Biomedical Science Letters 2017, 23(2): 111~117 http://dx.doi.org/10.15616/bsl.2017.23.2.111 eissn : 2288-7415 Original Article Comparison of Efficiency between Pre-storage and Post-storage filtration by Leukoreduction Blood Filter Geon Sik Shin 1,2,, Bohee Kim 1,, Sung Hoon Kim 1, Ki-Jong Rhee 1 and Yoon Suk Kim 1, 1 Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University, Wonju, Gangwon-do 26493, Korea 2 Blood Transfusion Research Institute, Korean Red Cross, Wonju, Gangwon-do 26465, Korea Leukocytes in blood components are involved in diverse adverse transfusion reactions such as febrile non-hemolytic transfusion reactions. Therefore, leukocyte reduction is required to reduce these adverse reactions. The objective of this study was to compare the efficiency of pre-storage filtration and post-storage filtration. Filtration time, residual leukocyte count, RBC recovery, and hemolysis were assessed after pre-storage or post-storage filtration. Compared to pre-storage filtration, filtration time was prolonged and hemolysis was dramatically increased when post-storage filtration was performed. Residual leukocytes count and RBC recovery after post-storage filtration were similar with those obtained after pre-storage filtration. These results suggest that pre-storage filtration has better efficiency than post-storage filtration. These are thought to contribute to the production of better quality of leukoreduction blood components. Key Words: Leukoreduction blood filter, Adverse transfusion reaction, Pre-storage filtration, Filtration efficiency 서론수혈의필요성이증가하고있어혈액제제의수요는계속증가하고있다 (Menitove et al., 1982). 이러한혈액제제들에있는백혈구에의해이식편대숙주병 (graft-vs-host disease) 등다양한수혈부작용 (transfusion reaction) 이유발된다고알려져있다 (Evans and Gordon, 1974; Herzig et al., 1975). 이를예방하기위해백혈구를제거한혈액제제에대한연구가진행되어왔다 (Saarinen et al., 1990; Lane et al., 1992; Bruil et al., 1995; Kwon, 1995; Zoon, 1996; Vamvakas, 2006). 그결과많은국가에서헌혈후백혈구를제거하는정책 (Universal Leukocyte Reduction; ULR) 이시행중이다 (Engelfriet et al., 2001; Vamvakas and Blajchman, 2001; Bassuni et al., 2008). 현재백혈구제거혈액제제의사용이꾸준히증가하고있다. 백혈구제거평가기준으로미국 Food and Drug Administration (FDA) 및유럽 Council of Europe (CE) 의기준등이있는데 FDA 기준은잔여백혈구수 5.0 10 6 /unit 미만 (Guidances, 2012), 적혈구회수율 85% 이상이며 CE 기준은잔여백혈구수 1.0 10 6 /unit 미만 (Medicines, 2013), 적혈구회수율은 85% 이상이다. 적혈구용혈률은 FDA 기준 1% 미만, CE 기준 0.8% 미만이다. 현재국내에서는백혈구제거혈액제제제조시에저장전여과법과저장후여과법으로백혈구를제거하고있으며두여과법의사용률은비슷하다고보고되어있다. 저 * Received: June 22, 2017 / Revised: June 27, 2017 / Accepted: June 28, 2017 These authors contributed equally to this manuscript Corresponding author: Yoon Suk Kim. Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University,Wonju, Gwangwon 26493, Korea. Tel: +82-33-760-2860, Fax: +82-33-760-2195, e-mail: yoonsukkim@yonsei.ac.kr C The Korean Society for Biomedical Laboratory Sciences. All rights reserved. CC This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. - 111 -
장전여과는의료기관에공급하기전에혈액원에서채혈 후 8 시간내에백혈구를제거하여백혈구제거혈액제제를 제조하는것을의미하며, 저장후여과는혈액원이여과 하지않은혈액제제를공급후의료기관에서수혈직전에 백혈구를제거하는것을말한다. 최근과같은한정된혈 액수급환경에서더욱효율적인수혈을위해서는저장전 여과법과저장후여과법의여과효율을비교분석하여 고효율의여과법을제시하여사용을권장하는것이필요 하다. 본연구에서는저장전여과와저장후여과의여과 효율을비교분석하여저장전여과법이저장후여과법 보다여과효율이더우수함을확인하였고, 이결과를토 대로저장전여과법사용을권장하여보다질좋은백혈 구제거혈액제제제조에기여하고자한다. 혈액여과및여과시간측정 재료및방법 본실험은대한적십자사생명윤리심의승인을득한후 ( 안전관리팀 -513, 2017. 02. 08) 실시하였다. 채혈후 4 시간 이내에농축적혈구를제제하였고코오롱인더스트리 ( 주 ) 에서개발된농축적혈구용백혈구제거혈액필터를이용하 여여과를실시하였다. 여과시점효율성비교연구를위해 저장전여과는채혈후 8 시간이내에여과를실시하였으 며, 저장후여과는농축적혈구제조후 4 일동안 4 로 보관한농축적혈구제제로여과를실시하였다. 여과시간은 여과시작부터혈액백내혈액이모두비워지기까지의시 간으로정하여측정하였다. 잔여백혈구수및백혈구제거율측정, 계산 잔여백혈구수는 Fluorescence Activated Cell Sorter (FACS) 장비를이용하여 LeucoCOUNT kit (BD bioscience, San Jose, CA, USA) 를사용한 Bead-based flow cytometry 검사법으로 측정하였다. TruCOUNT tube 에검체 100 μl 를첨가후 LeucoCOUNT reagent 400 μl 를첨가하여 5 분간반응후 FACSCanto II (BD bioscience, San Jose, CA, USA) 장비를 이용하여 Bead events 수 (R1) 와 WBC events 수 (R2) 를각 각측정하여아래의공식에있는식에의해여과후잔 여백혈구수와백혈구제거율을계산하였다 (Barclay et al., 1998; Gomez-Arbones et al., 1999). 잔여백혈구수 ( 개 /μl) = 백혈구수 (R2) Bead 수 (R1) 총 Bead 수 혈액량 (μl) 백혈구제거율 (%) = 100 적혈구회수율, 적혈구용혈률측정 저장전여과와저장후여과전 후에혈액검체 1 ml 을채취하여적혈구수와적혈구용적률 (Hct) 을자동혈구 계산기 (KX-21N, Sysmex, Kobe, Japan) 로측정하였다. 적혈 구회수율은아래의공식을이용하여산출하였다. 혈장 혈색소 (Hb) 측정을위하여혈액백에서채취한 2 ml 혈액 검체를 3,000 rpm 10 분간원심분리후분리한혈장 200 μl 와 Na 2 CO 3 용액 2 ml 을혼합후 415 nm, 450 nm, 700 nm 파장에서각각흡광도를측정하였다. 혈장혈색소 농도와적혈구용혈률은아래의공식을이용하여계산하 였다 (Fairbanks et al., 1992). 적혈구회수율 (%) = 혈장 Hb (mg/dl) = (155.0 A 415 ) - (130.0 A 450 ) - (124.0 A 700 ) 적혈구용혈률 (%) = 결 과 저장전여과법과저장후여과법의여과시간비교. 백혈구제거농축적혈구제제는저장전에백혈구를제거 하거나저장후에백혈구를제거하는두가지방법으로 제조한다. 최근과같이혈액수급상황이점차로안좋아 지는상황에서여과시점에따른여과효율을비교연구하 여보다효율적인여과시점을확인하는것은제한된혈액 을보다효과적으로사용하는데매우중요하다. 따라서 본연구에서는백혈구제거혈액필터를이용하여저장전 여과와저장후여과의효율을비교평가하였다. 채혈후 8 시간이내여과를저장전여과로채혈후 4 일간냉장 보관된농축적혈구제제의여과를저장후여과로하여연 구를진행하였다. ( 여과후백혈구수 ) ( 여과전백혈구수 ) ( 여과후용량 ) ( 여과후 Hct) ( 여과전용량 ) ( 여과후 Hct) 혈장 Hb (100 % Hct) Total Hb 100 100 100-112 -
Table 1. Comparison of parameters evaluated after post-storage and pre-storage filtration * Filtration time Residual leukocyte ( 10 6 /unit) Parameters Leukoreduction (%) RBC recovery (%) Hemolysis (%) at day 0 after filtration Hemolysis (%) at day 28 after filtration 농축적혈구는본래 4 냉장상태에서보관하지만백혈 구제거를위한여과는상온에서실시하고있다. 따라서 여과시간이길어지면상온에노출되는시간이증가하여 적혈구손상을야기할가능성이있어여과시간을평가하 는것이중요하다. 따라서저장전여과와저장후여과의 여과시간을먼저평가하였다. Table 1 과 Fig. 1 에서보는바 와같이여과시간은저장전여과의경우평균 12 분, 최대 16 분 33 초, 최소 7 분 16 초였으며, 저장후여과의경우평 균 59 분 44 초, 최대 107 분 30 초, 최소 29 분 50 초였다. 이 결과를통해저장전여과가저장후여과에비해여과시 간이짧은것을확인할수있었으며, 이를통해저장전 여과중에적혈구손상가능성이저장후여과보다적을 것임을추론할수있었다. Post-storage filtration (n=10) Pre-storage filtration (n=10) Ave 59m 44s 12m Min 29m 50s 7m 16s Max 107m 30s 16m 33s Ave 0.003 0.098 Min 0 0 Max 0.03 0.2 Ave 99.999 99.996 Min 99.999 99.989 Max 100 100 Ave 88.4 90.6 Min 85.3 88.2 Max 90.5 92.3 Ave 0.3 0.02 Min 0.21 0.01 Max 0.53 0.04 Ave 0.69 0.09 Min 0.51 0.06 Max 1.01 0.15 * For this study to compare the efficacy of pre-storage filtration and post-storage filtration, domestic filters, FINECELL developed by KOLON INDUSTRIES, INC., (Gumi, Korea) were used as test filters. assessed immediately after filtration. assessed at day 35 after filtration. 저장전여과법과저장후여과법의잔여백혈구수비교다음은저장시점에따른잔여백혈구수를확인하였다. 결과 (Table 1, Fig. 2) 에서보는바와같이저장전여과의경우잔여백혈구수는평균 0.098 10 6 /unit, 최대 0.2 10 6 /unit, 최소 0 10 6 /unit 였으며, 저장후여과의경우는평균 0.003 10 6 /unit, 최대 0.03 10 6 /unit, 최소 0 10 6 /unit로측정되어두방법모두유럽기준 (<1 10 6 / unit) 및미국기준 (<5 10 6 /unit) 을만족하는백혈구제거능을보였다. 저장전여과의백혈구제거율은평균 99.996%, 최대 100%, 최소 99.989% 였으며, 저장후여과의경우는평균 99.999%, 최대 100%, 최소 99.999% 로계산되었다 (Table 1). 이결과를통해저장전여과와저장후여과의백혈구제거율은비슷함을확인할수있었다. 저장전여과법과저장후적혈구회수율비교. 적혈구회수율은결과 (Table 1, Fig. 3) 에서보는바와같이저장전여과의경우평균 90.6%, 최대 92.3%, 최소 88.2% 였고저장후여과의경우평균 88.4%, 최대 90.5%, 최소 85.3% 여서두여과법모두국제기준 ( 85%) 을만족하는적혈구회수율을보였다 (Table 3, Fig. 3). 이결과로저장전여과와저장후여과에서적혈구회수율은큰차이가없음을확인하였다. 저장전여과법과저장후적혈구용혈률비교. 다음으로적혈구용혈률을비교확인하였는데, 결과 (Table 1, Fig. 4) 에서보는바와같이저장전여과법으로여과한직후의적혈구용혈률은평균 0.02%, 최대 0.04%, 최소 0.01% 였으며, 저장후여과법으로여과한직후의적혈구용혈률은평균 0.3%, 최대 0.53%, 최소 0.21% 였다. 저장전여과법으로여과한후 28일이지나적혈구용혈률을측정한결과는평균 0.09%, 최대 0.15%, 최소 0.06% 였으며, 저장후여과법으로여과후 28일이지나측정한적혈구용혈률은평균 0.69%, 최대 1.01%, 최소 0.51% 였다. 저장전여과와저장후여과에의한적혈구용혈률은여과직후에평가시 15배정도의차이 ( 평균 0.02% 대평균 0.3%) 를보였다. 저장후여과법으로여과후 28일이지나측정한적혈구용혈률평균 ( 약 0.7%) 은유럽 (< 0.8%) 과미국제한기준 (< 1%) 에가까운근사치를나타냈으며, 1개의실험군혈액은국제적제한기준을초과하는 1.01% 의적혈구용혈률을보였다 (Table 1, Fig. 4). 이러한결과를통하여저장전여과가저장후여과에비해적혈구용혈 - 113 -
Fig. 1. Comparison of filtration time between post-storage and pre-storage filtration. Filtering for leukoreduction was carried out at room temperature within 8 hours of blood collection for prestorage filtration or at day 4 after blood collection for post-storage filtration. (A) Filtration time assessed after post-storage filtration (n=10), (B) Filtration time assessed after pre-storage filtration (n=10), (C) Comparison of filtration time between post-storage filtration (n=10) and pre-storage filtration (n=10). Data are expressed as the mean ± SD. P-values were determined by the Student's t-test (***P<0.001). Fig. 2. Comparison of residual leukocyte count between poststorage and pre-storage filtration. Residual leukocyte count was measured by bead-based flow cytometry. Filtration was carried out at room temperature at day 4 after blood collection for post-storage filtration. (A) Residual leukocyte count evaluated after post-storage filtration (n=10). (B) Residual leukocyte count evaluated after prestorage filtration (n=10). (C) Comparison of residual leukocyte count between post-storage filtration (n=10) and pre-storage filtration (n=10). Data are expressed as the mean ± SD. Dashed lines represent CE guideline (<1.0 10 6 /unit) for residual leukocyte count. - 114 -
이적은것을확인할수있었고, 저장전여과를하는것이적혈구에손상을적게주어장시간경과후 (28일 ) 확인했을때저장후여과의경우보다적혈구용혈이적은것을확인하게되어적혈구안정도를위해서는저장전여과가더좋은방법임을추론할수있었다. 고 찰 Fig. 3. Comparison of RBC recovery between post-storage and pre-storage filtration. RBC count and hematocrit were measured by Automated Hematology Analyzer. RBC recovery was calculated from the RBC count and hematocrit according to the formula described in Materials and methods. (A) RBC recovery assessed after post-storage filtration (n=10). (B) RBC recovery assessed after pre-storage filtration (n=10). (C) Comparison of RBC recovery between post-storage filtration (n=10) and pre-storage filtration (n=10). Data are expressed as the mean ± SD. Dashed lines represent CE guideline ( 85%) for RBC recovery. 해외에서는혈액의저장전백혈구제거가여러면에서더유용하다고보고되고있다 (King et al., 2004; Yazer et al., 2004; Bassuni et al., 2008). 저장후여과는여러단위의혈액을수혈하는경우규정된단위마다필터를교환해야하는번거로움이있으며이러한필터교환시와여과시에오염의위험이더크다. 혈액제제에서저장전에백혈구를제거하는경우, 저장후백혈구제거시보다백혈구에서분비하는싸이토카인과같은체액성물질에의한부작용을줄일수있다. 또한혈액보관시파괴되는백혈구파편에의한동종면역빈도도감소한다고보고되어있다. 따라서모든 ULR 도입국가는저장전여과를실시하고있다. 반면국내에서는저장전여과와저장후여과두방법모두백혈구제거에사용되고있다. 따라서저장전여과와저장후여과를비교분석하여더효율적인여과법을확인하고이를권장하는것이혈액관련정책에서매우중요하다고판단되어본연구에서는여과시점에따른여과효율성비교연구를진행하였다. 현재혈액원에서저장전여과시채혈후 8시간이내에실시하기때문에채혈후 8시간이내여과를저장전여과로규정하였다. 또한의료기관이혈액을직접여과후수혈할목적으로혈액원으로부터여과하지않은혈액을공급받을경우, 보통채혈후 4일이경과한혈액을공급받기때문에채혈후 4일경과한혈액여과를저장후여과로규정하여본연구를진행하였다. 먼저저장전여과와저장후여과시소요되는시간을평가한결과저장후여과의여과시간이저장전여과시간보다약 40분이상증가함을확인하였다 (Table 1, Fig. 1). 따라서저장후여과시상온에서의여과시간이증가하여적혈구활성도에좋지않은영향을미칠것으로추측된다. 또한저장후여과의적혈구용혈률이저장전여과의적혈구용혈률보다높은수치를보였다 (Table 1, Fig. 4). 특히, 저장후여과의경우여과한지 28일이지난후적혈구용혈률은국제제한기준 (0.8% 미만 ) 에근접한용혈률을나타냈으며그중 1개실험군에서는적혈구용혈률 1.01% 로국제적인제한 - 115 -
Fig. 4. Comparison of hemolysis between post-storage and prestorage filtration. Filtered RBC units were stored at 1~6 for 28 days. To calculate the hemolysis, plasma hemoglobin was measured before filtration, immediately after filtration, and at day 28 after filtration. (A) Hemolysis assessed after post-storage filtration (n=10). (B) Hemolysis assessed after pre-storage filtration (n=10). (C) Comparison of hemolysis between post-storage filtration (n=10) and pre-storage filtration (n=10). Data are expressed as the mean ± SD. P-values were determined by the Student's t-test (**P<0.01, ***P<0.001). Dashed lines represent CE guideline (< 0.8%) for hemolysis. 기준을초과하는결과를보였다 (Fig. 4). 이러한결과를통하여저장후여과가저장전여과에비해적혈구용혈이증가함을확인할수있었다. 결과적으로백혈구제거를위한농축적혈구여과시저장전여과를하는것이적혈구의안정도를위해서더효율적이라는것을다시한번확인할수있었다. 본연구를통해저장후여과보다저장전여과가적혈구안정도를더잘유지한다는사실을확인하였는데이러한결과는향후백혈구제거농축적혈구제제제조시저장전여과를실시할것을권장하는데기초자료로이용할수있을것으로생각한다. CONFLICT OF INTEREST The authors have no conflicts of interest to disclose. REFERENCES Barclay R, Walker B, Allan R, Reid C, Duffin E, Kane E, Turner M. Flow cytometric determination of residual leucocytes in filter-depleted blood products: an evaluation of Becton- Dickinson's LeucoCOUNT system. Transfusion Science. 1998. 19: 399-403. Bassuni WY, Blajchman MA, Al-Moshary MA. Why implement universal leukoreduction? Hematology/Oncology and Stem Cell Therapy. 2008. 1: 106-123. Bruil A, Beugeling T, Feijen J, van Aken WG. The mechanisms of leukocyte removal by filtration. Transfusion Medicine Reviews. 1995. 9: 145-166. Engelfriet CP, Reesink HW, Pietersz RN, Schwartz DW, Mayr WR, Blajchman MA, Goldman M, Decary F, Sher G, Georgsen J, Sprogoe-Jakobsen U, Kekomaki R, Kuhnl P, Seitz R, Maniatis A, Pinter J, Baroti K, Shinar E, Rebulla P, Greppi N, Sirchia G, Faber JC, Flanagan P, Brand A, Letowska M, Nel T, Argelagues E, Martin-Vega C, AuBuchon JP, Williamson L, Wallington T. Universal leucocyte-depletion of blood components: cell concentrates and plasma. Vox Sanguinis. 2001. 81: 56-77. - 116 -
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