대한수혈학회지 : 제 29 권제 3 호, 2018 The Korean Journal of Blood Transfusion Vol. 29, No. 3, 291-300, December 2018 https://doi.org/10.17945/kjbt.2018.29.3.291 pissn 1226-9336 eissn 2383-6881 Original Article Haemonetics ACP 215 를이용한적혈구의동결과세척 윤경원 1 ㆍ최경영 2 ㆍ이선아 3 ㆍ민혁기 1 ㆍ김재현 1 대한적십자사혈액수혈연구원 1, 대한적십자사중부혈액검사센터 2, 명지성모병원진단검사의학과 3 Freezing and Washing of Red Blood Cells Using Haemonetics ACP 215 Kyoung Won Youn 1, Kyoung Young Choi 2, Sun Ah Lee 3, Hyuk Ki Min 1, Jaehyun Kim 1 Blood Transfusion Research Institute, Korean Red Cross 1, Wonju, Jungbu Blood Laboratory Center, Korean Red Cross 2, Daejeon, Department of Laboratory Medicine, Myongji St. Mary's Hospital 3, Seoul, Korea Background: The use of a functionally closed system for the glycerolization and deglycerolization of red blood cells (RBCs) allows for prolonged post-thaw storage for more than 24 hours. The aim of this study was to assess glycerolization and deglycerolization processing for RBCs using a high glycerol method in the automated, closed system provided by Haemonetics ACP 215. Methods: Thirty-five packed RBCs were glycerolized using the ACP 215 to a final concentration of 40% (wt/vol). The units were either frozen as such (n=30) or excess glycerol was removed (n=5) before freezing. After storage at 80 o C, the units were thawed, deglycerolized and resuspended in SAG-M. The frozen-thawed RBCs were stored at 4 o C, and analyzed for their stability and in vitro quality. Results: No prefreeze excess glycerol removal units showed significantly less potassium leakage during post-thaw storage compared to the prefreeze excess glycerol removal units. All measurements of the stability and in vitro quality of thawed RBCs prepared from frozen RBCs without the prefreeze removal of excess glycerol during post-thaw storage at 4 o C for 7 days were acceptable to the American Blood Bank Association s standards and European standards. Conclusion: RBCs frozen without prefreeze removal of excess glycerol and the ACP 215 simplifies cryopreservation procedure and increases the stability of frozen-thawed RBCs. This increases the practical applicability of cryopreserved RBCs in blood transfusion practice. (Korean J Blood Transfus 2018;29:291-300) Key words: Red blood cells, Cryopreservation, Glycerolization, Deglycerolization 서론 적혈구를동결하여장기간보존하는방법은 1950년 Smith [1] 에의해동결보호제인글리세롤을첨가하여최초로고안된이후많은연구가이루어졌다. Valeri 등 [2] 은 2005년에적혈구에글리 Received on September 7, 2018. Revised on October 16, 2018. Accepted on October 30, 2018 Correspondence to: Jaehyun Kim Blood Transfusion Research Institute, Korean Red Cross, 50 Hyeoksin-ro, Wonju 26465, Korea Tel: 82-33-811-0221, Fax: 82-33-811-0240, E-mail: kimjh@redcross.or.kr, ORCID: http://orcid.org/0000-0002-9494-156x This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Copyright C 2018 The Korean Society of Blood Transfusion - 291 -
Korean J Blood Transfus Vol. 29, No. 3, 291-300, Dec. 2018 세롤의최종농도가약 40% (wt/vol) 가되도록첨가하여동결하는고농도글리세롤 (high glycerol method) 법과 Haemonetics ACP 215 (Haemonetics, Braintree, MA, USA) 장비를이용하여폐쇄계 (closed system) 에서자동으로글리세롤을주입하여적혈구를동결하고, 탈글리세롤 (deglycerolization) 하여세척하는시스템을보고한이후동결해동적혈구의실용성및유용성이높아졌다. 적혈구의동결과해동, 세척이개방계 (open system) 에서이루어지는경우그유효기간은 24 시간이지만, 폐쇄계에서적혈구를자동으로동결하고해동하여세척하는경우에는 saline-adenineglucose-mannitol (SAG-M) 을보존액으로사용할때해동후 7일, additive solution-3 (AS-3) 를사용할때 14일까지보존기간연장이가능하다 [3-6]. 현재많은해외선진혈액사업국가에서는희귀혈액의공급또는전시ㆍ사변, 대규모자연재해등국가적비상사태시필요한혈액제제를공급하기위하여동결혈액프로그램을운영하고있다. 따라서국내에서도희귀혈액의공급이나국가적비상사태에대비하여혈액을동결하여장기보관할필요성이있을것으로생각된다. 본연구에서는향후국내에동결혈액보관시스템이도입될것을대비하여고농도글리세롤법과동결적혈구제조및해동의자동화기기인 ACP 215를이용한동결해동적혈구제조의프로토콜확립과여러가지기술적인문제점을점검하여동결혈액관리의기초자료로활용하고자하였다. 대상및방법 1. 연구용혈액연구용혈액은전혈 400 ml에서분리된농축적혈구제제로헌혈혈액선별검사결과 ALT 65 IU/L 이상으로폐기대상인혈액중채혈후 6일이경과하지않은혈액 35단위를사용하였으며, 본연구는대한적십자사생명윤리심의위원회로부터승인을받은후진행하였다 (IRB No. 17-1차- 정-8). 2. 동결적혈구의제조냉장보관해두었던농축적혈구제제를 37 o C 진탕항온수조에서 10분간두어표면온도가적외선온도계로측정하여 20 30 o C가되도록방치하였다. 혈액의온도가등온화되면농축적혈구가포함되어있는혈액백을무균백줄봉합기 (sterile connection device, SCD) 를사용하여 2 L 용량의 RBC Freezing Bag (Fresenius Kabi, Lake Zurich, IL, USA) 과연결하고, 혈액을 RBC Freezing Bag 으로옮겨담았다. ACP 215 장비에 glycerolization disposable set LN225 (Haemonetics, Braintree, MA, USA) 와 57.1% 글리세롤용액 (S.A.L.F. S.p.A. Laboratorio Farmacologico, Bergamo, Italy) 을장착하고, SCD를이용하여무균적으로혈액과연결한후혈액은 ACP 215와연결되어있는진탕기위에올려놓았다. ACP 215에혈액의무게를입력하고혈액무게에따라장비가자동적으로글리세롤을최종농도 40±4% (w/v) 로주입하도록미리설정되어있는프로그램을구동하여장비를작동하였다. 글리세롤주입이완료된 30단위의혈액은 Lelkens 등 [7] 의방법에의하여동결전잔여글리세롤을제거하지않고초기 24시간동안은 80 o C 냉동고의바닥에넓은면이닿도록하여냉동시키고 (no prefreeze volume reduction frozen red blood cell, no-pfvr FRBC) 그이후에는보관용트레이에넣어포개어보관하였다. 또한글리세롤주입이완료된혈액중 5단위는 Valeri 등 [2] 의방법에따라동결전잔여글리세롤을제거하기위하여 RBC - 292 -
윤경원외 : Haemonetics ACP 215 를이용한적혈구의동결과세척 Freezing Bag에 600 ml waste bag을무균적으로연결한후 22 o C에서 1,248x g에서 10분간원심분리하고, 혈장추출기를사용하여상층의글리세롤을 waste bag으로옮겨제거하였다. 제조한혈액은초기 24시간동안은 80 o C 냉동고의바닥에넓은면이닿도록하여냉동시키고 (prefreeze volume reduction frozen red blood cell, PFVR FRBC) 그이후에는보관용트레이에넣어포개어보관하였다. 3. 동결적혈구의해동및세척 80 o C에서 120일보존한동결적혈구는 32±2 o C 진탕항온수조에서 35분간두어혈액제제표면의온도가적외선온도계로측정하여 30 34 o C 되면적합한것으로판단하고세척을실시하였다. 동결해동적혈구의세척을위해우선 ACP 215에 deglycerolization disposable set LN235 (Haemonetics, Braintree, MA, USA), 12% NaCl 용액 (Bioluz Laboratoire Pharmaceutique, Saint-Jean-de-Luz, France), 0.9% saline/0.2% dextrose 용액 (Bioluz Laboratoire Pharmaceutique, Saint-Jean-de-Luz, France), SAG-M preservative solution (Haemonetics, Braintree, MA, USA) 을장착하였다. SCD를이용하여 LN235와항온수조에서해동한적혈구를무균적으로연결한후혈액은 ACP 215와연결되어있는진탕기위에올려놓았다. ACP 215에해동적혈구의무게와적혈구용적률 (hematocrit) 을입력하고, 입력된값에따라장비가자동적으로 12% NaCl, 0.9% saline/0.2% dextrose, SAG-M 용액을단계적으로주입하여세척하도록미리설정되어있는프로그램을구동하여장비를작동하였다. 동결해동적혈구의세척을위해서우선 12% NaCl 용액 50 ml을주입하고이어서 PFVR FRBC 의경우 ACP 215 세척프로그램의기본설정값대로 0.9% saline/0.2% dextrose 용액 340 ml을 60 ml/min의유속 (flow rate) 으로, PFVR FRBC의경 우에는 0.9% saline/0.2% dextrose 용액 600 ml 을 110 ml/min의유속으로 1차희석하고농축하는등총5회의세척과정을수행하였다. 마지막세척단계에서 Color Comparator (Haemonetics, Braintree, MA, USA) 의표시색상중 5번이하가되면세척이완료된것으로판정하고 SAG-M용액 240 ml 을주입하였다. 4. 동결해동적혈구의품질평가 동결해동적혈구의품질은동결적혈구를해동ㆍ세척한직후, 세척후 3일, 7일이경과한시점에서측정하였다. 1) 적혈구회수율및용혈률 적혈구냉동전및해동적혈구세척직후일반혈액검사 (CBC, complete blood count) 용검체 1 ml을무균적으로채취하였다. 적혈구수와적혈구용적률은자동혈구계산기 Sysmex KX-21N (Sysmex Corporation, Kobe, Japan) 장비로측정하고다음과같이적혈구회수율및용혈률을산출하였다. RBC recovery rate (%)= 100 Hemolysis (%)= 2) 혈장 K +, 혈장 Hb 및삼투압측정 혈장 K +, Hb 은동결해동적혈구세척직후, 세척후 3일및 7일이경과한후에측정하였다. 동결해동적혈구상층액에서 K + 농도와삼투압은검사를시행하는수탁검사기관에검사를의뢰하여측정하였다. SAG-M 용액에글리세롤의최종농도가 0.5 5% 되도록첨가한실험을 5회반복한후삼투압을측정하여 SAG-M 용액에서글리세롤농도에따른삼투압을계산하였다. 혈장 Hb - 293 -
Korean J Blood Transfus Vol. 29, No. 3, 291-300, Dec. 2018 측정을위하여혈액백에서 sampling site coupler (Baxter Healthcare corporation, Deerfield, IL, USA) 를통해무균적으로채취한 2 ml 검체를 2,000 g에서 10분간원심분리후, 분리한혈장 200 L 와 0.942 mol/l Na 2CO 3 용액 2 ml을혼합 (1:10 희석 ) 하여 Spectra Max Plus 384 (Molecular Devices, CA, USA) 장비를이용하여 415 nm, 450 nm, 700 nm 파장에서각각흡광도를측정하였다. 혈장 Hb의농도는 Fairbanks 등 [8] 의방법에따라아래와같은수식으로계산하였다. Plasma Hb (mg/dl)=155a 415 130A 450 124A 700 3) 가상수혈 (simulated transfusion) 동결해동적혈구의실제수혈시적혈구의파괴정도를예측하기위한가상수혈은세척이완료된해동적혈구 0.5 ml을생리식염수 10 ml에혼합하고 2,000 g에서 10분간원심분리후, 분리한상층액을 Spectra Max Plus 384 장비를이용하여 415 nm, 450 nm, 700 nm 파장에서각각흡광도를측정하였다. 상층액의자유혈색소 (free hemoglobin) 는혈장 Hb의농도와동일한방법으로 Fairbanks 등 [8] 의수식에따라계산하고, 용혈률을산출하였다. 4) 미생물배양검사세척이완료된직후동결해동적혈구에서 10 ml의검체를무균적으로채취하여 40 ml의 BacT/ALERT SA 배양액과 40 ml의 BacT/ ALERT SN 배양액 (biomérieux, Marcy I'Etoile, France) 에각각 5 ml씩접종하고 BacT/ALERT 3D 에서 14일간배양하여검사하였다. 5. 통계분석통계학적분석을위해서는 Microsoft Excel 2007 (Microsoft Corporation, Redmond, WA, USA) 소프트웨어를사용하였다. 동결전잔여글리세롤을제거한그룹과제거하지않은그룹간의비교는 paired t-test를사용하였고, P<0.05를통계학적으로유의한차이가있는것으로간주하였다. Table 1. Comparison of (de) glycerolized red blood cells with and without prefreeze volume reduction by the removal of excess glycerol after glycerolization Parameters 결과 동결해동적혈구 35 단위모두에서동결, 해동 No-PFVR 1 units (n=30) PFVR 2 units (n=5) Prefreeze RBC Volume (ml) 229.6±10.6 242.8±15.6 Total Hb (g) 56.3±3.1 60.1±3.4 Glycerolized RBC Volume (ml) 644.8±36.1 287.6±21.5* Hematocrit (%) 30.5±1.6 70.4±4.3* Total Hb (g) 53.3±2.8 56.4±3.4 Deglycerolized RBC Volume (ml) 307.3±2.4 308.3±1.6 Hematocrit (%) 45.3±2.5 50.8±1.7* Total Hb (g) 43.9±2.8 48.5±3.0* Plasma Hb (mg/dl) 4.4±1.9 4.5±2.1 RBC recovery (%) 81.5±4.7 85.6±2.4 Supernatant osmolarity 339.2±22.3 347.8±14.1 (mosm/kgㆍh 2O) Simulated transfusion 0.18±0.08 0.16±0.05 (%) Sterility No growth No growth 1 No-PFVR, No prefreeze volume reduction; 2 PFVR, prefreeze volume reduction; RBC, red blood cells; Hb, hemoglobin. *Data are reported as mean±sd. *Significantly different (P<0.05, Student s t-test) from the values obtained with prefreeze removal of excess glycerol. - 294 -
윤경원외 : Haemonetics ACP 215 를이용한적혈구의동결과세척 및세척과정상혈액의유출이나혈액백의손상은발견할수없었고, 미생물배양검사결과는모두음성이었다. No-PFVR FRBC의동결전평균용량은 229.6± 10.6 ml, 총혈색소함량은 56.3±3.1 g 이었고, PFVR FRBC의동결전평균용량은 242.8±15.6 ml, 총혈색소함량은 60.1±3.4 g 이었다. 적혈구에동결보호제인글리세롤을투여한뒤적혈구의용량은예상했던대로 no-pfvr FRBC는 644.8± 36.1 ml으로 PFVR FRBC의 287.6±21.5 ml에비해크게늘어났으며적혈구용적률은 PFVR FRBC 의 70.4±4.3% 에비해 no-pfvr FRBC에서 30.5± 1.6% 로낮게나타났다. 또한세척용액에의한혈색소손실이 no-pfvr FRBC에서더많이발생하여, 결과적으로세척이완료된동결해동적혈구의혈색소함량과적혈구용적률이 no-pfvr 방법의동결해동적혈구가 PFVR 방법의동결해동적혈구보다낮게나타났다 (Table 1). 세척이완료된해동적혈구의세척정도를평가하기위한지표로서삼투압은혈액을보존하는용액에따라그기준이달라질수있기때문에본연구에서사용된혈액보존액인 SAG-M 용액에서글리세롤농도에따른삼투압을측정하였다. 그결과글리세롤이전혀들어있지않은 SAG-M 용액의삼투압이 344.8±1.5 mosm/kgㆍh 2O 이었으며, SAG-M 용액에글리세롤이최종농도 1% 로포함되어있을때 454.2±4.0 mosm/kgㆍh 2O 이었다 (Fig. 1). 세척이완료된해동적혈구상층액의삼투압은 no-pfvr FRBC 의경우 339.2±22.3 mosm/kgㆍ H 2O, PFVR FRBC 의경우 347.8±14.1 mosm/kgㆍ H 2O 이었으며, 두그룹모두잔여글리세롤농도 1% (wt/vol) 에해당하는삼투압 454.2±4.0 mosm/ KgㆍH 2O을초과하는혈액은없었다. 동결해동적혈구에서평균적혈구회수율은 no-pfvr FRBC 의경우 81.5±4.7% ( 범위 : 76.8 86.2%) 이었고 PFVR FRBC 의경우 85.6±2.4% ( 범위 : 83.2 88.0%) 이었다. 한편가상수혈시 no-pfvr 방법의동결해동적혈구는상층액의자유혈색소가 14.6±6.4 mg/dl로서 0.18±0.08% 의용혈을보였으며, PFVR 방법의동결해동적혈구는상층액의자유혈색소가 16.2±5.4 mg/dl 로서 0.16±0.05% 의용혈을보였다 (Table 1). 세척이완료된후 SAG-M 용액에부유한동결해동적혈구를 4 o C에서보존하면서세척직후, 세척후 3일및 7일이경과한시점에서측정한 K + 및용혈률은, no-pfvr 방법으로동결하고해동한동결해동적혈구에서세척직후 K + 농도는 3.5±0.8 mol/gㆍhb (0.5±0.1 mmol/l), 보존 3일및 7일뒤는 43.1±7.5 mol/gㆍhb (6.2±1.4 mmol/l) 및 77.3±11.9 mol/gㆍhb (11.3±2.0 mmol/l), 용혈률은세척직후 0.1±0.0%, 보존 3일및 7일뒤는 0.3±0.1% 및 0.6±0.2% 로증가하였다. 반면 PFVR Fig. 1. Standard curve for the osmolarity of glycerol in SAG-M. A standard curve was prepared by making serial dilutions of glycerol in SAG-M within a range of concentrations near the expected concentrations of the unknown samples and tested triplicate. - 295 -
Korean J Blood Transfus Vol. 29, No. 3, 291-300, Dec. 2018 방법으로동결하고해동한동결해동적혈구에서는세척직후 K + 농도가 5.5±1.5 mol/gㆍhb (0.9±0.2 mmol/l), 보존 3일및 7일뒤는 112.2±16.8 mol/g ㆍ Hb (18.0±3.3 mmol/l) 및 160.6±22.8 mol/gㆍhb (26.1±4.3 mmol/l), 용혈률은세척직후 0.0±0.0%, 보존 3일및 7일뒤는 0.2±0.1% 및 0.6±0.1% 로증가하여용혈률증가양상은두방법이동일하였으나, 보존기간경과에따른 K + 유출은 no-pfvr 방법으로동결하고해동한동결해동적혈구보다 PFVR 방법으로동결하고해동한동결해동적혈구에서증가폭이크게나타났다 (Fig. 2). 고찰 CPDA-1 항응고보존액에보존한적혈구는채혈후 1 6 o C에서 35일간보존할수있으며, 적혈 구를동결하여보관할경우적혈구의보존기간을 10년이상최대 37년까지연장할수있는것으로보고되고있다 [9]. 그러나적혈구를동결하여보관하면적혈구는화학적, 열화학적, 기계적힘에노출되어수혈후산소운반능력에영향을받을수있다. 고농도글리세롤법에의해동결된동결적혈구의품질은동결전적혈구의보존기간, 해동후의보존기간및사용한보존액이중요한영향을미친다. 또한글리세롤을동결보호제로하여적혈구를동결하고, 동결적혈구를해동할때가장중요한기술적요구는탈글리세롤 (deglycerolization), 즉세척과정인데글리세롤이완전히제거되지않은혈액을인체에수혈하면세포내에고농도로존재하는글리세롤에의하여혈장내의물이흡수되어용혈이일어날수있어용혈성수혈부작용과신부전 (renal failure) 을유발할수있기때문이다 Fig. 2. In vitro quality of frozen-thawed red blood cells during storage in SAG-M. Red blood cells were either frozen with ( ) or without ( ) prefreeze removal of excess glycerol. After thawing and deglycerolization of frozen red blood cells, the red blood cells were resuspended in SAG-M, stored at 4 o C and analyzed for in vitro quality parameters: (A) hemolysis and (B) supernatant potassium. The results given are the mean and standard deviation of 5 units for without prefreeze removal and 30 units for prefreeze removal of excess glycerol. *Significantly different (P<0.05, Student s t-test) from the values obtained with prefreeze removal of excess glycerol. - 296 -
윤경원외 : Haemonetics ACP 215 를이용한적혈구의동결과세척 [10,11]. 한편동결해동적혈구의세척과정은동결보호제로사용한글리세롤을제거하는것뿐만아니라유해한생리활성지질 (bioactive lipids), 미립자 (microparticles), 사이토카인 (cytokines), 칼륨, 자유혈색소및백혈구도제거하는것으로보고되고있다 [12-14]. 동결해동적혈구의세척과정이완벽한지여부를확인하는방법으로는삼투압측정, 가상수혈 (simulated transfusion), 비중측정등의방법이있다. 해동후세척한적혈구의상층액에대해비중측정과삼투압측정을시행하여비중이 1.3384 이하, 잔존글리세롤의양이 1% 미만임을보증하기위해삼투압이 400 mosmol/l 이하면수혈용으로적합하다고판단하고있다. 과거유럽연합의동결해동적혈구품질관리기준에는전체해동된혈액의 1% ( 최소월 4단위 ) 를무작위표본검사하여삼투압이 340 mosm/kgㆍh 2O 이하가되어야한다고규정하였으나 [15], 최근에개정된기준에는삼투압측정에따른잔존글리세롤의평가기준이혈액을보존하는용액에따라달라질수있기때문에혈액을보존하는용액의삼투압보다최대 20 mosm/kgㆍh 2O 가초과되지않도록개정되었다 [16]. 따라서본연구에서사용한동결해동적혈구보존액인 SAG-M의삼투압이 344.8±1.5 mosm/kgㆍh 2O 인점을감안한다면 364 mosm/kgㆍh 2O 이하면탈글리세롤조작이잘이루어진것으로판단할수있겠는데, 본연구결과 no-pfvr 및 PFVR 방법으로제조된동결적혈구의해동ㆍ세척후삼투압은모두 364 mosm/kgㆍh 2O 이하로측정되었다. 또한실제수혈후적혈구의파괴정도를예측하는가상수혈은탈글리세롤된농축적혈구 0.5 ml을생리식염수 10 ml에섞어원심분리한후상층액의혈색소치를측정하여 3% 이하의용혈을보이면탈글리세롤조작이잘이루어진것으로판단한다. 한 편세척조작이충분히이루어졌는지의지표로서 K + 은 1.5 meq/l (1.5 mmol/l) 이하이어야하고, 세척과정중마지막상층액의혈색소치가 150 mg/dl 이하의값을보이면세척이충분히이루어진것으로판단한다. 본연구결과 no-pfvr 및 PFVR 방법으로제조된동결적혈구의해동ㆍ세척후삼투압및 K + 이모두이기준치이하였으며, 가상수혈결과허용기준이 3% 이하인데본연구결과 no-pfvr 방법으로제조된동결해동적혈구는 0.18±0.08%, PFVR 방법으로제조된동결해동적혈구는 0.16±0.05% 로기준치에비해상당히낮게나타나실제수혈시적혈구의파괴정도가매우낮을것으로예측되었다. 동결해동적혈구의용량및적혈구회수율의미국혈액은행협회 (American Association of Blood Banks, AABB) 의평가기준 [17] 은적혈구의회수율이동결전적혈구의 80% 이상이어야한다. 유럽연합의기준은해동된모든적혈구의용량이 185 ml 이상, 상층액의혈색소치가혈액제제 1 단위당 0.2 g 이하, 적혈구용적률 35 70% 이어야한다고규정하고있다 [16,17]. 본연구결과에서는 no-pfvr 방법으로제조된동결적혈구의해동ㆍ세척후평균용량은 307.3±2.4 ml, 적혈구회수율 81.5±4.7%, 적혈구용적률 45.3±2.5% 이고 PFVR 방법으로제조된동결적혈구의해동ㆍ세척후평균용량은 308.3±1.6 ml, 적혈구회수율 85.6±2.4%, 적혈구용적률 50.8±1.7% 로서모두기준에적합하였다. 또한상층액의혈색소치도두가지방법에서모두 0.2 g 이하로기준에적합하였다. ATP는모든생물의세포내에존재하여에너지대사에매우중요한역할을한다. 또한적혈구세포막안쪽의골격단백질인스펙트린 (spectrin) 을조절하여세포구조를유지하는데중요한역할을하는데, 적혈구내의 ATP 감소는적혈구세포 - 297 -
Korean J Blood Transfus Vol. 29, No. 3, 291-300, Dec. 2018 막의경직등세포막의변형을유발하여적혈구의생존력을저하시킨다 [18,19]. 그러므로일반적으로채혈직후적혈구내의 ATP의농도가평균 4±1 mol/gㆍhb 인데적혈구수혈후 24시간생존률이 75% 이상이려면적혈구내의 ATP 함량이최소한 2.7 mol/gㆍhb가되어야하고 [20], 동결해동적혈구에서높은수준의 ATP의양을기대하기위해서는적혈구동결전보존시간을제한하는것이중요하다 [4,21]. 현재고농도글리세롤법에의한적혈구의동결방법으로는 Valeri 등 [2] 의방법에의해동결전잔여글리세롤을제거하는방법이가장널리사용되고있으나, 2012년 List 등 [22] 과 2015년 Lelkens 등 [7] 이동결전잔여글리세롤을제거하지않은동결적혈구의해동후보존성이우수하고잔여글리세롤을제거하기위한원심분리과정이생략됨에따라동결과정의간소화, 혈액백파손의위험이경감된다는것이보고된이후유럽과아시아ㆍ태평양지역을중심으로적용이확대되고있다. 본연구에서는고농도글리세롤법과 ACP 215 장비를이용하여적혈구를동결하고세척하였는데, 동결해동적혈구의품질평가결과가 AABB 및유럽연합의기준에모두적합하여향후우리나라에서희귀혈액공급및국가적위기상황에대비하기위한 동결혈액보관시스템 구축시동결적혈구제조및사용에기술적인문제는없을것으로생각된다. 그러나본연구결과및 Lelkens 등 [7] 의결과에서도혈액동결을위해동결보호제인글리세롤을적혈구에첨가할때포화되지않은잔여글리세롤을제거하지않고바로동결하는 no-pfvr 방식의동결법은동결적혈구의해동ㆍ세척이후 K + 등의지표로보아해동후보존성이기존의 PFVR 방식보다우수하고, 적혈구동결과정의간소화및원심분리등에의해혈액에가해지는물리력을감소시킬수있는장점이있으 나, 적혈구회수율등에서는오히려기존의 PFVR 방식이보다우수한결과를나타내었다. 또한 no-pfvr 방식의동결적혈구부피가 PFVR 방식의동결적혈구부피보다상당히크므로동결혈액의보존관리의용이성측면에서불리한점이있다. 따라서본연구의결과와국내에서이미보고된연구결과 [23,24] 를종합하여볼때동결혈액보관시스템이국내에도입될경우밸리데이션절차를통하여검증한다면두방법중어느방법을선택하여도무방할것으로보인다. 적혈구동결및세척에사용된 ACP 215 장비는작동방법이비교적간단하고자동화되어있으나동결해동적혈구의안전성에는제조, 해동및세척에작업자의기술및숙련도역시중요한요인으로작용할것으로생각되어작업자의기술및숙련도를일정한수준이상으로유지하여야할것으로생각된다. 요약배경 : 적혈구를폐쇄계에서동결하고세척하는경우동결해동적혈구의보존기간을 24시간이상으로연장할수있다. 본연구에서는고농도글리세롤법과자동화장비인 Haemonetics ACP 215 를이용한폐쇄계에서적혈구의동결과세척과정을평가하였다. 방법 : ACP 215를이용하여 35단위의적혈구에글리세롤을최종농도가 40% (wt/vol) 가되도록첨가하고 30단위는즉시동결하였으며, 나머지 5 단위는잔여글리세롤을제거한다음동결하였다. 동결한적혈구는 80 o C에보관후해동ㆍ세척하여 SAG-M 보존액에부유하고, 4 o C에서보존하면서안정성및품질관리지표를분석하였다. 결과 : 잔여글리세롤을제거하지않은동결적혈구는잔여글리세롤을제거한동결해동적혈구에비해해동후보존기간동안칼륨유출이현저 - 298 -
윤경원외 : Haemonetics ACP 215 를이용한적혈구의동결과세척 히낮게나타났다. 잔여글리세롤을제거하지않은동결해동적혈구를해동후 4 o C에서 7일간보존하는동안측정한모든안정성및품질지표는 AABB와유럽연합의기준에모두적합하였다. 결론 : ACP 215를사용하여동결전잔여글리세롤을제거하지않고바로적혈구를동결하는방법은동결보존과정이단순하고동결해동적혈구의안정성이향상되어향후수혈을위한동결적혈구에현실적으로적용가능성이높은것으로생각된다. 감사의글 본연구를위해연구용장비와시약을공급하여주신헤모네틱스코리아 ( 주 ) 및애크미메디칼관계자분들께감사의뜻을표합니다. 논문의저자들은본연구에외부지원연구비및편견이개입되지않았으며이해관계충돌의여지가없음을밝힙니다. References 1. Smith AU. Prevention of haemolysis during freezing and thawing of red blood-cells. Lancet 1950;2:910-1 2. Valeri CR, Ragno G, Van Houten P, Rose L, Rose M, Egozy Y, et al. Automation of the glycerolization of red blood cells with the high-separation bowl in the Haemonetics ACP 215 instrument. Transfusion 2005;45:1621-7 3. Bandarenko N, Hay SN, Holmberg J, Whitley P, Taylor HL, Moroff G, et al. Extended storage of AS-1 and AS-3 leukoreduced red blood cells for 15 days after deglycerolization and resuspension in AS-3 using an automated closed system. Transfusion 2004;44:1656-62 4. Bohonek M, Petrás M, Turek I, Urbanová J, Hrádek T, Chmátal P, et al. Quality evaluation of frozen apheresis red blood cell storage with 21-day postthaw storage in additive solution 3 and saline-adenine-glucose-mannitol: biochemical and chromium-51 recovery measures. Transfusion 2010;50:1007-13 5. Lagerberg JW, Truijens-de Lange R, de Korte D, Verhoeven AJ. Altered processing of thawed red cells to improve the in vitro quality during postthaw storage at 4 degrees C. Transfusion 2007;47:2242-9 6. Sen A, Khetarpal A. Comparative study of automated cryopreservation of red blood cells. Med J Armed Forces India 2013;69:345-50 7. Lelkens CC, de Korte D, Lagerberg JW. Prolonged post-thaw shelf life of red cells frozen without prefreeze removal of excess glycerol. Vox Sang 2015;108:219-25 8. Fairbanks VF, Ziesmer SC, O'Brien PC. Methods for measuring plasma hemoglobin in micromolar concentration compared. Clin Chem 1992;38:132-40 9. Valeri CR, Ragno G, Pivacek LE, Cassidy GP, Srey R, Hansson-Wicher M, et al. An experiment with glycerol-frozen red blood cells stored at -80 degrees C for up to 37 years. Vox Sang 2000;79:168-74 10. Cregan P, Donegan E, Gotelli G. Hemolytic transfusion reaction following transfusion of frozen and washed autologous red cells. Transfusion 1991;31:172-5 11. Bechdolt S, Schroeder LK, Samia C, Schmidt PJ. In vivo hemolysis of deglycerolized red blood cells. Arch Pathol Lab Med 1986;110: 344-5 12. Vlaar AP, Kulik W, Nieuwland R, Peters CP, Tool AT, van Bruggen R, et al. Accumulation of bioactive lipids during storage of blood products is not cell but plasma derived and temperature dependent. Transfusion 2011;51: - 299 -
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