대한수혈학회지 : 제 25 권제 3 호, 2014 The Korean Journal of Blood Transfusion Vol. 25, No. 3, 235-242, December 2014 ISSN 1226-9336 Original Article ABO 및 RhD 혈액형검사와항체선별검사를위한혈액은행자동화장비 Galileo NEO 와 QWALYS-3 의평가 장미애ㆍ오종원ㆍ이승태ㆍ서지영ㆍ김대원 성균관대학교의과대학삼성서울병원진단검사의학과 Evaluation of the Automated Blood Bank Systems Galileo NEO and QWALYS-3 for ABO-RhD Typing and Antibody Screening Mi-Ae Jang, Jong Won Oh, Seung-Tae Lee, Ji Young Seo, Dae-Won Kim Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea Background: An automation system for ABO-RhD typing and antibody screening has been developed and its use is increasing. We compared the results of ABO-RhD typing and antibody screening tests using the manual (ABO-RhD typing) or semiautomated (antibody screening) method and with the automation instruments Galileo NEO (Immucor Gamma, Norcoss, USA) and QWALYS-3 (DIAGAST, Loos Cedex, France). Methods: A total of 332 blood samples were tested for ABO-RhD typing in comparison with routine manual tests, and 236 samples for antibody screening in comparison with DS-Screening II (Bio-Rad Laboratories, 1785 Cressier FR, Switzerland). We evaluated the performance of Galileo NEO and QWALYS-3 in terms of concordance, carryover, and sensitivity test for ABO-RhD typing and antibody screening. Results: The concordance rates of ABO-RhD typing results between the manual methods and the two instruments were 99.4% for Galileo NEO and 99.1% for QWALYS-3, respectively. On antibody screening tests, a concordance rate of 97.9% was observed between the semiautomated method and Galileo NEO or QWALYS-3, because of discordance in five specimens. The carryover was not observed for ABO-RhD typing and antibody screening. The overall sensitivity of the two automation instruments appears to be parallel with that of DS-Screening II except for anti-e. Conclusion: The Galileo NEO and QWALYS-3 system showed good performance, it can be used with confidence for routine pre-transfusion testing in the blood bank. (Korean J Blood Transfus 2014;25:235-242) Key words: Galileo NEO, QWALYS-3, ABO-RhD typing, Antibody screening, Automation Received on November 21, 2014. Revised on December 12, 2014. Accepted on December 15, 2014 Correspondence to: Dae-Won Kim Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, 81 Irwon-ro, Gangnam-gu, Seoul 135-710, Korea Tel: 82-2-3410-2701, Fax: 82-2-3410-2719, E-mail: dwsmc.kim@samsung.com 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. Copyright C 2014 The Korean Society of Blood Transfusion - 235 -
Korean J Blood Transfus Vol. 25, No. 3, 235-242, Dec. 2014 서론 ABO-RhD 혈액형검사와비예기항체선별검사는수혈전검사중에서가장기본적이고중요한검사로서안전한수혈을위해필수적인검사이다. 1) 다른검사분야와달리아직까지수혈의학의검사항목들은수기법에치중도가높은상황이다. 그이유는숙련된검사인력에대한의존도가높고고가의자동화장비를도입하기어려웠기때문이다. 그러나최근에는여러자동화장비가개발, 소개되어유럽이나미국에서는이들장비를혈액은행에서널리이용하고있는추세이다. 2) 자동화장비의도입은사무적착오의발생을예방할수있고, 혈액형검사건수가많은기관에서는검사시간과비용을줄일수있는효과가기대되므로, 3) 우리나라에서도차츰혈액은행자동화장비의도입이증가하고있다. 혈액은행자동화장비인 Galileo NEO (Immucor Gamma, Norcoss, USA) 는마이크로플레이트법 (microplate method) 을기반으로샘플과시약의항원-항체복합체의응집반응에따른결과를사진으로캡쳐해서판정하는방법이며, 4) QWALYS-3 (DIAGAST, Loos Cedex, France) 는적혈구에 Magnetic 입자를부탁시킨후자성에의해침강시켜항원항체의반응여부를확인하는 Erythrocyte Magnetized Technology를이용하는장비이다. 5) 이에본연구에서는 Galileo NEO 자동화장비와 QWALYS-3 자동화장비로 ABO-RhD 혈액형검사와비예기항체선별검사를시행하고, 그결과를슬라이드법과시험관법을사용한 ABO-RhD 혈액형검사결과및원주응집법 (column agglutination technology) 을이용한비예기항체선별검사결과와비교하여장비의유용성을평가하였다. 대상및방법 2014년 6월부터 8월까지삼성서울병원진단검사의학과혈액은행검사실에의뢰된검체중무작위로선정한 332검체를대상으로 Galileo NEO 와 QWALYS-3 자동화장비를이용하여 ABO-RhD 혈액형검사를시행하였다. 기존에사용하던수기법은첨가제가없는채혈관혈액을사용하였고, 자동화장비에는동일한환자의 EDTA 혈액을사용하였다. EDTA 혈액은혈액은행검사와일반혈액검사가동일한날의뢰된경우에일반혈액검사를시행하고남은잔여검체를이용하여검사하였다. 비예기항체선별검사평가를위해서는기존의항체선별및동정검사에서양성, 음성결과가확인된 236건의검체를이용하였다. 그중 24건의양성검체는냉동보존되었으며, 검사하는과정에서바로해동하여사용하였다. 나머지 212건의음성검체는냉장보존된상태였다. 반응을위한시약과검체의첨가량, 반응시간등은각장비제조사의지침에따라진행하였다. 검사결과는현재검사실에서시행중인방법을기준검사 (reference method) 로설정하여두자동화장비와의결과를비교하였다. 본평가는면역혈액학적검사 (immunohematological testing) 를위한자동화장비검사평가지침인 CLSI guideline I/LA33-A에따라 ABO-RhD 혈액형검사와비예기항체선별검사의결과일치율, Carryover 검사, 비예기항체희석검사결과를평가하였다. 1. ABO-RhD 혈액형검사기준검사인수기법 ABO-RhD 혈액형검사는현재검사실에서시행중인슬라이드법과시험관법으로이중검사하였다. 슬라이드법에는 BIOSCOT Anti-A (Millipore, Livingston, UK), BIOSCOT Anti-B (Millipore), BIOSCOT Anti-D (Millipore) 시 - 236 -
장미애외 : 혈액은행자동화장비 Galileo NEO 와 QWALYS-3 의평가 약을이용하였고시험관법에는 Anti-A BioClone (Ortho Clinical Diagnostics, Raritan, NJ, USA), Anti-B BioClone (Ortho Clinical Diagnostics), Anti-D BioClone (Ortho Clinical Diagnostics) 시약을이용하였으며, ABO 혈청형검사는 Affirmagen A1, B cell (Ortho Clinical Diagnostics) 을사용하였다. Galileo NEO 장비의 ABO-RhD 혈액형검사는혈구형검사와혈청형검사결과가일치하는경우에만혈액형을판정하고불일치하는경우에는 NTD (No Type Determined) 로판정하였다. QWALYS-3 장비는반응시약이건조되어도포되어있는 ABDLys microplate (DIAGAST) 와 magnetic 입자를입힌 HemaLys A1, B (DIAGAST) 혈구를사용하여 ABO-RhD 혈액형검사를시행하였다. 결과분석은기준검사인수기법의슬라이드법과시험관법을모두시행한후종합하여혈구형과혈청형검사결과가일치하는경우에혈액형을판정하여두자동화장비결과와비교하였고불일치하는경우에는 판독불가 (indeterminate) 로판정하였다. 판독불가 인경우그에대한원인분석및두자동화장비간의결과를비교하였다. 2. 항체선별검사비예기항체선별검사는원주응집법 (column agglutination technology) 으로 DC-Screening II (BioRad Laboratories, 1785 Cressier FR, Switzerland) 과동정용혈구인 ID-DiaCell I, II (BioRad Laboratories) 을이용하여시행하였다. Galileo NEO는응집괴의크기및형태에따라반응강도를판정하며, 패널혈구 Capture-R Ready-Screen I and II (Immucor) 중하나라도반응강도가 +/ 이상인경우선별검사양성으로판정하였다. QWALYS-3는 HEMASCREEN 1, 2, 3 (DIAGAST) 와항글로불린혈청이고정되어있는 ScreenLys microplate (DIAGAST) 를이용하여비예기항체선별검사를시행하였다. 3. Carryover 검사 ABO 혈액형검사는수기법으로시행한검사에서결과가확인된잔여환자검체를이용하여 B 형, O형, AB형순서대로 20회측정하여결과를확인하였다. RhD 혈액형검사의경우제조회사에서제공하는컨트롤물질인 Hema CQI (DIAGAST) 을이용하여양성물질 Hema CQI-4를 4회연속으로측정후이어서음성물질 Hema CQI-1을 4회연속으로측정하는순서로검사하여양성물질이음성물질의결과에영향을미치는지확인하였다. 비예기항체선별검사는 Anti-D BioClone (Ortho Clinical Diagnostics) 시약을생리식염수에 200배희석하여양성물질로, 생리식염수를음성물질로이용하여양성물질을 4회연속으로검사하고, 이어서음성물질을 4회연속으로검사하여양성물질이음성물질의결과에영향을미치는지확인하였다. 4. 비예기항체희석검사적혈구비예기항체의검출민감도를비교하기위해항체희석검사를시행하였다. 비예기항체동정검사를위해시약용으로나온항체를사용하였으며, 그종류는 anti-d, anti-le a, and anti-e이었다. 이들원액을생리식염수로계대희석 (1:128-1:4096) 하여두대의자동화검사법으로비예기항체선별검사를시행하여각항체별검출민감도를확인하였다. 결과 ABO 혈액형검사는총 322 검체중 A+형은 117검체 (36.3%, 117/322), B+형은 90검체 (28.0%, - 237 -
Korean J Blood Transfus Vol. 25, No. 3, 235-242, Dec. 2014 90/322), O+형 81검체 (25.2%, 81/322), AB+형 32 검체 (9.9%, 32/322), B 형 1검체 (0.3%, 1/322), O 형 1검체 (0.3%, 1/322) 의분포를나타내었다. Galileo NEO 자동화장비의경우총 322건중 320 건에서수기법으로시행한결과와일치하였으며 (99.4%, 320/322), QWALYS-3 자동화장비의경 우총 322건중 319 건에서수기법으로시행한결과와일치하였다 (99.1%, 319/322) (Table 1). 수기법과일치하지않은결과를보였던 5검체 (Galileo NEO; 2건, QWALYS-3; 3건 ) 모두장비분석결과혈구형과혈청형간의결과가불일치하여각각 NTD (Galileo NEO) 또는? (QWALYS-3) 의결 Table 1. Comparison of the ABO-RhD typing results between the manual test and automated methods (Galileo NEO, QWALYS-3) ABO group Manual test*, number (%) Galileo NEO QWALYS-3 Concordance Discordance Concordance Discordance A+ 117 (36.3) 116 1 114 3 B+ 90 (28.0) 90 0 90 0 O+ 81 (25.2) 81 0 81 0 AB+ 32 (9.9) 31 1 32 0 B 1 (0.3) 1 0 1 0 O 1 (0.3) 1 0 1 0 Total 322 (100.0) 320/322 (99.4%) 2/322 (0.6%) 319/322 (99.1%) 3/322 (0.9%) *Slide and tube methods were tested simultaneously; A total of 5 results were discrepant in front or back typing due to weak reaction, which were flagged as NTD (No Type Determined) in Galileo NEO or? in QWALYS-3. Table 2. Indeterminate cases in ABO-RhD typing with manual test and automated methods (Galileo NEO, QWALYS-3) Case Manual test* Galileo NEO QWALYS-3 anti-a anti-b anti-d A1 cell B cell anti-a anti-b anti-d A1 cell B cell anti-a anti-b anti-d A1 cell B cell Comment 1 1+ mf 4+ - 1+ 4+ 4+ 4+ - 2+ 4+ 4+ 4+ - - A 2B 3+ 2 4+ 4+ 4+ 3+ - 4+ 4+ 4+? - 4+ 4+ 4+ - - PBSCT 3 mf - 4+ - 4+? - 4+ - 4+ 3+ - 4+ - 4+ PBSCT 4 4+ +/ 4+ +/ +/? - 4+ - -? - 4+ - - PBSCT 5 - - 4+ 3+ - - - 4+ 3+ - - - 4+ 4+ - PBSCT 6 4+ mf 4+ - 2+ 4+ 4+ 4+ - 1+ 4+ 4+ 4+ 4+? A 2B 3+ 7 mf - 4+ +/ 3+? - 4+ 1+ 3+ - - 4+ - 4+ PBSCT 8 4+ - 4+ - +/ 4+ - 4+ - - 4+ - 4+ - - Infant 9 mf 4+ 4+ - - - 4+ 4+ - - - 4+ 4+ - - PBSCT 10 mf - 4+ +/ +/ 4+ - 4+ -? 4+ - 4+ - - PBSCT Abbreviations: mf, mixed field agglutination; PBSCT, peripheral blood stem cell transplantation. *Slide and tube methods were tested simultaneously; A 15-month-old infant. - 238 -
장미애외 : 혈액은행자동화장비 Galileo NEO 와 QWALYS-3 의평가 과를보였던경우로수기법과다른 ABO 혈액형결과로잘못보고한예는없음을감안할때수기법과의혈액형판정율은 100% 이었다. 결과의반응강도를수기법과비교하였을때두대의장비와같은강도를보이거나, 1 grade 이내의차이를보였다. 기준검사인수기법으로시행한 ABO 혈액형검사에서혈구형과혈청형이서로일치하지않아 판독불가 로판정된검체는총10건으로, ABO 혈액형아형, 특이병력없는영아그리고말초혈액조혈모세포이식후생착과정중에있는환자로부터얻은검체이었다 (Table 2). 비예기항체선별검사평가에사용된총 236건의검체중음성검체는 212건, 양성검체는 24건이었다. 양성검체중 anti-c는 1건 (4.2%, 1/24), anti-di b 는 1건 (4.2%, 1/24), anti-e는 6건 (25.0%, 6/24), anti-jk a 는 2건 (8.3%, 2/24), anti-jk b 는 2건 (8.3%, 2/24), anti-le a 는 2건 (8.3%, 2/24), anti-le b 는 2건 (8.3%, 2/24), anti-m은 1건 (4.2%, 1/24), anti-s 은 1건 (4.2%, 1/24), anti-e와 anti-s가함께동정된예가 1건 (4.2%, 1/24), anti-e와 anti-fy b 가함께동정된예가 1건 (4.2%, 1/24), unidentified 4건 (16.7%, Table 3. Comparison of the antibody screening results between the manual test and automated methods (Galileo NEO and QWALYS-3) Manual test* Galileo NEO and QWALYS-3 Positive Negative Total Positive 19 5 24 Negative 0 212 212 Total 19 217 236 *The antibody screening identified by DC-Screening II (BioRad Laboratories, 1785 Cressier FR, Switzerland); Anti-JK b (1), anti-le a (1), anti-le b (2), and unidentified (1). The numbers in parentheses refer to specimen numbers. 4/24) 의분포를나타내었다. 음성검체의경우수기법과두자동화장비간의결과가모두일치하였다 (Table 3). 양성검체의경우 Galileo NEO와 QWALYS-3 자동화장비모두 24건중 19건에서수기법으로시행한결과와일치하여, 음성또는양성검체를모두포함한결과일치율 (concordance rate) 은 97.9% (231/236) 로확인되었다. 자동화장비에서위음성결과를보인 5검체는 anti-jk b 1건, anti-le a 1건, anti-le b 2건, 특이성을알수없는 (unidentified) 항체 1건의분포를나타내었다. ABO 혈액형검사의 carryover 확인을위해 B 형, O형, AB형순서대로 20회측정한결과 Galileo NEO와 QWALYS-3 장비모두해당순서의혈액형을모두정확히판정하였다. RhD 혈액형검사의경우양성검체 4회연속측정후이어서음성검체를 4회연속측정하는순서로 6회측정한결과두자동화장비모두 RhD 양성검체의결과가뒤의음성검체의결과에영향을주지않는것으로확인하였다. 비예기항체선별검사의 carryover 확인을위해양성검체 4회연속측정후이어서음성검체를 4회연속측정하는순서로 8회측정한결과두자동화장비모두항체양성검체의결과가뒤의항체음성검체의결과에영향을주지않는것으로확인하였다. Table 4. Comparison of unexpected antibody titration results between manual test and automated methods (Galileo NEO, QWALYS-3) Ab specificity Manual test* Galileo NEO QWALYS-3 Anti-D 1:4,096 1:4,096 1:4,096 Anti-Le a 1:64 1:64 1:128 Anti-E 1:4,096 1:128 1:128 *The antibody specificity identified by LISS-Coombs Card (Bio-Rad Laboratories, 1785 Cressier FR, Switzerland). - 239 -
Korean J Blood Transfus Vol. 25, No. 3, 235-242, Dec. 2014 비예기항체희석검사는총 3종의비예기항체 anti-d, anti-le a, and anti-e에대해시행하였다. Anti-D의경우두자동화장비모두수기법과동일한검출민감도를보이는것으로확인되었다. Anti-Le a 은 QWALYS-3 장비에서수기법과 Galileo NEO 장비에비해한단계희석배율정도검출민감도가우수하였으나, anti-e의경우두자동화장비모두수기법으로수행한결과보다낮은검출민감도를보였다 (Table 4). 고찰 ABO-RhD 혈액형검사에서기존수기법과두자동화장비간의결과일치율은 Galileo NEO의경우 99.4% (320/322), QWALYS-3의경우 99.1% (319/322) 로우수하였는데이는기존의보고결과와유사한결과이었다. 4-6) 수기법의혈구형과혈청형의불일치로인한혈액형판정불가는 10건 (3.0%, 10/332) 으로, ABO 혈액형불일치말초혈액조혈모세포이식 (peripheral blood stem cell transplantation, PBSCT) 을받은경우가 7건으로가장흔하였고, A 2B 3 혈액형인경우가 2건, 나머지는특이병력없는 15개월영아인경우 1건이었다 (Table 3). Galileo system의경우혈구형또는혈청형중한쪽이약화된경우에대해수기법보다더민감하게이상반응을나타내는것으로기존에보고된바있다. 4) 본연구에서는 Galileo NEO 장비뿐아니라 QWALYS-3 장비결과에서도수기법에서약한항원성을보이거나혼합반응 (mixed filed agglutination) 을보이는경우에도민감하게검출하는것으로확인되었다. 이는 ABO아형의항원을반드시검출하여야하는혈액원의경우에는유용할수있으나, 아형의약한항원을약한항원그대로검출하여수혈용혈액을결정하여야만하는의료기관의경우에는해석에주의를필 요로한다. 비예기항체선별검사에서기존수기법과두자동화장비간의결과일치율은 97.9% (231/236) 로기존에보고되었던결과와유사한결과를얻었다. 4-7) 불일치한결과는모두자동화장비에서위음성으로판정된경우로서각각 anti-le a 가 1건, anti-le b 가 2건, anti-jk b 가 1건, 특이성을알수없는 (unidentified) 항체 1건이었다. Galileo NEO 장비의경우비예기항체중에서임상적으로의의가있는 IgG 항체만검출하기때문에항-Lewis 항체에대한불일치결과는가능한경우로생각되었다. QWALYS-3는기존의 QWALYS-2와는다르게 IgM 항체도검출가능한장비로알려져있지만, 본연구에서항-Lewis 항체에대해여전히위음성결과를나타내었다. 항-Lewis 항체에대한 QWALYS-3장비의위음성결과는기존에보고된연구에서도동일하게문제점으로제시된바있어, 6) 위음성문제에대해지속적인개선이필요함을시사하였다. 비예기항체선별검사의불일치에대한또다른원인으로서본연구에서사용된양성검체가냉동보존검체로서항체의역가가저하되었을가능성을생각해볼수있다. QWALYS-3 자동화장비를평가한기존연구결과에서도임상검체에비해냉동보관검체의경우항체의역가가떨어져보다낮은예민도를보이는것으로보고한바있으며, 7) Galileo system 자동화장비의경우수기법에비해한랭항체에대한검출률이떨어지는것으로보고된바있다. 4) 따라서검체보관상태에따른추가연구가향후더필요할것으로사료되었다. 또한본연구를포함한그동안보고되었던자동화장비의비예기항체선별검사평가의경우평가에포함된양성검체수가음성검체수에비해적고제한적임을감안할때향후좀더다양한종류의비예기항체를포함하면서충분한수의검체를포함한연구가필요할것으로 - 240 -
장미애외 : 혈액은행자동화장비 Galileo NEO 와 QWALYS-3 의평가 생각된다. 본연구에서평가한 Galileo NEO, QWALYS-3 자동화장비이외에도 gel column을이용한원주응집법을자동화한장비인 IH-1000 (Bio-Rad Laboratories, Hercules, CA, USA) 과 microbead column을이용한원주응집법을기반으로한 AutoVue Innova (Ortho, Raritan, NJ, USA) 장비등다양한자동화장비에대한평가결과들이보고되고있다. 8-10) 자동화장비의경우수기법과비교하여신뢰할만한높은일치율을보여주고있으며검사의전과정을표준화하여사무적착오를감소시키고인력재배치등의검사실운영의효율성을증가시키는등의장점을공통적으로가지고있다. 특히 Galileo NEO는다른자동화장비에비해 V, C w, K p, Di a 항원등비예기항체선별검사에사용되는패널혈구가좀더다양한장점이있다. Di a 항원은신생아용혈성질환및용혈성수혈부작용과관련하여임상적으로중요한항체로서한국인을비롯한아시아인에서비교적흔하게발견되는것으로알려져있다. 그러나, 장비의구입가격이고가라는점과처리할검사수가많지않은소규모검사실에서는검사소요시간이나비용절감에대한운영효율성이떨어질수있으므로, 3) 자동화장비의도입전에적절한사전평가를통해검사실실정에맞는적절한자동화장비를결정하는것이필요하리라생각된다. 요약 배경 : 혈액형검사와비예기항체선별검사의자동화장비가개발되어사용이증가추세이다. 본연구에서는혈액은행자동화장비인 Galileo NEO (Immucor Gamma, Norcoss, USA) 와 QWALYS-3 (DIAGAST, Loos Cedex, France) 장비를기존수기법과비교평가하고자하였다. 방법 : 본원검사실에의뢰된 ABO-RhD 혈액형검사 332검체와비예기항체선별검사평가를위한 236검체를대상으로수기법과두혈액은행자동화장비를이용하여각검사의결과일치율, carryover 확인검사, 비예기항체희석검사항목에대해비교하였다. 결과 : ABO 및 RhD 혈액형검사에서 Galileo NEO와 QWALYS-3 자동화장비결과와수기법의결과일치율은각각 99.4%, 99.1% 였다. 236건의비예기항체양성또는음성검체에대한결과일치율은 97.9% 였다. Carryover 확인검사결과신뢰할만한결과를보여주었으며, 비예기항체희석검사에서는일부항체를제외하고수기법과비슷한검출민감도를보였다. 결론 : Galileo NEO와 QWALYS-3 자동화장비는전체적인정확도면에서신뢰할만한결과를보여주었다. 따라서수혈전검사에서사용할수있을것으로생각된다. References 1. Han KS, Park KU, Song EY. Transfusion medicine. 4th ed. Seoul: Korea Medical Book Publisher, 2014:269-77 2. Malomgré W, Neumeister B. Recent and future trends in blood group typing. Anal Bioanal Chem 2009;393:1443-51 3. Song YK, An TK, Hwang JY, Shim HE, Lee DH, Kong SY. Comparison of effectiveness between blood bank automation system and manual method for abo-rhd blood typing and antibody screening test in a single center. Korean J Blood Transfus 2014;25:93-8 4. Joo SY, Han KS, Kwak YK, Park KU. Evaluation of an automated solid-phase cell adherence assay in the galileo system (immucor) for routine pretransfusion tests. Korean J Blood - 241 -
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