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1 대한수혈학회지 : 제 21 권제 2 호, 2010 냉동보관전제대혈세포의생존율평가 이대영 1 ㆍ허지영 1 ㆍ강명서 1,2 차의과학대학교진단검사의학교실 1, 차병원제대혈은행 2 Assessment of Cell Viability in Umbilical Cord Blood before Cryopreservation Dae Young Yi 1, Ji Young Huh 1, Myung Seo Kang 1,2 Department of Laboratory Medicine, CHA University 1, Pocheon, CHA Medical Center Cord Blood Bank 2, Seongnam, Korea Background: The viability of cord blood is an important measure of product quality. Trypan blue (TB) stain is the most commonly and conveniently used method to measure the viability of the cord blood. Recently, cytometric analysis using 7-Aminoactinomycin D (7-AAD) was introduced. Staining with 7-AAD is more sensitive in detecting cellular damage than staining with TB. In addition to this, 7-AAD allows specific measurement of the viability of total nucleated cells (TNC), mononuclear cells (MNC) and CD34+ cells. In this study, we compared the viability of TNC between the TB and 7-AAD method, as well as analyzing the viability of each cell population. Methods: From February to July 2010, 102 cord blood units were collected and assessed for the viability of TNC by the TB and 7-AAD methods. The viability of mononuclear cells (MNC) and CD34+ cells was assessed by 7-AAD method. Results: The TB and 7-AAD methods were used to assess the viability of TNC, which was 90.1±5.7% and 68.4±8.0%, respectively. The viability of MNC and CD34+ cells measured by the 7-AAD method was 91.8±4.3% and 93.4±5.1%, respectively. Conclusion: The TNC viability of 7-AAD method was significantly lower than that of TB method. In 7-AAD method, the viabilities of MNC and CD34+ cells were significantly higher than that of TNC. As those are important prognostic factors and measures for successful engraftment after the transplantation, the measurement of the viabilities of MNC and CD34+ cells by 7-AAD method would be helpful to the quality control of the cord blood product. (Korean J Blood Transfus 2010;21: ) Key words: Viability, Trypan blue, 7-aminoactinomycin (7-AAD), Cord blood 접수일 :2010년 7월 5일, 수정일 :2010년 8월 19일, 승인일 :2010년 8월 20일책임저자 : 강명서 서울시강남구역삼 1동 650-9번지차의과학대학교강남차병원진단검사의학과 TEL: 02) , FAX: 02) , olive@chamc.co.kr

2 Assessment of Cord Blood Cell Viability 서론 제대혈에는조혈줄기세포가풍부하게함유되어있어골수이식이필요한환자들에게조직적합성항원이일치하는형제나기증자가없을경우조혈모세포이식의공급원으로사용되고있다. 이를위해 1993년뉴욕제대혈은행이설립된이후현재까지전세계적으로다수의제대혈은행이설립되어운영되고있고, 이를통해제대혈을이용한조혈모세포이식이활발하게이루어지고있다. 1,2) 국내에서는 1999년에최초로소아에서의제대혈조혈모세포이식이성공하였다. 3) 이후다수의제대혈은행들이설립되어운영되었으나, 각각의은행에서보관하고있는제대혈의정보가통합되어서제공되지않았기때문에제대혈의이용에어려움이있었다. 2006년부터는한국조혈모세포은행협회 (Korea Marrow Donor Program, KMDP) 에서국내 7개제대혈은행과협약을통해한국제대혈네트워크를구성하였고이를통해제대혈이식이활발히시행되고있다. 4) 국내외의제대혈은행을이용한제대혈이식을보다활성화시키고이식성적을향상시키기위해서는제대혈제제에대한엄격한품질관리가필요하다. 이를위해 FACT (Foundation for the Accreditation of Cellular Therapy) 와 NetCord에서는제대혈의품질관리항목으로총유핵세포수, CD34 양성세포수, 세포생존율 (viability) 등을제시할것을권장하고있다. 5) 이중세포생존율은제대혈이식에서생착과예후에영향을미치는중요한요인으로, 채취한제대혈의보관여부를결정하는기준항목중하나이다. 이에대한국제기준으로미국제대혈이식연구회 (cord blood transplantation study, COBLT) 에서는제대혈의보관기준으로총유핵세포생존율 90% 이상을제시하고있으며, 6) 국내에서는 2005 년에보건복지부에서마련한 제대혈은행표준업무지침 에서총유핵세포의생존율이 80% 이상일경우를보관기준으로하고있다. 7) 일반적으로제대혈의생존율평가에는 0.4% trypan blue 용액으로총유핵세포를염색하여염색된세포를제외하는방법이사용되고있으며, 제대혈의보관기준으로제시된생존율도이방법에기초하고있다. 6,7) 최근에는유세포분석기를이용하여 CD34 양성세포수를측정하면서 7- aminoactinomycin D (7-AAD) 염색으로생존율측정을동시에시행하는방법이상용화되어있다. 7-AAD를이용한유세포분석법은총유핵세포, 단핵구 (mononuclear cell, MNC), CD34 양성세포군에서각각의생존율을측정할수있는장점이있을뿐아니라 trypan blue 염색법에비해세포사멸을예민하게반영한다. 8,9) 저자들은제대혈세포의생존율평가를위해 trypan blue 염색법과 7-AAD를이용한유세포분석법으로측정한세포의생존율을비교하였다. 또한총유핵세포와단핵구, CD34 양성세포들의생존율을비교하여총유핵세포의생존율이단핵구나 CD34 양성세포의생존율을잘반영하는지평가하고자하였다. 1. 대상 대상및방법 2010년 2월부터 7월까지차병원제대혈은행에보관이의뢰되어저장처리가완료된공여또는개인보관제대혈 102단위를대상으로하였다. 대상제대혈은기증또는보관동의서에자발적으로동의및서명한한국인산모들로부터채취되었다

대한수혈학회지 : 제 21 권제 2 호 2. 방법 1) 제대혈의수집및공정과정분만을담당한산부인과의사가태아만출직후제대를결찰하고항응고제 (citrate phosphate dextrose adenine, CPDA-1) 24.

채취된제대혈은 10% pentastarch로처리하여적혈구를제거하고원심분리하여단핵구를모은후냉동보존제인 dextran-40/dmso를첨가하여 196 o C의액체질소탱크에보관하였다.

희석된제대혈 10μL를 trypan blue 시약 (0.4%, Gibco, Grand Island, USA) 10μL와혼합후 100개세포를계수하였고푸르게염색된세포를사멸이일어난세포로간주하였다.

Engineering) 법에따라 CD34 양성세포수를산정하였고, 7-AAD 염색을시행하여총유핵세포와단핵구구역세포, CD34 양성세포의생존율을측정하였다.

3 대한수혈학회지 : 제 21 권제 2 호 2. 방법 1) 제대혈의수집및공정과정분만을담당한산부인과의사가태아만출직후제대를결찰하고항응고제 (citrate phosphate dextrose adenine, CPDA-1) 24.5 ml가포함된채취백에제대혈을채취하였으며, 채취된제대혈은운송담당자가실온 24시간이내에제대혈은행으로운반하였다. 채취된제대혈은 10% pentastarch로처리하여적혈구를제거하고원심분리하여단핵구를모은후냉동보존제인 dextran-40/dmso를첨가하여 196 o C의액체질소탱크에보관하였다. 2) 세포생존율분석공정과정을거친제대혈에냉동보존제를넣기전검체에대해 trypan blue 염색법과 7-AAD염색법으로새포생존율측정을시행하였다. (1) Trypan blue 염색 : 제대혈을생리식염수로 1:7에서 1:10의비율로희석하였다. 희석된제대혈 10μL를 trypan blue 시약 (0.4%, Gibco, Grand Island, USA) 10μL와혼합후 100개세포를계수하였고푸르게염색된세포를사멸이일어난세포로간주하였다. (2) 7-AAD 염색유세포분석법 : Stem-Kit Reagents (Beckman Coulter, Fullerton, USA) 를이용하여 ISHAGE (International Society of Hematotherapy and Graft Engineering) 법에따라 CD34 양성세포수를산정하였고, 7-AAD 염색을시행하여총유핵세포와단핵구구역세포, CD34 양성세포의생존율을측정하였다. 유세포분석장비는 FACS- Calibur (BD Biosciences, San Jose, USA) 를사용하였고 CellQuest Pro (BD Biosciences) 소프트웨어를이용하여분석하였다. 제대혈 100μL에 CD45-fluorescein isothiocyan- Fig. 1. Strategy for flow cytometric analysis to determine the viabilities of total nucleated cells (TNC) and CD34+ cells. The viabilities of TNC (A) and of CD34+ cell are 100 viable TNC (F)/TNC (A) and 100 viable CD34+ cells (E)/CD34+ cells (D), respectively

세포생존율분석을위해서총유핵세포와 CD34 양성세포구역을각각 7-AAD/SSC plot으로분석하였고각각의생존율을 100 7-AAD low cells/(7-aad low+7-aad high) cells 공식을이용하여구하였다 (Fig. 1).

4 Assessment of Cord Blood Cell Viability ate (FITC)/CD34-phycoerythrin (PE) 시약 20μL와 7-AAD 시약 20μL를넣고잘혼합한후실온에서 20분간반응시켰다. NH 4Cl lysing solution 2 ml를첨가하여실온에서 10분간두어적혈구를용혈시킨후 100μL의 Stem-Count Fluorosphere를첨가하여 5초간혼합하고 1시간이내에분석하였다. 세포생존율분석을위해서총유핵세포와 CD34 양성세포구역을각각 7-AAD/SSC plot으로분석하였고각각의생존율을 AAD low cells/(7-aad low+7-aad high) cells 공식을이용하여구하였다 (Fig. 1). 또한, SSC와 CD45 강도에따라 (low-intermediate SSC, intermediate-high CD45) 림프구와단구 (monocyte) 구역의세포를단핵구로간주하여생존율을계산하였다 (Fig. 2). 3. 통계분석 Trypan blue와 7-AAD로측정한총유핵세포의생존율비교를위해쌍체 T검정 (paired t-test) 을사용하였다. 7-AAD로측정한총유핵세포, 단핵구, CD34 양성세포들간의생존율비교에는일원분산분석 (one-way ANOVA) 을사용하였고 Tukey 방법으로사후검정을하였으며 P-value가 0.05 이하일경우통계적으로유의하다고판정하였다. 통계프로그램으로는 SAS 9.1 (SAS Institute Inc., Cary, USA) 을사용하였다. 결과채취후제대혈의세포생존율을 trypan blue 염 Fig. 2. Strategy for flow cytometric analysis to determine mononuclear cells (MNC). The viability of MNC (A) is 100 viable MNC (B)/MNC (A). Table 1. Characteristics of samples and comparison of viabilities measured by trypan blue and 7-AAD method* Method Time (hr) from sampling to test TNC count ( 10 8 /unit) CD34+ cells count ( 10 6 /unit) Mean viability (%) TNC MNC CD34+ cell P-value Trypan blue 90.1±5.7 NA NA < ±7 6.0± ±1.3 7-AAD 68.4± ± ±5.1 < Abbreviations: AAD, aminoactinomycin D; TNC, total nucleated cell; MNC, mononuclear cell; NA, not applicable. *Data are mean±sd, Paired t-test for TNC viabilities of trypan blue and 7-AAD, One-way ANOVA with post hoc analysis by the Tukey method for 7-AAD viabilities of TNC, MNC, and CD34+ cells (The difference of viability between MNC and CD34+ cells is not significant)

5 대한수혈학회지 : 제 21 권제 2 호 색법과 7-AAD를이용한유세포분석법으로동시에측정하기까지는평균 34±7시간이소요되었다. 제대혈검체들의총유핵세포수는평균 /unit이었고, CD34 양성세포수는평균 /unit이었다(table 1). Trypan blue와 7-AAD로측정한총유핵세포의평균생존율은각각 90.1±5.7% 와 68.4±8.0% 로 trypan blue 염색법으로측정한경우가월등히높았다 (P<0.0001)(Table 1). 각각의세포군에대해 7-AAD로측정한평균생존율은단핵구, CD34 양성세포에서각각 91.8±4.3%, 93.4±5.1% 를보여 CD34 양성세포>단핵구>총유핵세포순으로높은생존율을보였으나 (P<0.0001) CD34 양성세포와단핵구생존율의차이는통계적으로는유의하지않았다 (Table 1). 고찰 제대혈제제의품질관리를위해 FACT와 Net- Cord, COBLT 등에서제시한국제적인지침들이나국내보건복지부에서발표한지침들에서는모두제대혈의냉동보관여부를결정하는데있어반드시생존율을측정하도록하고있다. 5-7) 이와같은지침들이보관기준으로제시하는생존율은일반적으로 trypan blue로측정한총유핵세포의생존율을기준으로하고있다. 6,7) Trypan blue 염색을이용한세포생존율의측정은비교적간단하고신속하게생존율을측정할수있는장점이있으나, 검사자에따른주관적판독및세포사멸의초기단계에서는염색이되지않아세포손상을민감하게반영하지못하는단점이있다. 8,9) 또한, 총유핵세포의생존율만을확인할수있는데, 실제골수이식의생착과예후에중요한 CD34 양성세포나조혈모세포는유핵세포중극히일부이므로정확하게반영되지않을가능성이있다 ) 본연구에서는기존에사용하던 trypan blue와이후소개된 7-AAD 염색법을이용해총유핵세포의생존율을비교하였고, 7-AAD법으로유핵세포중단핵구와 CD34 양성세포의생존율을각각확인하였다. Trypan blue로염색한경우총유핵세포의생존율은 90.1±5.7% 로서 Lee 등 13) 이보고한결과보다낮은수치를보였으나, 이는본연구에서생존율을측정하기까지시간이더소요되었기때문일가능성이있다. 7-AAD로측정한총유핵세포의생존율은 68.4±8.0% 로보건복지부에서제대혈보관기준으로제시한 80% 에미치지못했으나, 단핵구와 CD34 양성세포의생존율은각각 91.8± 4.3%, 93.4±5.1% 로 80% 이상이었다. 총유핵세포의생존율이 trypan blue와 7-AAD법에서크게차이를보인이유는 7-AAD가보다민감하게조기사멸단계에있는세포들을검출할수있기때문으로생각된다. 7-AAD법으로검사하였을때단핵구및 CD34 양성세포의생존율은비교적높게측정되었기때문에총유핵세포의낮은생존율은제대혈유핵세포의 40 70% 를차지하는과립구의 14) 생존율을반영한것으로추정된다. 과립구의경우, 채혈후 12 24시간이내에세포사멸이시작되는반면림프구나단구같은단핵구들은시간이지나도과립구에비해생존율이높게유지되는것으로알려져있다 ) 7-AAD로측정한 CD34 양성세포의생존율은 trypan blue로측정한총유핵세포의생존율보다높았으며 (P<0.0001), 이는 Humpe 등의보고와일치하였다. 18) 현재국내에서는 10여개이상의제대혈은행들이운영되고있으며한국제대혈네트워크에는 2010년 6월기준으로 21,000여단위이상의공여제대혈이등록되어있다. 19) 대부분의제대혈은행은 COBLT에서제시한국제적인제대혈은행관리기준 6) 을따르고있으나아직이에대한국가

6 Assessment of Cord Blood Cell Viability 가제시하는체계적인관리기준이없어이에대한필요성이커지고있다. 최근국가에서는이러한문제들을해결하기위해 2010년 3월에 제대혈관리및연구에관한법률 을공표하였고, 이는제대혈제제와은행들의국가차원에서의관리를위한등록및허가등에대한내용을담고있다. 20) 제대혈제제의보관전생존율의측정은필수적이므로이에대한기준도앞으로보완되어야할것이며, 생존율측정방법및보관기준을결정하는데있어본연구결과를참고할수있을것으로생각한다. Trypan blue 염색법과 7-AAD 염색법으로세포생존율을측정할경우검사결과에차이를보이므로, 세포생존율에따른제대혈의보관기준을결정할때에는시약의종류에따른고려가필요하다고생각된다. 또한, 7-AAD 염색법을시행할경우에는총유핵세포의생존율뿐만아니라단핵구와 CD34 양성세포의생존율도같이측정하여기록하는것이유용할것으로판단된다. 저자들은기존에생존율측정에사용되어온 trypan blue와 7-AAD를이용한세포생존율결과를비교하였다. 총유핵세포의생존율은 trypan blue 염색법의생존율이 7-AAD 보다유의하게높았으며, 이는 7-AAD가세포사멸의초기단계부터염색되기때문인것으로생각되었다. 7-AAD법을이용할경우단핵구군과 CD34 양성세포의생존율을따로측정할수가있었는데총유핵세포보다는단핵구군과 CD34 양성세포에서유의하게높은세포생존율을보였다. 단핵구군과 CD34 양성세포는제대혈이식후생착및예후에중요한영향을미치는요인으로알려져있어총유핵세포생존율에더하여단핵구군과 CD34 양성세포의생존율을같이측정할경우제대혈의품질관리에도움이될수있을것으로기대한다. 요약배경 : 제대혈의생존율측정은제대혈제제의질관리에있어중요한지표가된다. 세포생존율측정법은 trypan blue 염색법이간편하여가장흔히사용되는방법이다. 최근소개된 7-aminoactinomycin (7-AAD) 을이용한유세포분석법은 trypan blue에비해세포손상을예민하게반영하고총유핵세포, 단핵구, CD34 양성세포군에서각각생존율의특정이가능한장점이있다. 저자들은 trypan blue와 7-AAD 염색법을이용하여측정한생존율을비교하고, 총유핵세포와각세포군별로측정한생존율을비교하고자하였다. 방법 : 2010년 2월에서 7월동안수집된제대혈 102단위를대상으로각각 trypan blue 염색법과 7-AAD를이용한유세포분석법으로총유핵세포의생존율을측정하였다. 또한 7-AAD 염색법으로는총유핵세포뿐아니라단핵구, CD34 양성세포에대해생존율을각각측정하였다. 결과 : Trypan blue와 7-AAD로측정한총유핵세포의생존율은각각 90.1±5.7% 와 68.4±8.0% 였다. 7-AAD로측정한단핵구및 CD34 양성세포의생존율은각각 91.8±4.3%, 93.4±5.1% 였다. 결론 : 총유핵세포의생존율은 7-AAD 방법이 trypan blue 염색법으로측정한것보다유의하게낮았다. 7-AAD법으로측정한경우, 단핵구나 CD34 양성세포의생존율은총유핵세포의생존율보다유의하게높았다. 제대혈이식후의생착과예후에중요한것은단핵구나 CD34 양성세포의생존율이므로향후 7-AAD법으로이들의생존율을같이측정한다면제대혈제제의품질관리에도움이될것으로사료된다

7 대한수혈학회지 : 제 21 권제 2 호 참고문헌 1. Gluckman E, Broxmeyer HA, Auerbach AD, Friedman HS, Douglas GW, Devergie A, et al. Hematopoietic reconstitution in a patient with Fanconi's anemia by means of umbilical-cord blood from an HLA-identical sibling. N Engl J Med 1989;321: Rubinstein P, Taylor PE, Scaradavou A, Adamson JW, Migliaccio G, Emanuel D, et al. Unrelated placental blood for bone marrow reconstitution: organization of the placental blood program. Blood Cells 1994;20: Lee YH, Cho NC, Je KH, Han H, Han JY, Kim JS, et al. Successful sibling cord blood stem cell transplantation for relapsed acute mixed lineage leukemia. Korean J Hematol 1999; 34: KMDP. Introduction for Korea Marrow Donor Program (KMDP). [Online] (last visited on 21 June 2010) 5. FACT and NetCord. NetCord-FACT International Standards for Cord Blood Collection, Banking, and Release for Administration. h_edition_cord_blood_standards_4.0.pdf [Online] (last visited on 22 June 2010) 6. COBLT. Cord Blood Bank Standard Operating Procedures. jb/sjb030301vw.jsp?par_menu_id=03&me NU_ID= &BOARD_ID=1003&BOARD _FLAG=02&CONT_SEQ=34903&page=1 [Online] (last visited on 25 June 2010) 7.The Korean Society of Blood and Marrow Transplantation. The Standard Manual for Cord Blood Banking. kr/front/jb/sjb030301vw.jsp?par_menu_id =03&MENU_ID= &BOARD_ID=1003 &BOARD_FLAG=02&CONT_SEQ=34903&pag e=1 [Online] (last visited on 19 June 2010) 8. Darzynkiewicz Z, Li X, Gong J. Assays of cell viability: discrimination of cells dying by apoptosis. Methods Cell Biol 1994;41: van der Pol MA, Broxterman HJ, Westra G, Ossenkoppele GJ, Schuurhuis GJ. Novel multiparameter flow cytometry assay using Syto16 for the simultaneous detection of early apoptosis and apoptosis-corrected P-glycoprotein function in clinical samples. Cytometry B Clin Cytom 2003;55: Solomon M, Wofford J, Johnson C, Regan D, Creer MH. Factors influencing cord blood viability assessment before cryopreservation. Transfusion 2010;50: Broxmeyer HE, Smith FO. Cord blood hematopoietic cell transplantation. In: Appelbaum FR, Forman SJ, Negrin RS, Blume KG. Thomas hematopoietic cell transplantation. 3rd ed. Oxford: Blackwell Publishing, 2004: Barker JN, Wagner JE. Umbilical cord blood transplantation. In: Hoffman R, Benz E, Shattil SJ, Furie B, Cohen HJ, Silberstein LE. Hematology Basic principles and practice. 4th ed. Edinburgh: Elsevier Churchill Livingston, 2005: Lee HR, Roh EY, Yoon JH, Han KS, Kim BJ, Hwang KR, et al. Analysis of maternal and neonatal factors affecting hematopoietic parameters of cord blood. Korean J Blood Transfus 2009;20: Lee HR, Shin S, Yoon JH, Kim BJ, Hwang KR, Kim JJ, et al. Complete blood count reference values of donated cord blood from Korean neonates. Korean J Lab Med 2009;29: Colotta F, Re F, Mantovani A. Granulocyte transfusions from granulocyte colony-stimulating factor-treated donors: also a question of cell survival? Blood 1993;82: Dale DC, Liles WC, Price TH. Renewed interest in granulocyte transfusion therapy. Br

8 Assessment of Cord Blood Cell Viability J Haematol 1997;98: Dale DC, Liles WC, Summer WR, Nelson S. Review: granulocyte colony-stimulating factor--role and relationships in infectious diseases. J Infect Dis 1995;172: Humpe A, Beck C, Schoch R, Kneba M, Horst HA. Establishment and optimization of a flow cytometric method for evaluation of viability of CD34+ cells after cryopreservation and comparison with trypan blue exclusion staining. Transfusion 2005;45: KMDP. The coordination records for hematopoietic stem cell transplantation. kmdp.or.kr [Online] (last visited on 21 June 2010) 20. Ministry of Health and Welfare, Republic of Korea. The law for cord blood administration and study. do?lsnm=&cptofi= &searchtype=&lsk ndcd=&p_spubdt=&p_epubdt=&p_spubno=& p_epubno=&pageindex=1&chridx=6&sortidx= 0&lsiSeq= [Online] (last visited on 24 June 2010)

하는방법에대해서는구체적으로제시되어있지않지만, 일반적으로제대혈은행에서는 trypan blue법으로총유핵세포의생존율을검사하여저장전제대혈보관의적합유무를판정하고있다. 최근에는유세포분석기를이용하여 CD34 양성세포수측정과 7-aminoactinomycin (7-AAD) 생존율을

하는방법에대해서는구체적으로제시되어있지않지만, 일반적으로제대혈은행에서는 trypan blue법으로총유핵세포의생존율을검사하여저장전제대혈보관의적합유무를판정하고있다. 최근에는유세포분석기를이용하여 CD34 양성세포수측정과 7-aminoactinomycin (7-AAD) 생존율을 원저 Lab Med Online Vol. 4, No. 1: 1-7, January 214 http://dx.doi.org/1.3343/lmo.214.4.1.1 진단혈액학 7-AAD/annexin V 동시염색법을이용한제대혈의세포생존율평가 Assessment of Cell Viability in Umbilical Cord Blood by Using 7-AAD/annexin