing the net weight of the RBCs by the density (1.055).13 Total Hb, Hct, RBC counts, and the MCV were performed on a cell counter (Advia 120, Bayer Diagnostics, Munich, Germany; now Siemens Medical Solutions, Erlangen, Germany). Hb in the supernatant solution was determined by measuring the absorption at 540 nm using Drabkin's cyanohemoglobin method (Drabkin's solution, Merck, Darmstadt, Germany) and an automatic analyzer (Cobas Fara, Roche, Basel, Switzerland). The rate of hemolysis was calculated from the supernatant Hb and expressed as a percentage of the total amount of Hb in the RBC suspension.
Potassium, glucose, and the activity of LDH were determined according to standard methods using an automatic analyzer (Olympus AU 640, Olympus Europe, Hamburg, Germany). The limit of detection of potassium ions was 200 mmol per L. pH values were measured at 37°C on an automatic blood gas system (ABL520, Radiometer Copenhagen, Willich, Germany).
ATP and DPG were analyzed using commercially available assays (Roche) and determining absorption at 340 nm on an automatic analyzer (Cobas Fara, Roche).
We analyzed data with statistical software (SPSS for Windows, Release 15.0, SPSS, Chicago, IL). Results were tested for normal distribution by means of the Lilliefors and the Shapiro-Wilks tests. If variables were not distributed normally, statistical analysis was performed using nonparametric methods. Comparisons between groups were done with the U test. If results were distributed normally, a t test for paired or unpaired data was used when appropriate. We considered differences to be significant when p values were less than 0.05. Linear associations between two variables were ascertained with Pearson's r test. p Values of less than 0.05 were considered to indicate a significant correlation.
Mean values of volume, Hct, Hb content, and ATP and 2,3-DPG levels of the four groups of examined RBCs on Day +1 are shown in Table 1. There were significant differences between 40 RBC units that had been leukoreduced after component separation (“LR of RBCs”) and 40 RBC units from leukoreduced whole blood (“LR of WB”) with respect to volume (287.4 ± 16.6 mL vs. 325.8 ± 17.4 mL), Hct (59.8 ± 3.0 L/L vs. 61.0 ± 3.4 L/L), Hb per unit (54.7 ± 5.3 g vs. 63.4 ± 6.6 g), and glucose (34.3 ± 1.0 mmol/L vs. 31.5 ± 0.9 mmol/L). A surprising finding was the lower Hct of the units that had been leukoreduced as RBCs in comparison to the other groups whereas the Hb content per unit did not show the same difference. This observation remained unexplained. All 80 units were collected from the same population and prepared by the same manufacturer. There was an interval of 1 to 2 weeks between the collection of the 20 units of each of the four groups. All 80 units were examined for residual WBCs by using a Nageotte chamber. No residual WBCs were found in any unit.
Table 1. Volume, Hct, and Hb content and ATP and 2,3-DPG levels of the four groups of examined RBCs on Day +1 after collection
|Volume (mL)||289.9 ± 17.9†‡||284.8 ± 15.2†‡||326.3 ± 18.0§‖||325.2 ± 17.2§‖|
|Hct (L/L)||0.621 ± 0.020‖||0.574 ± 0.016†‡§||0.604 ± 0.044‖||0.616 ± 0.020‖|
|Hb (g/L)||192 ± 10||187 ± 7||195 ± 14||194 ± 9|
|Hb per unit (g)||55.9 ± 6.0†‡||53.4 ± 4.4†‡||63.7 ± 7.8§‖||63.0 ± 5.2§‖|
|Glucose (mmol/L)||34.2 ± 1.1†‡||34.4 ± 0.9†‡||31.6 ± 0.9§‖||31.4 ± 0.9§‖|
|ATP (µmol/g Hb)||5.46 ± 1.06‡||5.62 ± 0.54‡||5.63 ± 0.75‡||6.16 ± 0.90†§‖|
|2,3-DPG (µmol/g Hb)||12.05 ±3.14†‡||10.96 ± 2.73‡||9.71 ± 1.65§||8.07 ± 4.49§‖|
From each of both groups of 40 RBC concentrates, 20 were gamma-irradiated on Day +14 after collection. At this time, there were no significant differences between RBCs to be irradiated and RBCs that served as nonirradiated controls with respect to the rate of hemolysis, the potassium levels, ATP per g Hb, and 2,3-DPG per g Hb. Only the MCV was, obviously by chance, significantly lower in both control groups.
Table 2 shows in vitro quality indicators of the four examined groups of RBCs on Day +28 after collection, which was Day +14 after irradiation in the irradiated components. In both component groups, LR of RBCs and LR of WB, there were significant signs of irradiation-induced leakage of potassium ions and LDH in the irradiated components. In the LR of RBCs group, the mean rate of hemolysis was already significantly higher in the irradiated components than in the nonirradiated ones. In the LR of WB group, this effect was not yet distinguishable.
Table 2. In vitro quality indicators of the four groups of examined RBCs on Day +28 after collection (in the groups LR of RBCs, gamma-irradiated, and LR of WB, gamma-irradiated the irradiation was performed on Day +14)
|Glucose (mmol/L)||22.3 ± 1.8‡||23.2 ± 2.3‡||21.3 ± 2.1||19.7 ± 1.8§‖|
|Phosphate (mmol/L)||3.1 ± 0.5†‡||4.9 ± 0.7‡||5.4 ± 0.5§||5.6 ± 0.4§‖|
|Potassium (mmol/L)||37.9 ± 4.5‡‖||47.9 ± 5.2†§||37.2 ± 4.4‡‖||48.0 ± 4.0†§|
|LDH (IU/L)||59 ± 12†‡‖||112 ± 35§‡||97 ± 35§‡||164 ± 54†§‖|
|Rate of hemolysis (%)||0.06 ± 0.01‡‖||0.10 ± 0.03§||0.09 ± 0.04||0.11 ± 0.04§|
|MCV (fL)||99.8 ± 3.4‡||102.5 ± 3.8||100.6 ± 4.3||106.2 ± 4.6§|
|ATP (µmol/g Hb)||5.16 ± 0.86†‡||4.70 ± 0.52||4.25 ± 0.92§||4.32 ± 0.81§|
|2,3-DPG (µmol/g Hb)||0.79 ± 0.42†‡||0.54 ± 0.27‡||0.40 ± 0.15§||0.30 ± 0.10§‖|
|pH||6.48 ± 0.04||6.48 ± 0.03||6.48 ± 0.03||6.47 ± 0.03|
|Lactate (mmol/L)||27.3 ± 2.7‡||27.0 ± 2.7‡||28.4 ± 2.9||30.2 ± 3.1§‖|
Table 3 shows the same series of quality indicators of the four component groups on Day +42 after collection, that is, on Day +28 after irradiation in the irradiated components. At this time, the level of potassium ions in the supernatants was no longer significantly enhanced in the irradiated subgroups of units. In the LR of RBCs and LR of WB groups, potassium levels had increased more in the nonirradiated components than in the irradiated ones, which means that the levels had approximated between Days +28 and +42. However, on Day +42 the rate of hemolysis and the mean MCV values in irradiated components were significantly higher than in nonirradiated ones.
Table 3. In vitro quality indicators of the four groups of examined RBCs on Day +42 after collection (in the groups LR of RBCs, gamma-irradiated, and LR of WB, gamma-irradiated the irradiation was performed on Day +14)
|Glucose (mmol/L)||20.4 ± 2.1‡||20.5 ± 2.4‡||19.1 ± 2.2‡||16.9 ± 2.2†§‖|
|Phosphate (mmol/L)||5.8 ± 0.4‡||5.8 ± 0.6||6.1 ± 0.4||6.2 ± 0.4§|
|Potassium (mmol/L)||44.8 ± 6.6||49.5 ± 5.9‡||44.9 ± 3.1‖||47.7 ± 4.7|
|LDH (IU/L)||92 ± 28†‡‖||196 ± 51†§||142 ± 64‡‖||307 ± 111†§|
|Rate of hemolysis (%)||0.09 ± 0.03‡‖||0.18 ± 0.07‡§||0.14 ± 0.06‡||0.35 ± 0.12†§‖|
|MCV (fL)||102.1 ± 3.6‡‖||109.0 ± 4.0†§||103.5 ± 4.4‡‖||108.3 ± 4.8†§|
|ATP (µmol/g Hb)||3.14 ± 0.49†||3.06 ± 0.65†||4.74 ± 1.90‡§‖||2.63 ± 0.68†|
|2,3-DPG (µmol/g Hb)||0.47 ± 0.11†‡‖||0.36 ± 0.15†§||0.17 ± 0.09‡§‖||0.26 ± 0.07†‖|
|pH||6.34 ± 0.04||6.40 ± 0.03||6.40 ± 0.04||6.34 ± 0.03|
|Lactate (mmol/L)||33.0 ± 4.0||31.8 ± 3.3||32.3 ± 2.9||33.6 ± 3.3|
The results of our investigation add substantial information to the knowledge of the effects of gamma irradiation on leukoreduced RBCs. For the first time, we provide data reflecting the worst-case scenarios of US and of European recommendations and guidelines on the latest time, when irradiation of RBCs should be performed after a defined anteceding storage period, combined with the maximal recommended storage period after irradiation.1,13 These data are the measurements on Day +28, when the RBCs had been stored 14 days before and another 14 days after gamma irradiation (the European worst-case scenario),13 and the measurements on Day +42, when they had been stored 14 days before and another 28 days after gamma irradiation (the FDA worst-case scenario).1
Already from Day +21 until the end of the examined storage period, irradiation caused a distinctive leakage of potassium ions and LDH. On Day +28, the difference in potassium levels in the supernatants between irradiated and nonirradiated units was higher than on Day +42, indicating that the early leakage of potassium ions by irradiation is followed by a period of slower leakage of the remaining potassium in leukoreduced RBCs. The in vitro hemolysis rate on Day +28 was only slightly enhanced in irradiated units whereas the enhancement of the in vitro hemolysis rate was clearly significant and remarkable on Day +42. It should be mentioned that RBCs have been irradiated with 30 Gy, which is the standard irradiation dose in Germany to fulfill the European recommendation that the irradiation dose should be not less than 25 Gy and not more than 40 Gy on any given place in the bag.13,20 This is more than the US recommendation that the irradiation dose should be 25 Gy in the center of the bag and at least 15 Gy on any given place in the bag.1
Our studies confirm previous findings in leukoreduced RBCs that have been irradiated earlier than on Day +14 and nevertheless have been stored until Day +42. There is one such study presenting in vivo recovery measurements,18 and on reporting in vitro findings.19 Both studies arrived at the conclusion that it is possible to perform irradiation of leukoreduced RBCs on any day after the collection during the usual storage period without changing the maximal shelf life of the component. Our data do not contain findings that allow differing conclusions.
Nevertheless, our study as well as the cited ones underline the early effect of gamma irradiation on the leakage of potassium ions. Therefore, we recommend that the irradiation of RBCs should not be performed until the units are clinically needed without any subsequent storage whenever the logistic situation allows doing so, especially for patient groups at enhanced peril by increased levels of free potassium ions. On the other hand, our study is the third one providing data on irradiated RBC components, which allow the conclusion that the European regulation that irradiated RBCs must not be stored for more than 14 days after the irradiation is outdated after the implementation of general leukoreduction into the processes of collecting and manufacturing RBCs.
There was a remarkable difference between RBCs prepared from whole blood and filtered thereafter on the one hand and RBCs prepared from leukoreduced whole blood on the other. The latter ones contained significantly more RBCs and Hb per unit. The difference averaged to 16 percent! This underlines the postulation by Davenport21 that blood components should be labeled for content. On the other hand, both subgroups of components contained significantly more Hb per unit than a series of outdated RBCs that we examined earlier.22 In the present study, RBCs that had been leukoreduced after component separation contained 54.7 ± 5.3 g Hb per unit, and RBCs from leukoreduced whole blood even 63.4 ± 6.6 g. In the earlier series, we had measured between 43.0 ± 10.7 and 50.9 ± 13.2 g Hb per unit in subgroups of RBCs from different manufacturers. Thus, it seems as if manufacturers in Germany today pay more attention to optimal production processes than some years ago.
Nevertheless, the 16 percent mean difference of the Hb content per unit between the examined groups of RBCs, which depends on the time in the production process when leukoreduction is performed, is remarkable. If a RBC unit, a plasma unit, and a PLT unit shall be separated from one whole blood donation, it is necessary to perform a filtration of the RBCs after the component separation. If PLTs are mainly collected by apheresis, whole blood filtration with subsequent component separation is preferable. Unfortunately, the side effect of pool PLT preparation from whole blood donations on the mean Hb content of RBCs prepared from the same donations is only rarely taken into account in discussions on the preferable method of PLT collection.23
Unexpectedly, we found on the other hand that the in vitro hemolysis rate in the nonirradiated and in the irradiated subgroups of RBCs from filtered whole blood was significantly higher than in both subgroups of units that had been leukoreduced after component separation. This higher-than-average increase of the in vitro hemolysis rate in RBCs from leukoreduced whole blood occurred not earlier than between the Days +35 and +42. Nevertheless, in this study we found no component the in vitro hemolysis rate of which reached the limiting value of 0.8 percent. This is in accordance with the findings of Wagner and Myrup,19 but differs from another in vitro study by Leitner and coworkers8 who found altered intracellular purine nucleotides in leukoreduced gamma-irradiated RBCs and presented hemolysis rates at the end of shelf life, which exceeded 0.8 percent in irradiated units. However, the latter study examined aliquots of RBCs, which had been divided into two equal parts. Therefore, the finding by Leitner and coworkers of critically enhanced in vitro hemolysis in stored irradiated units probably resembles the well-known enhancement of the in vitro hemolysis rate in underfilled components.24 Other reports on quality control of blood irradiation also divided RBC units into unusually small aliquots, which do not resemble the normal filling status of RBCs to study differences between groups without the bias of biologic variabilities.25
In conclusion, this study presents data on the in vitro quality of leukoreduced RBCs in the AS SAG-M that have been irradiated with 30 Gy on Day +14 after collection and subsequently stored until Day +42. Our findings, together with recent results of other investigations18,19 on the effect of gamma irradiation on leukoreduced RBCs, allow the proposal that a storage time up to 28 days after irradiation is allowable.