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

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1 원저 Lab Med Online Vol. 4, No. 1: 1-7, January 진단혈액학 7-AAD/annexin V 동시염색법을이용한제대혈의세포생존율평가 Assessment of Cell Viability in Umbilical Cord Blood by Using 7-AAD/annexin V Dual Staining 김경미 1 허지영 1 강명서 1 정상희 2 Kyeong Mi Kim, M.D. 1, Ji Young Huh, M.D. 1, Myung Seo Kang, M.D. 1, Sang Hee Jung, M.D. 2 차의과학대학교분당차병원진단검사의학과 1, 산부인과 2 Departments of Laboratory Medicine 1, Obstetrics & Gynecology 2, CHA Bundang Medical Center, CHA University, Seongnam, Korea Background: The quality of cord blood largely depends on cell viability. Viability assessments using trypan blue or 7-aminoactinomycin (7-AAD) staining, which are commonly used methods, may not reflect early apoptosis of cord blood cells. We aimed to investigate early apoptosis in cord blood cells following elapsed time after collection using double staining with annexin V and 7-AAD and to compare the result with that of viability evaluation using trypan blue or 7-AAD staining. Methods: Umbilical cord blood samples were obtained from 3 pregnant women at the time of delivery between July 212 and March 213. Viability of cord blood cells was determined at (T), 24, and 48 hr after collection by using trypan blue exclusion assay, 7-AAD staining, and 7-AAD/ annexin V staining. Results: Viabilities defined by 7-AAD/annexin V staining at T, 24, and 48 hr after collection were respectively as follows: total nucleated cells, 92.8±4.5%, 78.4±7.8%, and 65.5±8.1%; mononuclear cells, 94.4±1.7%, 9.8±4.2%, and 84.2±6.7%; and CD34-positive cells, 92.4± 3.%, 9.7±4.7%, and 89.3±7.%. The viability using trypan blue was more than 9% until 48 hr after collection. Conclusions: The mean viability of total nucleated cells using 7-AAD/annexin V staining decreased to less than 8% at 24 hr after collection; however, the viability of CD34-positive cells was more than 85% until 48 hr. Our study s data will provide useful information for the assessing the quality of cord blood products. Key Words: Cord blood, Viability, Trypan blue, 7-aminoactinomycin, Annexin V 서론 제대혈 (umbilical cord blood) 은조혈줄기세포 (hematopoietic stem cell) 가풍부하게포함되어조혈모세포이식의공급원중하 Corresponding author: Myung Seo Kang Department of Laboratory Medicine, CHA Bundang Medical Center, 59 Yatap-ro, Bundang-gu, Seongnam , Korea Tel: , Fax: , olive@chamc.co.kr Received: December 14, 212 Revision received: May 28, 213 Accepted: May 28, 213 This article is available from 214, Laboratory Medicine Online This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 나로널리사용되고있다 [1-3]. 이식성적을향상시키기위해서는제대혈에대한엄격한품질관리가필요하다. 제대혈품질에관련된여러가지요소중세포생존율은제대혈이식의생착과예후에중요한요소이며, 제대혈채취후공정까지소요되는시간은세포생존율에가장큰영향을미친다 [4, 5]. 따라서제대혈의품질관리를위해미국및유럽의경우채취후보관처리전까지의시간이나세포생존율을제대혈보관여부를결정하는기준에포함시켜제시하고있다. Cord Blood Transplantation Study (COBLT) 와 NetCord-Foundation for the Accreditation of Cellular Therapy (FACT) 에서는제대혈채취부터동결저장까지의시간을 48시간이내로, 세포생존율을 9% 이상으로규정하고있다 [6, 7]. 최근시행된국내의 제대혈관리및연구에관한법률 에서는채취후 36시간이내에보관처리를시행하고, 8% 이상의세포생존율을보관기준으로명시하고있다 [8]. 세포생존율을측정 eissn

2 하는방법에대해서는구체적으로제시되어있지않지만, 일반적으로제대혈은행에서는 trypan blue법으로총유핵세포의생존율을검사하여저장전제대혈보관의적합유무를판정하고있다. 최근에는유세포분석기를이용하여 CD34 양성세포수측정과 7-aminoactinomycin (7-AAD) 생존율을동시에시행하는방법이상용화되어있다. 그러나 trypan blue나 7-AAD를이용한세포생존율측정은진행된세포사멸단계를검출하며, 초기세포사멸은반영하지못한다 [9, 1]. 즉, 제대혈의세포생존율이기존의방법으로높은결과를보였더라도초기세포사멸이진행된세포가상당수있을수있고, 이는조혈모세포이식의성적에직접적으로영향을미칠수있다 [11]. 따라서, 조혈모세포이식의성적을보다정확하게예측하고향상시키기위해서는초기세포사멸이일어난세포를배제하여정확한세포생존율을측정하는것이필요하다. 현재국내에서는초기세포사멸을검출할수있는민감한방법을이용한제대혈의세포생존율측정에대한연구가미흡한실정이다. 이에저자들은 7-AAD에초기세포사멸을검출할수있는 annexin V를추가하여제대혈채취후시간경과에따른세포생존율을측정하고이를기존의 trypan blue 및 7-AAD 만을이용한생존율과비교하고자하였다. 대상및방법 1. 연구대상 212년 7월부터 213년 3월까지분당차병원에서만삭에분만한산모 3명에서채취한제대혈을대상으로하였다. 본연구는분당차병원의학연구윤리심의위원회의심의를통과하였으며 (BD212-96D), 모든대상산모들에게연구참가동의서를받은후시행되었다. 2. 방법 1) 검체수집태아만출직후제대를결찰하고 EDTA 채혈관에제대혈 3 ml 를채취하였다. 채취된제대혈은즉시검사실로운반하였고, 검사전까지실온보관하였다. 2) 세포생존율측정제대혈은채취후 6시간이내에 trypan blue, 7-AAD 단독및 7-AAD/annexin V를이용하여각각세포생존율을측정하고 (T), 이후 24 및 48시간경과시점에서세포생존율을반복측정하였다. (.4%, Gibco, Grand Island, NY, USA) 1 μl와혼합후 1개의세포를계수하여푸르게염색된세포를사멸이일어난것으로판정하여백분율로나타내었다. (2) 유세포분석기를이용한 7-AAD/annexin V 염색법상품화된 Stem-Kit Reagents (Beckman Coulter, Fullerton, CA, USA) 에 annexin V-allophycocyanin (APC; BD Biosciences, San Jose, CA, USA) 을추가하여 CD34양성세포수와 7-AAD/annexin V 를이용하여세포생존율을동시에측정하였다 [12]. 유세포분석기는 FACSCalibur (BD Biosciences) 를사용하였고 CellQuest Pro (BD Biosciences) 프로그램을이용하여분석하였다. 제대혈 1 μl에 CD45-fluorescein isothiocyanate (FITC)/ CD34-phycoerythrin (PE) 시약 2 μl를넣고실온에서 2분간반응시켰다. NH4Cl lysing solution 2 ml를첨가하여실온에서 1분동안적혈구를용혈시킨후 2, rpm으로 5분간원심분리하여상층액을제거하였다. Annexin V 염색을위하여 annexin V binding buffer (BD Biosciences) 5 μl를넣어만든부유액에annexin V-APC 1 μl와 7-AAD 1 μl를첨가한후잘혼합하여 15분간실온에서반응시켰다. 유세포분석전 Stem-Count Fluorosphere 1 μl를첨가하여 5초간혼합한후분석하였다. 세포생존율분석을위해서총유핵세포, 단핵구및 CD34 양성세포구역을 7-AAD/annexin V plot에서분석하였다. 7-AAD 생존율은각구역의세포중 7-AAD 음성인세포의분획을퍼센트로나타내었다. 7-AAD/annexin V 생존율은각구역의세포중 7-AAD와 annexin V에서동시에음성을보이는세포의분획을퍼센트로나타내었다. 분석시 Stem-Kit Reagents (Beckman Coulter) 를이용해 CD34 양성세포측정만시행하고 7-AAD 및 annexin V로는염색하지않은시험관을음성대조로하여각세포군별양성과음성을구분하는 cut-off를결정하였다 (Fig. 1). 3) 통계분석총유핵세포, 단핵구및 CD34 양성세포분획들간의생존율비교및각염색법에따른생존율비교를위해일원분산분석 (oneway ANOVA) 를사용하였고, Tukey 방법으로사후검정을하였으며 P-value가.5 이하일경우통계적으로유의하다고판정하였다. 통계프로그램으로는 SPSS 2 (SPSS Inc., Chicago, IL, USA) 을사용하였다. 결과 (1) Trypan blue 염색법 생리식염수로 1:1 비율로희석한제대혈 1 μl 를 trypan blue Trypan blue, 7-AAD 및 7-AAD/annexin V 를이용해측정한시 간경과에따른제대혈의세포생존율은 Table 1 에제시하였다. 모 2

1 1 1 1 2 1 3 1 4 1 1 1 1 2 1 3 1 4 1 1 1 1 2 1 3 1 4 CD45 FITC CD34 PE CD45 FITC 1 4 1 4 1 4 2 4 6 8 Forward scatter Annexin V 1 2 Annexin V 1 2 Annexin V 1 2 1 1 1 1 1 2 1 3 1 4 1 1 1 1 1 2 1 3 1 4

The 7-aminoactinomycin D (7-AAD) viabilities (%) of TNC (A), MNC (E), and CD34+ cells (D) are 1 viable TNC (F+G)/TNC (A), viable MNC (H+I)/MNC (E), and viable CD34+ cells (J+K)/CD34+ cells (D),

1 1 Viability using 7-AAD/annexin V (%) 9 8 7 6 5 Viability using 7-AAD (%) 9 8 7 6 5 4 T T+24 T+48 Time (hr) of exposure A 4 T T+24 T+48 Time (hr) of exposure B Fig. 2.

Data shown in this figure were obtained using flow cytometric analysis with 7-aminoactinomycin D (7-AAD)/annexin V (A) and 7-AAD (B) and analyzed at T, T+24, and T+48 hr.

The + sign indicates the additional time of incubation (hr) at room temperature after receipt in the laboratory. Error bars represent SD.

3 CD45 FITC CD34 PE CD45 FITC Forward scatter Annexin V 1 2 Annexin V 1 2 Annexin V AAD 7-AAD 7-AAD Fig. 1. Flow cytometric analysis results in determining the viabilities of total nucleated cells (TNC), mononuclear cells (MNC), and CD34+ cells. The 7-aminoactinomycin D (7-AAD) viabilities (%) of TNC (A), MNC (E), and CD34+ cells (D) are 1 viable TNC (F+G)/TNC (A), viable MNC (H+I)/MNC (E), and viable CD34+ cells (J+K)/CD34+ cells (D), respectively. The 7-AAD/annexin V viabilities (%) of TNC, MNC, and CD34+ cells are 1 viable TNC (G)/TNC (A), viable MNC (I)/MNC (E), and viable CD34+ cells (K)/CD34+ cells (D), respectively. 1 1 Viability using 7-AAD/annexin V (%) Viability using 7-AAD (%) T T+24 T+48 Time (hr) of exposure A 4 T T+24 T+48 Time (hr) of exposure B Fig. 2. Time-dependent alterations in the viability of total nucleated cells (TNCs) ( ), mononuclear cells (MNCs) ( ), CD34+ cells ( ). Data shown in this figure were obtained using flow cytometric analysis with 7-aminoactinomycin D (7-AAD)/annexin V (A) and 7-AAD (B) and analyzed at T, T+24, and T+48 hr. T represents data obtained immediately after receipt in the laboratory, which was within 6 hr after collection. The + sign indicates the additional time of incubation (hr) at room temperature after receipt in the laboratory. Error bars represent SD. 든측정시점에서총유핵세포의생존율은통계학적으로유의하게 trypan blue 염색법이가장높았고, 7-AAD, 7-AAD/annexin V 순이었으며, 단핵구및 CD34 양성세포의생존율은 7-AAD법이 7-AAD/annexin V법보다유의하게높았다 (P <.1). 7-AAD/annexin V를이용해측정한총유핵세포의생존율은채취직후, 24 및 48시간경과시점에서각각 92.8±4.5%, 78.4±7.8%, 65.5±8.1% 로 24시간경과시점이후부터평균 8% 이하를나타내었다. 단핵구의시간경과에따른세포생존율은각각 94.4±1.7%, 9.8±4.2%, 84.2±6.7% 로 48시간경과시점까지평균 8% 이상을유지하였다. CD34 양성세포의생존율은측정시간순서대로각각 92.4±3.%, 9.7±4.7%, 89.3±7.% 로 48시간경과시점에도 평균 85% 이상의생존율을보였다. 채취직후에는총유핵세포, 단핵구및 CD34 양성세포의생존율이유의한차이를보이지않았으나, 24시간경과시점부터는 CD34 양성세포와단핵구가총유핵세포보다높은세포생존율을보였고, 48시간경과시점부터는 CD34 양성세포, 단핵구, 총유핵세포순으로통계학적으로유의하게높은세포생존율을보였다 (Fig. 2A). 7-AAD를이용해측정한세포생존율도 7-AAD/annexin V를이용한결과와유사하게시간경과에따라감소하였으며, 각시점에서총유핵세포, 단핵구및 CD34 양성세포의생존율차이도 7-AAD/ annexin V와동일한양상을보였다 (Fig. 2B). 초기세포사멸단계의세포 (7-AAD 음성, annexin V 양성 ) 의분획은 % 였고, 시간경 3

4 Table 1. Comparison of cell viabilities (%) measured using the trypan blue, 7-AAD, and 7-AAD/annexin V methods following elapsed time after collection Elapsed time Trypan blue 7-AAD 7-AAD/annexin V Early apoptosis (7-AAD-/annexin V+) T* TNC 99.6± ± ± ±1.3 MNC NA 97.3± ± ±1.2 CD34+ cell NA 97.2± ±3. 4.8±2.4, P NA <.1 T+24 hr TNC 95.5± ± ± ±1.2 MNC NA 94.1± ± ±1.4 CD34+ cell NA 95.8± ± ±2.9 P NA <.1 <.1.21 T+48 hr TNC 9.1± ± ± ±1.5 MNC NA 9.1± ± ±2.3 CD34+ cell NA 95.6± ±7., 6.4±4.4 P NA <.1 < *T represents data obtained immediately after receipt in the laboratory, which was within 6 hr after collection. Statistical comparisons were made by one-way ANOVA techniques with post hoc analysis by the Tukey s method. P <.5 vs TNC at each time; P <.5 vs MNC at each time. Abbreviations: 7-AAD, 7-aminoactinomycin D; TNC, total nucleated cells; MNC, mononuclear cell; NA, not applicable. 과에따라증가하는경향을보였다 (Table 1). Trypan blue로측정한총유핵세포의생존율은채취직후에 99.6%, 24시간에 95.5% 이었으며, 48시간경과시점까지 9.1% 의높은생존율을유지하였다. 고찰 성공적인제대혈이식을위해서는제대혈에포함된조혈모세포수도중요하지만제대혈세포의기능적생존율 (functional viability) 을반영하는집락형성단위 (Colony forming unit, CFU) 가이식성적을더정확하게예측한다고알려져있다 [13, 14]. 그러나집락형성단위의측정은 2주이상의기간이소요되고표준화가어려워실제임상에서사용하기어려우므로이를대체할정확한세포생존율의측정이필요하다. 세포생존율은제대혈의품질과관련된중요한지표이지만, 검사방법에따라민감도의차이가있어상당한생존율의차이를보일수있다. 통상적으로대부분의제대혈은행에서는신속하고간편한 trypan blue법을사용하여총유핵세포의생존율을측정하고있다. 그러나 trypan blue법은검사자간에주관적인오차가있을수있고, 적은수를세므로정확도가낮으며, 염색후짧은시간경과에도결과의차이를보이고, 세포사멸이완전히일어난세포만이염색되므로초기단계를검출하지못하는단점이있다. 더구나총유핵세포의생존율을측정하므로실제골수이식의생착과예후에중요한 CD34 양성조혈모세포의생존율은정확하게반영되지않을가능성이있다 [1, 15]. 7-AAD를이용해유세포분석을시행할경우 trypan blue보다민감하게세포사멸을검출할수있고, CD34 양성세포측정과 7-AAD 염색을같이시행할경우총유핵세포, 단핵구및 CD34 양성세포군에따른각각의생존율을알수있는장점이있다 [4]. 그러나세포사멸은세포막의변화, 세포질과핵의응축, DNA 분절등의순서로진행되는데 [16, 17], 7-AAD는핵산염색물질이므로초기세포막의변화를검출하지는못한다. 초기세포사멸단계의세포를검출하기위해서는 Syto16이나 annexin V 등의표지자가흔히이용되는데 Syto16이 annexin V 보다민감도가높은것으로알려져있다 [18, 19]. 여러연구자들에의하면진행된세포사멸을검출하는 7-AAD나 propidium iodide로염색되지않는조혈모세포중에상당수가초기세포사멸을검출하는 Syto16 혹은 annexin V에염색이되며, 초기세포사멸이진행된세포는골수생착력및집락형성능이현저히감소된다고보고하였다 [11, 19, 2]. 따라서, 제대혈품질관리중세포생존율의기준은측정방법에따른특성을고려할필요가있으며, 이를위해서는각방법에대해제대혈채취후시간경과에따른생존율자료가필요하다. 본연구에서는기존에사용되던 trypan blue 및 7-AAD 생존율을초기세포사멸을반영한 7-AAD/annexin V 생존율과비교하였으며, 채취후시간경과에따라세포군별생존율의변화를검토하였다. 가장낮은민감도를보이는 trypan blue의경우모든측정시간에서가장높은세포생존율을나타내었는데, 채취직후에는 99.6% 였고, 48시간경과시점에는평균 9.1% 로 9% 이상의생존율을유지하였다. 이는 Solomon 등이보고한결과보다높았으나 [4], 이등의결과와는유사하였다 [21]. 7-AAD 및 7-AAD/annexin V 생존율의경우초기세포사멸정도를반영한 7-AAD/annexin V법이 7-AAD법에비해모든세포군들및측정시간에서 % 정도낮은생존율을보였다. 두방법모 4

5 두채취직후에는총유핵세포, 단핵구및 CD34 양성세포군사이에차이를보이지않았으나, 시간의흐름에따라세포군별로생존율차이를보였는데그중총유핵세포의생존율감소가가장빠르게, CD34 양성세포의생존율감소가가장느리게나타났고이런경향은이전의연구보고와일치하였다 [4]. 48시간경과시점에서총유핵세포의평균세포생존율은 71.5% (7-AAD 법 ) 및 65.5% (7- AAD/annexin V법 ) 로 8% 이하였으나, CD34 양성세포군은 95.6% (7-AAD법) 및 89.3% (7-AAD/annexin V법 ) 로비교적높은생존율을유지하였다. 총유핵세포의생존율감소가가장빠른것은제대혈의 4-7% 를구성하는과립구에서채혈후 12-24시간이내에가장빨리세포사멸이시작되기때문으로생각되었다 [22-24]. CD34 양성세포군은시간경과후에도다른세포군보다높은생존율을유지하였는데같은환경에노출되었을때다른단핵구들에비해항세포자멸 (anti-apoptosis) 유전자인 Bcl-2와 Bcl-x가높게발현되고세포자멸의표지자인 caspase-3의활성이낮기때문으로추정되고있다 [1]. 7-AAD/annexin V법으로제대혈의세포생존율을측정한결과기존의 trypan blue 또는 7-AAD법보다낮은세포생존율을보였으나, 48시간경과시점에서단핵구및 CD34 양성세포군의생존율은각각평균 84.2% 와 89.3% 였고, 36시간경과시점에서의세포생존율을유추해본결과각각 87.5% 와 9.% 였다. 따라서현재시행되고있는국내제대혈관리법의 8% 세포생존율기준및채취후보관처리전까지의 36시간기준을충분히충족시킬수있을것으로추정된다. 또한, 국내제대혈관리법은미국이나유럽등에서제시하는채취후보관처리전까지 48시간, 세포생존율기준 9% 에비해더짧은시간과낮은세포생존율을기준으로하고있는데통상적으로제대혈은행에서시행하고있는 trypan blue법을사용하여세포생존율을측정한다고가정하였을때외국의경우와같이보관처리까지의시간을 48시간으로연장하거나, 세포생존율기준을 9% 로설정하여도품질기준을충족시킬것으로판단된다. 현재국내제대혈은행에서는통상적으로보관전제대혈의세포생존율측정을위해 trypan blue법을사용하고있는데상용화된제품을이용해 CD34 양성세포수와 7-AAD법에의한세포생존율을동시에측정할경우 trypan blue법보다민감하게세포생존율을측정할수있고, 제대혈의품질에중요한단핵구및 CD34 양성세포의생존율을따로구할수있어제대혈의품질관리에도움이될것으로판단된다. 또한제대혈이식을위해보관제대혈을사용하는경우에는제대혈이식의성적예측및냉동보관된제대혈의품질평가를위해초기세포사멸을반영할수있는 7-AAD/annexin V법으로세포생존율을측정하는것이보다유용할것으로생각되며, 이때본연구결과에서제시하는보관전제대혈의세포생존율자료가냉동보관제대혈의품질을평가하는데이용될수있을것 으로기대한다. 본연구는제대혈채취시일반적으로사용하는 CPDA-1 항응고제가포함된채취백대신에 EDTA 시험관을사용하였으며, 보관처리를위한공정을실시하지않았으므로실제제대혈보관전세포생존율을측정하는검체의조건과는일부차이가있다. 이에저자들은항응고제종류에따른세포생존율의차이를확인하기위해추가로 1예의제대혈을 CPDA-1 항응고제백에채취하여세포생존율을측정한후본연구에서얻은 EDTA 시험관결과와비교하였다. Trypan blue법으로측정한총유핵세포와 7-AAD 및 7-AAD/annexin V법에의한단핵구및 CD34 양성세포의세포생존율은모든측정시점에서항응고제종류에따른차이는관찰되지않았다. 단, 7-AAD 및 7-AAD/annexin V법으로측정한총유핵세포의생존율은채취직후에는차이가없었으나, 시간경과에따라 EDTA 항응고제를사용한경우에서 CPDA-1 보다 % 낮게측정되어본연구결과를해석할때고려되어야할것으로생각된다. 저자들은본연구에서 7-AAD와초기세포사멸을검출할수있는 annexin V를이용하여제대혈채취후시간경과에따른세포생존율을측정하고이를기존의 trypan blue 및 7-AAD 염색만을이용한생존율과비교하였다. 7-AAD/annexin V법으로측정한세포생존율은모든측정시점및세포군별비교에서기존의검사법들에비하여통계적으로유의하게낮은결과를보였으나, 제대혈채취후 48시간경과시점까지 CD34 양성세포의생존율은평균 85% 이상을유지하였다. 7-AAD/annexin V법의경우기존의검사법들에비해제대혈이식시생착에가장중요한 CD34 양성세포의세포생존율을따로구할수있고, 초기세포사멸정도를측정하여냉동보관된제대혈의품질평가에이용할뿐아니라이식의성적을보다정확하게예측할수있는장점이있다. 저자들은본연구결과가향후제대혈의품질을평가하는기초자료로활용될수있을것으로기대한다. 요약 배경 : 세포생존율은제대혈의품질을결정하는중요한지표이다. 제대혈의세포생존율을측정하기위해 trypan blue나 7-aminoactinomycin (7-AAD) 를이용한방법이일반적으로사용되고있으나, 이들방법은초기세포사멸은검출할수없다. 저자들은 7-AAD 와초기세포사멸을반영하는 annexin V를동시에이용하여제대혈채취후시간경과에따른세포생존율을측정하고, 기존의 trypan blue 및 7-AAD를이용한방법과비교하고자하였다. 방법 : 212년 7월부터 213년 3월까지분당차병원에서출산한산모 3명에서채취한제대혈을대상으로하였다. 제대혈은채취직후, 24, 48시간이경과한시점에서 trypan blue, 7-AAD, 7-AAD/an

6 nexin V를이용하여세포생존율을반복측정하였다. 결과 : 7-AAD/annexin V를이용하여제대혈채취직후, 24, 48시간경과후에측정한총유핵세포의생존율은측정시간순서대로각각 92.8±4.5%, 78.4±7.8%, 65.5±8.1%, 단핵구는 94.4±1.7%, 9.8±4.2%, 84.2±6.7%, CD34 양성세포군은 92.4±3.%, 9.7± 4.7%, 89.3±7.% 였다. Trypan blue법으로측정한세포생존율은 48시간까지 9% 이상을보였다. 결론 : 7-AAD/annexin V를이용해측정한제대혈의세포생존율은총유핵세포의경우 24시간경과시점부터평균 8% 이하였으나, 제대혈이식에서생착에가장중요한 CD34 양성세포의생존율은 48시간경과시점까지 85% 이상을유지하였다. 저자들은본연구결과가향후제대혈의품질을평가하는기초자료로활용될수있을것으로기대한다. REFERENCES 1. Wagner JE, Barker JN, DeFor TE, Baker KS, Blazar BR, Eide C, et al. Transplantation of unrelated donor umbilical cord blood in 12 patients with malignant and nonmalignant diseases: influence of CD34 cell dose and HLA disparity on treatment-related mortality and survival. Blood 22;1: Laughlin MJ, Eapen M, Rubinstein P, Wagner JE, Zhang MJ, Champlin RE, et al. Outcomes after transplantation of cord blood or bone marrow from unrelated donors in adults with leukemia. N Engl J Med 24;351: Rocha V, Labopin M, Sanz G, Arcese W, Schwerdtfeger R, Bosi A, et al. Transplants of umbilical-cord blood or bone marrow from unrelated donors in adults with acute leukemia. N Engl J Med 24;351: Solomon M, Wofford J, Johnson C, Regan D, Creer MH. Factors influencing cord blood viability assessment before cryopreservation. Transfusion 21;5: Louis I, Wagner E, Dieng MM, Morin H, Champagne MA, Haddad E. Impact of storage temperature and processing delays on cord blood quality: discrepancy between functional in vitro and in vivo assays. Transfusion 212;52: COBLT. Cord Blood Bank Standard Operating Procedures. web.emm es.com/study/cord/sop.htm (Updated on Dec 2). 7. FACT and NetCord. NetCord-FACT International Standards for Cord Blood Collection, Banking, and Release for Administration. sit e.org (Updated on Jan 21). 8. Ministry of Health and Welfare. The Umbilical Cord Blood Management and Research Act. (Updated on Aug 212). 9. Darzynkiewicz Z, Li X, Gong J. Assays of cell viability: discrimination of cells dying by apoptosis. Methods Cell Biol 1994;41: Xiao M and Dooley DC. Assessment of cell viability and apoptosis in human umbilical cord blood following storage. J Hematother Stem Cell Res 23;12: Shim JS, Cho B, Kim M, Park GS, Shin JC, Hwang HK, et al. Early apoptosis in CD34+ cells as a potential heterogeneity in quality of cryopreserved umbilical cord blood. Br J Haematol 26;135: Duggleby RC, Querol S, Davy RC, Fry LJ, Gibson DA, Horton RB, et al. Flow cytometry assessment of apoptotic CD34+ cells by annexin V labeling may improve prediction of cord blood potency for engraftment. Transfusion 212;52: Prasad VK, Mendizabal A, Parikh SH, Szabolcs P, Driscoll TA, Page K, et al. Unrelated donor umbilical cord blood transplantation for inherited metabolic disorders in 159 pediatric patients from a single center: influence of cellular composition of the graft on transplantation outcomes. Blood 28;112: Page KM, Zhang L, Mendizabal A, Wease S, Carter S, Gentry T, et al. Total colony-forming units are a strong, independent predictor of neutrophil and platelet engraftment after unrelated umbilical cord blood transplantation: a single-center analysis of 435 cord blood transplants. Biol Blood Marrow Transplant 211;17: Yi DY, Huh JY, Kang MS. Assessment of cell viability in umbilical cord blood before cryopreservation. Korean J Blood Transfus 21;21: Cohen JJ, Duke RC, Fadok VA, Sellins KS. Apoptosis and programmed cell death in immunity. Annu Rev Immunol 1992;1: Kerr JF, Wyllie AH, Currie AR. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 1972;26: Sparrow RL and Tippett E. Discrimination of live and early apoptotic mononuclear cells by the fluorescent SYTO 16 vital dye. J Immunol Methods 25;35: Schuurhuis GJ, Muijen MM, Oberink JW, de Boer F, Ossenkoppele GJ, Broxterman HJ. Large populations of non-clonogenic early apoptotic CD34-positive cells are present in frozen-thawed peripheral blood stem cell transplants. Bone Marrow Transplant 21;27: de Boer F, Dräger AM, Pinedo HM, Kessler FL, Monnee-van Muijen M, Weijers G, et al. Early apoptosis largely accounts for functional impairment of CD34+ cells in frozen-thawed stem cell grafts. J Hematother Stem Cell Res 22;11: Lee HR, Roh EY, Yoon JH, Han KS, Kim BJ, Hwang KR, et al. Analysis 6

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untitled 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