Irradiation Effect On Aging Red Blood Cells*

Transcription

1 ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 21, No. 6 Copyright 1991, Institute for Clinical Science, Inc. Irradiation Effect On Aging Red Blood Cells* ELAINE K. JETER, M.D., RICHARD H. GADSDEN, Ph D., and JOHN C. CATE, IV, M.D. Department of Pathology/Laboratory Medicine, Medical University o f South Carolina, Charleston, SC ABSTRACT Irradiation of stored red blood cells (RBC) is increasingly utilized for patients who are im m unosuppressed or on chem otherapeutic regimens. W ith the growing dem and for irradiated cellular blood products, there has b een an increasing n eed for transfusion services to store previously irradiated blood until needed for transfusion. The effect of irradiation on aging stored RBC has not been studied to date. Five units each of group A, RBC collected in CPD-Adsol (AS-1) with a prior shelf-life of 10, 20, 30, and 40 days, respectively, w ere divided equally utilizing a sterile docking device and stored at 1 to 6 C. Baseline samples from each bag were obtained for the m easurem ent of extracellular potassium (K+), plasm a free hemoglobin (PFH), total lactate dehydrogenase (LD), and erythrocyte 2,3-DPG activity. One of each pair received 2,000 rads of gamma irradiation. Samples were obtained at 3 and 7 days post-irradiation, and m ultiples of 7 days until expiration. All irradiated units reached a state of K+ equilibrium at 60 to 70 mmol per L irrespective of the length of previous storage w ith an inverse relationship of RBC age at irradiation and the time required to reach the state of equilibrium. Increased K+ leakage from irradiated aging RBC suggests the need for including in vivo studies of cell survival to establish a post-irradiation storage life. Length of storage prior to irradiation had no effect on PFH, LD activity, and 2,3-diphosphoglycerate (2,3-DPG) activity com pared to paired controls. Introduction Recipients of blood from first degree relatives,11 neonates receiving exchange transfusions, and patients w ho are immu- * A ddress re p rin t re q u ests to E la in e K. Je ter, M.D., D epartm en t o f Pathology/L aboratory M edicine, T ransfusion M edicine Section, M edical U niv e rsity o f S o u th C a ro lin a, 171 A sh ley A v enue, C harleston, SC nosuppressed4 or on in ten siv e chem o therapeutic regim ens are candidates for irradiated blood or cellular blood products. W ith the growing dem and for irradiated blood products, there has been an increasing need by transfusion services, which often are not equipped with blood irradiators or w ith access to therapeutic irradiating devices, to store previously irradiated blood until needed for transfu /91/ $00.90 Institute for Clinical Science, Inc.

2 IRRADIATION EFFECT ON AGING RED BLOOD CELLS 421 sion. In many cases, this irradiated blood has been stored for some period of tim e by a blood center or transfusion service prior to irradiation. It has been shown that RBC, irradiated im m ediately following collection and subsequently stored, have significantly increased potassium (K+) concentrations compared to paired controls throughout their shelf-life,3 5 7 and that irradiated RBC CPDA-1 anticoagulant have significantly greater concentrations of K+ at comparable periods of post-irradiation storage than irradiated RBC an tico ag u lated w ith AS-1 (RBC AS-1).3 To our knowledge, one report of whole blood and RBC stored for 21 days (CPD anticoagulant) prior to 5,000 rads of irradiation had significantly d ifferent plasm a K + lev els com pared to n o n paired controls.1 R ed cell K + loss or e x tra c e llu la r K + gain in p rev io u sly stored, then irradiated RBC that are subsequently retu rn ed to storage has not b een reported. Data are presented on the in vitro postirradiation effects of 2,000 rads on RBC AS-1 that have been previously stored for 10, 20, 30, and 40 days, respectively, prior to irradiation. B aseline sam ples were obtained from the paired control and irradiated units im m ediately prior to irradiation; su b seq u en t sam ples w ere taken at designated intervals following return of units to routine storage until expiration (42 days). Materials and M ethods Five (5) units each of group A, RBC AS-1 with a shelf-life of 10, 20, 30, and 40 days, respectively, w ere provided by the C arolina L ow country A m erican R ed Cross (ARC) for this study. The hematocrit was approximately 60 percent in all units. Collection, preparation, storage, and transportation protocols follow ed standard ARC operating procedures. Contents of each of the 20 units were equally divided betw een the parent bag (controls) and a transfer bag (irradiated) via a sterile docking device; sterile samp le site c o u p le rs w e re a s e p tic a lly adapted to each bag. Baseline sampling from each bag was im m ediately p e r formed under a laminar flow hood. All units were stored at 1 to 6 C until expiration. Each of the complimentary transfer bags was irradiated w ith 2,000 rads of gamma irradiation.* Additional samples w ere obtained on post-irradiation days 3, 7, and weekly until expiration; sampling intervals for the 30-day and 40-day units w ere extended beyond the norm al expiration date for sufficient data points to d e m o n stra te tre n d s. C u ltu re s w ere obtained on the seventh day following irradiation and at expiration. All cultures showed no growth after seven days of incubation. After 30 gentle inversions, 6.5 m L of resu sp en d ed red blood cells w ere collected in 10 ml tubes adapted w ith 16 gauge needles, and separated into 5.6 ml and 1.0 ml aliquots. The supernatant, collected from the 5.6 ml aliquot, was assayed for potassium (K+), total lactate dehydrogenase (LD) activity and plasma free hem oglobin (PFH). T hree m L of eight percent trichloroacetic acid w ere added to the one ml aliquot, incubated at room tem perature for 10 m inutes and centrifuged at 3,200 rpm for 10 m inutes; the protein free supernatant used for 2,3- DPG determ ination was im m ediately frozen at 70 C and subsequently assayed in batch analysis. Supernatant K + d eterm in atio n was m ade by ion specific electro d e.t E le vated K+ levels were reanalyzed using the urine mode (analytical range: 2.0 to mmol per L). Supernatant PFH levels were determ ined by differential spectrophotom etric analysis calcu lated at * G am m acell 1,000, Atom ic E n erg y o f C anad a L im ited, R adiochem ical Co., Kanata, O ntario, C anada. t Astra-8, B eckm an In stru m en ts, B rea, CA

3 422 JETER, GADSDEN, AND CATE F i g u r e 1. M ean K + (m mol p e r L) p aired contro l (C T R L) a n d irra d i ated (IRRAD) RBC AS-1, equally d ivided and irrad ia te d a fte r 10, 20, 30, a n d 40 days o f sto rage, respectively. Irresp ectiv e o f th e p re v io u s sto rag e perio d, K + in cre ased in all irra d ia te d u n its to a m a x im u m o f 60 to 70 m m o l p e r L; th e o ld e r u n its a tta in e d th is lev e l in s i g n i f i c a n t l y l e s s tim e c o m p a r e d to th e fresher units. 1 Days delta 415 nm and 450 nm absorbance. Total L D was d eterm ined by the m odified procedure of Gay, M ccom b and Bow ers.t Protein free supernatants for 2,3-DPG determ inations w ere assayed according to the m ethod of Rose and Liebowitz with a reference range of 1.6 to 2.6 jjimol per ml. Aerobic cultures w ere performed using a non-radiom etric, autom ated bacterial detection system. 1 Analysis w ere perform ed with paired t-test at each assay tim e; significant levels w ere set at a = Results Serial m ean K + concentrations for paired control and irradiated RBC stored for 10, 20, 30, and 40 days, respectively, prior to irradiation are shown in figure 1. The m ean baseline K+ at day 10 was 19.4 t M o n arch 2000, In stru m e n ta tio n L ab o rato ry, L exington, MA Sigm a C hem ical Co., St. L ouis, M O P edi-p lu s/b actec H PS-660, B ecto n -D ick in so n D iagnostic In stru m en t System, T ow son, M D ± 2.3 mmol per L. On the third and seventh post-irradiation day, the K+ values in the irradiated units were 1.6 and 1.7 fold greater than the respective paired control units. Although extracellular K+ in the irradiated units continued to rise and w ere significantly greater than controls at subsequent testing intervals, the ratio of extracellular K+ in the irradiated units slowly decreased during storage to a steady state at 1.4 fold greater than the control units. At expiration, the m ean K+ concentration was 49.0 ± 5.0 mmol per L (CTRL) and 68.2 ± 7.6 m m ol p er L (IRRAD), respectively. B aseline m ean K+ in the units previously stored 20 days prior to irradiation was 29.4 ± 3.8 mmol per L. On the third and seventh post-irradiation day, K+ in the irradiated units was 1.6 and 1.8 fold greater than respective control units. At expiration, K+ was 1.7 fold greater than the controls; m ean K+ was 40.4 ± 5.7 mmol per L (CTRL) and 70.8 ± 9.5 mmol p er L (IRRAD), respectively. Units previously stored for 30 days prior to irradiation had baseline m ean K+ concentration of 38.2 ± 5.5 mmol per L. The K+ in the irradiated units was 1.2 fold greater than controls at all desig

4 IRRADIATION EFFECT ON AGING RED BLOOD CELLS 423 nated testing intervals. At expiration, the mean K+ concentrations were 50.8 ± 6.4 mmol per L (CTRL) and 63.6 ± 6.5 mmol per L (IRRAD). The baseline m ean K+ concentration for units previously stored for 40 days prior to irradiation was 47.0 ± 3.0 mmol per L. On the third post-irradiation day, K+ was 1.2 fold greater than the control units; m ean K+ was 50.0 ± 4.0 mmol per L (CTRL) and 62.8 ± 2.8 mmol per L (IRRAD), respectively. Concentrations of PFH, an indicator of hem olysis, in control and irradiated samples are presented in figure 2. Plasm a free hemoglobin increased over tim e in all samples. Baseline m ean PFH concentrations at 10, 20, and 30 days of prior storage were 47.2 mg per dl, 62.5 mg per dl, and 90.5 mg per dl, respectively. Baseline PFH in the 40 day control and irradiated units was 175 mg per dl and 235 mg per dl, respectively. Irrespective of the storage period prior to irradiation, F ig u r e 2. M ean P F H (m g p e r m L) in RBC AS-1 stored for 10(A), 20(B), 30(C), an d 40(D) days p rio r to irradiation. Plasm a free h em oglobin in creased in all units d esp ite th e previous p erio d o f storage p rior to irradiation. No significant differences exist b etw een th e control (CTRL) and irrad iated (IRRAD) u n its for each storage interval. F i g u r e 3. M ean L D (IU p e r L) in RBC AS-1 stored for 10(A), 20(B), 30(C), an d 40(D) days prio r to irradiation. L actate dehydro g en ase in creased in all units d esp ite th e previous p erio d of storage prio r to irradiation. N o significant d ifference exists b etw een th e control (CTRL) an d irrad iated (IRRAD) units for each storage interval.

5 424 JETER, G A D SD E N, A N D CATE no significant difference was observed betw een controls and irradiated pairs at all sam ple intervals. M ean LD activities in control and irradiated samples are presented in figure 3. Lactate dehydrogenase activity increased in all units regardless of the previous period of storage prior to irradiation. Mean baseline values of LD at 10, 20, and 30 days of prior storage were 209 IU per L, 326 IU per L, and 569 IU per L, respectively; baseline LD in the 40 day control and irradiated units were 940 IU per L and 1075 IU per L, respectively. Irrespective of the period of storage prior to irradiation, no significant differences were observed betw een control and irradiated pairs at all sam ple intervals. Baseline m ean 2,3-DPG activity in red cells previously stored for 10 days was / 0.38 xmol per ml. On the third p o st-irrad iatio n day, 2,3-D PG levels w ere less than 0.10 fimol per ml and w ere undetectable at all sam pling intervals to expiration. In units previously stored for 20, 30, and 40 days prior to irradiation, 2,3-D PG values w ere below detection lim its at all sam pling intervals. Discussion This study presents data documenting the effects of 2,000 rads of gamma irradiation on RBC previously stored for variable periods before irradiation and subsequently returned to storage. Irradiated units dem onstrated increased potassium perm eability w ith maximum values of approxim ately 60 to 70 m m ol p er L, obtained at variable intervals following irradiation. O n the other hand, at expiration (42 days), all controls contained approximately 40 to 50 mmol per L of K +. The age of RBC at irradiation shows an inverse relationship to the period of time required for the cells to reach maximum potassium concentrations. For example, RBC irradiated im m ediately following collection exceed 60 mmol per L after 21 days of storage3; units stored for 10, 20, and 30 days prior to irradiation exceeded 60 mmol per L w ithin 10, 7, and 11 days, respectively. It is our suggestion that the maximum potassium values obtained are a function of the N a+-k+ pum p, red cell porosity, and the m icroenvironm ent of the blood bags. W hile it is known that red cell m em brane N a+-k+ shift is tem peratu r e d e p e n d e n t a n d m a x im a l a t 4 C,2 6,10 13 irradiation leads to increased perm eability to N a+ and K+ by altering sulhydryl groups on m em brane surfaces and w ithin the cytoplasm.8,9 The slowed N a+-k+ shift in aged (greater than 30 days prior storage) RBC may reflect a ph dependent phenom enon. The age of the RBC at irradiation is inversely related to the period of time for the units to exceed 50 mmol per L, the expected level for K+ in RBC AS-1 at 42 days of shelf-life. For freshly collected, irradiated and stored units, K+ exceeds 50 m m o l p e r L a t 12 d a y s p o s t irradiation3; those irradiated after 10 and 20 days of storage exceed 50 mmol per L w ithin eight and four days, respectively. Moore and Ledford5 have shown that irradiation has little to no effect on red cell adenosine triphosphate, 2,3-DPG, and/or m ethem oglobin levels w ith prolonged storage. Plasma free hemoglobin follow ing irradiation is anticoagulant d e p e n d en t; RBC CPDA-1 sto red for more than seven days following irradiation have significantly increased levels of PFH compared to the paired controls.3 No significant difference in PFH has been noted for irradiated RBC collected in AS-1 and stored to expiration. Because extracellular K+ load may be a significant clinical problem in selected p a tie n t p o p u la tio n s, th e a d d itio n a l plasm a K+ concentration in aging, previously irrad iated RBC m ust b e considered. Issues regarding the efficacy of routinely washing irradiated red cells and the potassium load in small volume transfusions have been review ed.12 Although

6 IRRADIATION EFFECT ON AGING RED BLOOD CELLS 425 irradiation im m ediately prior to transfusion is ideal, this may be impractical and unavailable in many transfusion service settings. Some transfusion services have arbitrarily established a post-irradiation storage life not to exceed five or seven days, w hile others have not delineated a post-irradiation shelf-life. This study docum ents the enhanced K+ leakage in irradiated aging RBC and suggests that prior to the establishm ent of a definitive postirradiation storage life future investigation should include in vivo cell survival and red cell function studies after transfusion of variably aged, irradiated RBC. References 1. B u t t o n, L. N., D e w o l f, W. C., N e w b u r g e r, P. E., Ja c o b s o n, M. S., and Ke v y, S.V.: T he effects o f irra d ia tio n on b lo o d com p o n ents. T ransfusion 21: , G LYNN, I. M.: Sodium and potassium m ovem ents in h u m an red cells. J. Physiol. 134: , Je t e r, E. K., G a d s d e n, R. H., C a t e, J. C. IV: E ffects o f irrad iatio n on re d cells sto red in C PD A -1 a n d C P D -A dsol (AS-1). Ann. C lin. L ab. Sci. 22: , L e i t m a n, S. F.: U se o f blood cell irradiation in th e p reven tio n o f post-transfusion graft-vs-host disease. T ransfusion Sci. 2 0 : , M o o r e, G. L. and L e d f o r d, M. E.: E ffects o f rad irrad iatio n on th e in v itro storage p ro p e rtie s o f p a ck e d re d cells. T ran sfu sio n 2 5 : , M y e r s, D. K. a n d B i d e, R. W.: B io c h e m ic a l e ffe c ts o f x -irra d ia tio n o n e r y th r o c y te s. R adiat. R e s. 2 7 : , R a m i r e z, A. M., W o o l f i e l d, D. G., S c o t t, R., a n d M c L a c h l a n, J.: H igh potassium levels in stored irradiated blood. T ransfusion 2 7 : , S h a p i r o, B. an d Ko l l m a n, G.: T h e n atu re of th e m e m b ra n e in ju ry in irra d ia te d h u m a n erythrocytes. Radiat. Res. 34: , S h a p i r o, B., K o l l m a n, G., a n d A s n e n, J.: M echanism o f th e effect o f ionizing radiation on so d iu m u p ta k e b y h u m an e ry th ro c y te s. Radiat. Res. 2 7 : , S h e p p a r d, C. W. a n d B e y l, G. E.: C a tio n exchange in m am m alian erythrocytes iii. T h e prolytic effect o f x-rays on hum an cells. J. G en. Physiol. 3 4 : , SIMON, T. L.: M em orandum to AABB In stitu tional M em bers. A rlington, VA, A m erican Association o f B lood Banks, N o vem ber 6, S t r a u s s, R.: R outinely w ash ed irrad iated red cells b efo re transfusion seem s u n w a rran te d. T ransfusion 3 0 : , W a l l a s, C. H.: Sodium and potassium c hanges in b lo o d b a n k sto re d h u m an e ry th ro c y te s. T ransfusion 2 9 : , 1979.