Lipid Peroxidation in Platelet Concentrates: Effects of Leukocyte Removal by Filtration*

Transcription

1 ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 24, No. 1 Copyright 1994, Institute for Clinical Science, Inc. Lipid Peroxidation in Platelet Concentrates: Effects of Leukocyte Removal by Filtration* JOSEPH A. KNIGHT, M.D.,tt DOUGLAS A. SEARLES, B.S.,t and ROBERT C. BLAYLOCK, M.D.t fd epartment o f Pathology and Laboratory Medicine, Salt Lake VA Medical Center and fd epartm ent o f Pathology, University o f Utah School o f Medicine Salt Lake City, UT ABSTRACT Increased lipid peroxidation (LP) occurs in the presence of various transition metal ions, including copper and iron, as well as various heme moities. In addition, irradiation significantly increases LP. The degree of LP, as determined by the liquid chromatographic measurement of plasma malondialdehyde (MDA), can be significantly reduced by the addition of various metal chelating agents and other antioxidants to both stored red blood cells and platelet concentrates. They are also effective in reducing the degree of LP following irradiation. In the current study, leukocytes were effectively removed by filtration from platelet concentrates prepared by standard methods (mean ± SD before filtration, 460 ± 286 per fxl vs 0.3 ± 0.2 per jjll after filtration). The removal of the white blood cells significantly reduced the plasma MDA levels in the leukocyte-free platelet concentrates compared with concentrates containing leukocytes after storage for 4, 7, and 11 days (P < 0.001; P < 0.02; P < 0.05, respectively). In addition, plasma potassium levels were also significantly reduced in the leukocyte-free platelet concentrates (P < 0.001). Our results suggest that the early removal of leukocytes from platelet concentrates may be helpful in prolonging platelet viability and longevity. understood, various methods have been developed to improve platelet preparation and increase the acceptable storage time for platelet concentrates prior to transfusion. Thus, platelet suspensions are optimally stored at 20 to 24 C for up to five days with constant mild agitation in storage containers that allow adequate gas permeability /94/ $01.20 Institute for Clinical Science, Inc. Introduction The platelet storage lesion is a complex process involving various physical, chem ical, and m e tab o lic fa c to rs.4 Although these factors are not fully * Address reprint requests to: Joseph A. Knight, M.D., Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT

2 70 KNIGHT, SEARLES, AND BLAYLOCK Oxygen-derived free radicals, especially the hydroxyl free radical (HO ), are cytotoxic. In the presence of various transition metal ions, including copper and iron, as well as certain heme moities, HO is rapidly formed from hydrogen peroxide (H20 2); irradiation also results in greatly increased HO formation by the homolytic dissolution of water. This and other free radicals then initiate the process of lipid peroxidation (LP), an autocatalytic free radical chain reaction involving polyunsaturated fatty acids and phospholipids in cellular membranes.8 This process u ltim a te ly resu lts in decreased cell membrane deformability, increased perm eability, and eventual cell death.5 Okuma et al20 first compared LP in freshly collected platelets anticoagulated with ethylenediam inetetraacetic acid (EDTA) w ith a c id -citrate-d ex tro se (ACD). They noted that storage at both 4 C and 20 C resulted in a rapid increase and accum ulation of lipoperoxides; EDTA was more effective than ACD in reducing lipoperoxide production. In this regard, our findings w ere recently reported on LP in both irradiated14 and non-irradiated12,13 stored donor erythrocytes and platelets16 in which various added metal chelators and glutathione significantly reduced LP. In addition, a significant decrease was noted in LP in stored erythrocytes from donor volunteers supplemented for 10 days with vitamins C and E.15 Prophylactic irradiation of blood and blood components prior to transfusion is usually recommended to reduce the risk of graft-versus-host disease in high-risk patients. However, irradiation of erythrocy tes 14 and p la te le t concentrates 16 increase LP. More recently, leukocyte filters have been used in these cases,21 as well as for preventing the spread of various infectious agents.1,3,9 Therefore, the effects were studied of leukocytes on LP in stored platelet concentrates by measuring plasma MDA with and without prior leukocyte removal by filtration. Measurement was made of the extent of LP by quantifying plasma MDA levels by high performance liquid chromatography (HPLC) as previously reported.11,26 Also measured were plasma potassium (K+) levels to assess w hether or not K + changes were correlated with the degree oflp. Materials and Methods S p e c i m e n s /P r o c e d u r e s Groups of platelet concentrates were prepared from single units of whole blood antico agulated w ith c itratephosphate-dextrose-adenine (CPDA-1) taken from the blood bank inventory. This preparation method involved red cell and partial leukocyte separation within eight hours of collection with a light spin of2000 g for three minutes to produce platelet-rich plasma. Platelet concentration from the plasm a followed a heavy spin of 5,000 g for five minutes.24 Each platelet concentrate consisted of approximately 40 ml. A platelet filter* was attached to each sample bag and an I.V. sett was connected to the filter in order to control the filtration rate. The I.V. set was then attached to an empty five-day platelet b ag t via a catheter adapter. After filtering half of the platelet concentrate, the filter and tubing were removed and replaced with a sterile injection port. Both the control bags (unfiltered) and test samples (filtered) were weighed using an analytical balance to ensure that they contained equal amounts of concentrate (about 15 ml). * Sepacell PL-5A, Fenwall, Deerfield, IL t V " Flash Tube F2L086, McGaw Inc., Irvine, CA t PL 732 Fenwall, Deerfield, IL Becton-Dickinson, Rutherford, NJ

3 LIPID PEROXIDATION IN PLATELET CONCENTRATES 71 From each control bag, 0.5 ml of platelet suspension was removed for baseline MDA and potassium (K+) quantification. All p latelet concentrates w ere then stored between 20 to 24 C with continuous gentle agitation.25 In comparing leukocyte/platelet-free plasma with leukocyte-free plasma from filtered platelets, the leukocyte-free plasma was prepared as described previously. The platelet/leukocyte-free plasma was prepared by centrifuging the filtered platelet concentrates at 5,000 g for 10 m in utes. T he overly ing leukocyte/ platelet-free plasma was then removed and placed in an empty platelet bag. Both the leukocyte-free platelets and leukocyte/platelet-free plasm a w ere then stored at 20 to 24 C with gentle agitation as described previously. Both MDA and K+ were also quantified on days 0, 4, 7, and 1 1. Leukocytes were counted on the unfiltered samples using an automated cell analyzer. 1 The greatly decreased white cell counts in the filtered bags were determined using Turks solution for dilution6 and the N ageotte Bright Line counting chamber.h Duplicate plasma MDA levels were measured by high performance liquid chromatography (HPLC)11,26 on the overlying plasma from all platelet concentrates just prior to filtration (day 0), as well as on all control (unfiltered) and test (filtered) concentrates on days 4, 7, and 11. Plasma potassium (K+) levels were also quantified at the same time using a discrete chemistry analyzer according to the manufacturer s instructions.** On the days of analysis, 0.5 ml of platelet concentrate was removed from each bag and 1Technicon H -l System, Technicon Corporation, Terrytown, NY Hausser Scientific, Horsham, PA ** Astra 8, Beckman Instrum ents, Brea, CA, centrifuged at 2,500 g for five minutes. The overlying plasma was used for both MDA and K+ analyses. R e a g e n t s /S o l u t i o n s The phosphoric acid, thiobarbituric acid (TBA), 1,1,3,3-tetraethoxypropane (TEP), methanol-naoh solution, phosphate buffer, and mobile phase solutions for the MDA analyses were all purchased and prepared as previously reported.26 The reagents used for K+ determ inations were purchased from the instrument manufacturer. S t a t is t ic a l A n a l y s is D ata com putations in c lu d e d the means, standard deviations, and the Mann-Whitney test. A P value ^ 0.05 was considered statistically significant. Results In the preparation of platelet concentrates from whole blood, there is significant loss of leukocytes in the light spin step. Thus, the mean (SD) leukocyte count in the 11 platelet concentrates was 460 (286) per jxl compared with the postfiltration mean (SD) of 0.3 (0.2) per xl. Our results com paring the plasm a MDA levels between the control (nonfiltered) platelet concentrates with the filtered concentrates is shown in table I. Here, it is noted that the removal of leukocytes by filtra tio n sig n ific an tly reduced LP after all storage times compared with the controls. Interestingly, the most significant difference was after four days (P < 0.001); this difference remained significant but decreased over time. It is also noted that there was little difference in total MDA concentrations in controls between days 7 and 11, suggesting an initial rapid rate of LP that subsequently slowed, perhaps owing to early leukocyte death, especially with

4 72 KNIGHT, SEARLES, AND BLAYLOCK TABLE I Comparison of Plasma Maiondialdehyde Levels Between Control and Filtered Platelets Day n Control Umol/L Filtered \imol/l (Mean ±S D ) (0.31) 1.44 (0.31) (0.40) 1.64 (0.41 )a (0.73) 2.52 (0.60)b (0.54) 2.74 (0.64) Statistical significance, filtered platelet malondialdehyde (MDA) levels compared with corresponding controls by Mann-Whitney test: a = P < b = P < 0.02 c = P < 0.05 neutrophils. Levels of MDA also progressively increased in both filtered and nonfiltered platelets from day 0 through day 1 1. In table II it is shown that plasma K+ levels from the filtered platelet concentrates were also significantly reduced at all times compared with the unfiltered concentrates. However, K+ levels progressively increased in both filtered and nonfiltered concentrates from day 0 through day 1 1. Day TABLE II Comparison of Plasma Potassium Levels Between Control and Filtered Platelets n Control Filtered (Mean ±SD) (0.3) 4.0 (0.3) (0.5) 4.1 (0.4)a (0.5) 4.2 (0.3)a (0.4) 4.4 (0.3)a Statistical significance, filtered platelet potassium levels compared with corresponding controls by Mann-Whitney test: a = P < In order to address the question as to whether the measured MDA was primarily derived from plasm a lipid or platelet lipid, both MDA and K+ plasma levels were compared in leukocyte- and platelet-free plasma (cell-free plasma) with those in leukocyte-free platelet plasma (table III). Here, it is noted that in the cell-free plasma, both MDA and K+ levels were essentially unchanged during the entire 11 day period. Conversely, plasma levels from the leukocyte-free platelet concentrates increased significantly on days 4, 7, and 11. Plasma K+ was significantly increased on days 7 and 11, but not on day 4. The data indicate that the increased lipoperoxides were from platelet lipid and not from plasma lipid. Discussion Several successful procedures have been introduced over the past 20 years that have led to the currently recommended conditions for the preparation and storage of platelet concentrates for transfusion. These include im proved anticoagulant-preservative solutions, instructions for specific centrifugation, controlled storage temperature, use of gas-permeable plastic containers, constant gentle agitation, and specified storage time.19 Nevertheless, these improvements are still inadequate to prevent the platelet storage lesion, a process that involves biochemical, as well as physical and metabolic processes. As such, recent emphasis has been placed on the need for studies in the area of lipid peroxidation, including the effects of endogenous antioxidant depletion or exogenous antioxidant supplementation on platelet lipid composition and platelet storage and survival. 4 In this regard, Koerner et al17 recently reviewed the role of membrane lipid in the platelet storage lesion. They noted that studies of platelet factor 3 activity

5 LIPID PEROXIDATION IN PLATELET CONCENTRATES 73 TABLE III Comparison of Malondialdehyde and Potassium Levels in Platelet and Leukocyte-free Plasma Versus Leukocyte-free Platelet Concentrate Plasma* Day n MDA: +L-Free Plasma limol/l M D A.+P L - Free Plasma \imol/l K :+L-Free Plasma K: + P L - Free Plasma (0.37) 0.94 (0.37) 4.1 (0.3) 4.1 (0.3) (0.42) 0.97 (0.29)«4.3 (0.5) 4.1 (0.3)ns (0.58) 0.93 (0.20)a 4.4 (0.4) 4.0 (0.3)b (0.60) 0.98 (0.24)a 4.6 (0.5) 4.2 (0.3)b * Statistical significance, leukocyte-free and platelet-free plasma versus plasma from corresponding leukocyte-free platelet concentrates. + L - = leukocyte. P L- = platelet and leukocyte. A = P < b = P < 0.05 ns = not significant. and flow cytometry of stored platelets revealed that membrane lipid is lost through the process of microvesiculation. In addition to lipid depletion over time, there is a loss of over 50 percent of the potential capacity of lipid-dependent platelet function by the fifth storage day. They concluded that the most likely underlying mechanism for this loss of lipid and platelet functional capacity is lipid peroxidation (LP). There is considerable support for LP in this regard. For example, an early study18 demonstrated a marked response in platelet aggregation when platelets were exposed to peroxidized free arachidonic acid but not to unoxidized arachidonic acid. More recently, Fagiolo and associates7 reported a correlation between increased LP and functional platelet impairment in stored platelet concentrates. In addition, a recent report10 showed that nanomolar quantities of hemoglobin released from dam aged red blood cells can induce p la te le t aggregation. The proposed mechanism involves oxidation of oxyhemoglobin by platelet-derived hydrogen peroxide with subsequent generation of free radicals. Furtherm ore, platelet aggregation was completely blocked by catalase or radical scavengers, such as deoxyribose and mannitol. In addition, Burch and Burch2 reported that the concentrate of glutathione, a naturally occurring antioxidant, rapidly decreases in platelet concentrates stored at 22 C. Several recent studies have demonstrated that LP can be significantly reduced in stored erythrocytes,12 14 with and w ithout prior irradiation, and in stored platelet concentrates16 by the addition of various m etal chelating agents and other antioxidants. In addition, prior donor supplementation with vitam ins C and E, w ater and lipidsoluble free radical scavengers respectively, are also effective in reducing LP in stored red cells.15 Others17 have also suggested that the addition of antioxidants to platelet concentrates may be beneficial in preventing or at least slowing the events that make up the storage lesion. Irradiation of red cells and platelet concentrates prior to transfusion is common practice in order to reduce the risk of graft-versus-host disease in high risk patients. However, irradiation of these

6 74 KNIGHT, SEARLES, AND BLAYLOCK preparations increases LP.14,16 In addition, the release of hydrogen peroxide22 and possible superoxide ion (0 2 )23 or other products from the w hite cells destroyed by the irradiation or naturally released, may accelerate platelet LP. On the other hand, leukocyte filters have now been introduced to reduce the frequency and intensity of febrile transfusion reactions, as well as other potential benefits including (a) alloimmunization,21 (b) transmission of cytomegalovirus,1 (c) activation of human immunodeficiency virus Type l,3 and (d) bacterial contamination of cellular blood products.9 W hatever the mechanism(s), it has b een show n in this study that the removal of white blood cells by filtration significantly reduces LP in platelet concentrates, as determined by the HPLC measurement of malondialdehyde (table I). In addition, the removal of leukocytes resulted in decreased cellular loss of potassium (table II). Since LP decreases cell m em b ran e d efo rm a b ility and increases permeability, it may also negatively affect the K+ transport mechanism^) with resultant increased platelet K+ loss. Further studies are needed in this critical area. It has also been demonstrated by the current authors that the changes reported here are due to the effects of LP on platelet lipids and not plasma lipids (table III). In summary, LP probably plays an important role in the platelet storage lesion. Since LP is a natural phenomenon, the addition of various antioxidants to platelet concentrates may be beneficial in prolonging the viability and longevity of platelets by further slowing the various complex processes that make up the platelet storage lesion. Furthermore, early filtration of platelet concentrates to remove leukocytes may also be effective. References 1. B o w d e n, R. A., S l i c h t e r, S. J., S a y e r s, M. H., M o r i, M., C a y s, M. J., a n d M e y e r s, J. D.: Use of leukocyte-depleted platelets and cytomegalo-virus-seronegative red blood cells for prevention of prim ary cytomegalovirus infection after marrow transplant. Blood 78: , B u r c h, P. T. and B u r c h, J. W.: Alterations in glutathione during storage of human platelet concentrates. Transfusion 27: , B u s c h, M. P., LEE, T., and HEITMAN, J.: Allogeneic leukocytes but not therapeutic blood elements induce reactivation and dissemination of latent human immunodeficiency virus Type 1 infection: Implications for transfusion support of infected platelet. Blood 80: , C h e r n o f f, A. and Sn y d e r, E. L.: The cellular and m olecular basis of the platelet storage lesion: A symposium summary. Transfusion 32: , C h i u, D., Ku y p e r s, F., and L u b in, B. P.: Lipid peroxidation in human red cells. Semin. Hematol. 26: , D z i k, W. H., C u s a c k, W. F., and G a c e k, M. J.: Preparation of white cell-reduced platelet concentrates from whole blood during component preparation. Transfusion 31: , F a g io l o, E., L i p p a, S., M o r e s, N., O r a d e i, A., and A u r ELI, V.: Peroxidation events in stored p latelet concentrates. Vox Sang. 56:32 36, H a l l i w e l l, B. and G u t t e r i d g e, J. M. C.: Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem. J. 2J9.1-14, H O g m a n, C. F., G o n g, J., H a m b r a e u s, A., J o h a n s s o n, C. S., and E r ik s s o n, L.: The role of white cells in the transmission of Yersinia enterocolitica in blood components. Transfusion 32: , Iu l ia n o, L., Vi o l i, F., P e d e r s e n, J. Z., P r a- TICO, D., ROTILIO, G., and BALSANO, F.: Free radical-mediated platelet activation by hemoglobin released from red blood cells. Arch. Biochem. Biophys. 299: , Kn i g h t, J. A., S m i t h, S. E., Ki n d e r, V. E., and ANSTALL, H. B.: Reference intervals for plasma lipoperoxides: age-, sex-, and specimen-related variations. Clin. Chem. 33: , Kn i g h t, J. A., V o o r h e e s, R. P., M a r t in, L., and A n s t a l l, H.: Lipid peroxidation in stored red cells. Transfusion 32: , Kn i g h t, J. A., V o o r h e e s, R. P., and M a r t in, L.: The effect of metal chelators on lipid peroxidation in stored erythrocytes. Ann. Clin. Lab. Sci. 22: , Kn i g h t, J. A., S e a r l e s, D. A., and Bl a y l o c k, R. C.: The effect of metal chelators on lipid peroxidation in irradiated erythrocytes. Ann. Clin. Lab. Sci. 22:417-^22, Kn ig h t, J. A., Bl a y l o c k, R. C., an d Se a r l e s, D. A.: T h e effect o f v itam in s C an d E o n lip id p ero x id atio n in sto red eryth ro cytes. A nn. C lin. L ab. Sci. 23:51-56, Kn i g h t, J. A., B l a y l o c k, R. C., and Se a r l e s, D. A.: Lipid peroxidation in platelet concentrates: Effects of irradiation and metal chelators. Ann. Clin. Lab. Sci. 23: , 1993.

7 LIPID PEROXIDATION IN PLATELET CONCENTRATES Ko e r n e r, T. A. W., C u n n in g h a m, M. T., and ZHANG, D-S.: The role of membrane lipid in the platelet storage lesion. Blood Cells 1 8 : , M ic k e l, H. S. and HORBAR, J.: The effect of peroxidized arachidonic acid upon hum an platelet aggregation. Lipids 9 : , M o r o f f, G. a n d H o l m e s, S.: C o n cep ts about cu rren t conditions for th e preparation and storage o f platelets. T ransfus. M ed. Rev. 5 :48-59, O k u m a, M., S t e in e r, M., and Ba l d in i, M.: Studies on lipid peroxides in platelets. I. Method of assay and effect of storage. J. Lab. Clin. Med. 7 5 : , S a a r in e n, U. M., Ke k o m à k i, R., S iim e s, M. A., and M y l l y l à, G.: E ffective prophylaxis against platelet refractoriness in m ultitransfused patients by use of leukocyte-free blood components. Blood 7 5 : , S c h u b e r t, J. and W il m e r, J. W.: Does hydrogen peroxide exist free in biological systems? Free Radic. Biol. Med. ii: , THOM AS, M. J.: Urate causes the human polymorphonuclear leukocyte to secrete superoxide. Free Radic. Biol. Med. 12:89-91, W a l k e r, R. H., ed.: Technical Manual, 10th ed. Arlington, VA, American Association of Blood Banks, W id m a n n, F. K., ed.: Standards fo r Blood Banks and Transfusion Services, 14th ed. Arlington, VA, American Association of Blood Banks, W o n g, S. H. Y., Kn i g h t, J. A., H o p f e r, S. M., Z a h a r ia, O., L e a c h, C. N., J r, and S u n d e r - m a n, F. W. J r.: Lipoperoxides in plasma as m easured by liquid-chromatographic separation of m alondialdehyde-thiobarbituric acid adduct. Clin. Chem. 3 3 : , 1987.