임상검사와정도관리 J Lab Med Qual Assur 2010 ; 32:205-10 ISSN 1225-097X Evaluation of the Vacutainer Gel Plasma Tube for Common Clinical Chemistry Assays Gwan-Yong Seo, Hyun-Chul Lee, Yu Kyung Kim, and Ji-Hye Kim Department of Laboratory Medicine, Kyungpook National University Hospital, Daegu, Korea 일반임상화학검사에서의진공겔혈장시험관의평가 서관용 이현철 김유경 김지혜 경북대학교병원진단검사의학과 교신저자 : 김지혜우 ) 700-721 대구광역시중구삼덕동 2 가 50 번지, 경북대학교병원진단검사의학과전화 :053)420-5278 FAX:053)426-3367 E-mail:sophia7322@hanmail.net Background: Plasma specimens are recently used instead of serum in clinical chemistry to improve (test result turnaround time, TAT). We evaluated the performance of lithium heparin gel plasma separating tube in comparison to existing vacutainer gel serum tube in common clinical chemistry assays. Methods: Total 52 subjects who had visited the health promotion center were included in the study. Lithium heparin plasma tubes and serum tubes were tested for 30 clinical chemistry items and 3 cardiac markers. Test results were analyzed by calculation of mean bias and percent difference. Results: The performance of the lithium heparin plasma tube was considered to be clinically equivalent to the serum tube for all assays except for potassium. The difference of potassium levels between lithium heparin plasma tube and the serum tube was -7.32%. Test results of some analytes including glucose, lactate dehydrogenase, total CO 2 and potassium using the lithium heparin tube were significantly changed after 24 hours of storage. Conclusions: The lithium heparin plasma tube provided acceptable results comparison to the existing serum separating tube in common clinical chemistries and can be used for reducing, TAT. However, the stability of plasma gel tubes on 24 hours of storage was unstable in some analytes, it requires attention. Key Words:Plasma specimen, Vacutainer gel plasma tube, Serum gel separating tube 서론임상검사의검체로혈청이오랫동안널리사용되어왔지만최근혈장검체가혈청을대체하는경우가증가하고있다. 혈장검체사용의장점으로는항응고제를사용함으로써혈청분리를위해 30분이상의소요되는응고시간을줄여주어검사시간을단축할수있으며항응고약제를투여중인환자의검체에서응고가지연되는문제를해결하고섬유소응고물질에장비프로브 (probe) 가막히는현상을방지할수있다는점등이다 [1]. 혈장분리에사용되는항응고제 의종류에는여러물질이있으나리튬-헤파린 (lithium heparin) 이많이사용되고있다. 앞서다양한검사항목들에대해혈장과혈청검체간일치성을비교한연구들은상반된결과를보인다 [1-12]. 지질검사의경우에는임상의진료기준이혈청검체를바탕으로하여세워져있어사용이제한되기도한다. 혈장검체의가장큰단점은원심분리가지연되어혈액세포와의접촉시간이길어질때혈청에비해불안정성이크다는점이다 [2,13,14]. 그러나겔 (gel) 이포함된혈장분리시험관이상용화되어세포와혈장사이의장벽역할을통해시간에따른 205
206 서관용 이현철 김유경 김지혜 불안정성을줄일수있을것으로기대되며최근다양한일반화학과면역검사에서도혈장검체에대한평가가이루어지고있다 [15-17]. 따라서저자들은널리이용되는일반적임상화학항목에대해리튬-헤파린항응고제와겔이포함된혈장시험관을기존에사용되던겔이포함된혈청시험관과비교평가하였다. 또한기존에나트륨-헤파린 (sodium heparin) 혈장을사용하던몇항목에대해서도리튬-헤파린혈장과비교평가해보았다. 재료및방법 1. 대상및측정방법 2010 년 6월부터 7월까지건강검진을위해경북대학교병원에내원한환자 52명을대상으로하였다. 대상자들의전와정맥에서통상적방법으로채혈하여혈청용 (BD Vacutainer SST II Advance Plus Blood Collection Tubes, Becton Dickinson, Franklin lakes, NJ, USA), 리튬 - 헤파린혈장용 (BD Vacutainer PST Lithium Heparin Plus Blood Collection Tubes, Becton Dickinson) 및나트륨 -헤파린혈장용 (BD Vacutainer Sodium Heparin Plus Blood Collection Tubes, Becton Dickinson), 세종류의시험관에혈액을얻었다. 혈청시험관은 30분실온에서응고시켰고, 모든검체는 3000 g로 10분간원심하였다. 나트륨- 헤파린혈장용시험관에서는혈장을따로분리하였고, 혈청용및리튬-헤파린혈장용시험관은분리없이 2시간내에측정하였다. 모든검체는 24시간실온보관한후한번더측정하였다. 리튬-헤파린혈장시험관과혈청시험관의비교를위해일반화학, 전해질과심장표지자등총 33 종의종목을평가하였다. 28 종의화학검사종목은자동화학장비 Modular D2400 (Roche Diagnostics, Indianapolis, IN, USA) 을이용하여측정하였으며아밀라제, 리파제및 3종의심장표지자는면역장비 Dimension RXL (Siemens healthcare diagnostics, Tarrytown, NY, USA) 로측정하였다. 나트륨-헤파린혈장과리튬-헤파린혈장을비교하기위해 3종의심장표지자는면역장비 Dimension RXL 로, 암모니아는 VITROS 5,1 FS (Ortho-Clinical Diagnosis Inc., Raritan, NJ, USA) 로측정하였다. 2. 결과분석및통계측정된각시험관에대해항목별로처음과 24시간후결과에대해각각의평균과표준편차를구하였고, 시험관사이의비교를위해 SPSS 프로그램 (version 10.2, SPSS Inc., Chicago, IL, USA) 을이용하여 one sample paired T-test 를시행하였다. P<0.05 인항목에대한임상적유의성을판단하기위해두시험관평균값의차이 (mean bias) 를산출후백분율로차이를계산하여, 나트륨은 2%, 칼슘, 염소, 포도당및칼륨은 5%, 다른화학검사항목들은 10% 를허용기준으 로정하였다 [3]. 결과 2시간이내측정치에서리튬-헤파린혈장과혈청시험관을비교하여볼때두시험간통계적으로의미있는차이를보인항목은 33개의항목중 20개였다 (P<0.05). 그차이의평균은모든항목에서 10% 이내였고, 허용범위를넘는항목은칼륨으로, 혈장농도가혈청에비해 7.32% 낮았다 (Table 1). 실온에서의보관시나타나는불안정성에대한평가로리튬-헤파린혈장시험관과혈청시험관의 24시간후측정값을 2시간이내측정값과비교하였을때혈청시험관의경우총빌리루빈만이임상적으로의미있는차이를보인반면리튬-헤파린혈장시험관에서는포도당, lactate dehydrogenase (LD), 총이산화탄소와칼륨에서임상적으로의미있는변화가관찰되었다 (Table 2). 리튬-헤파린혈장과나트륨-헤파린혈장을비교한 3 종의심장표지자와암모니아결과는 2시간이내및 24시간후결과에서시험관간차이가없었다 (Table 3). 고찰저자는일반임상화학항목을대상으로널리사용되고있는진공혈청시험관과리튬-헤파린항응고제진공혈장시험관을비교분석하였다. 기존연구들은혈장과혈청검체의비교기준으로통계적인관련성보다각측정물질마다 significant change limit (SCL) 를설정하여이를벗어나는지를판단하였다. SCL 은정밀도, 정확도를포함한 total error (TE) 를이용하거나 [1] 해당검사실의수개월동안의정도관리에서얻어진평균적표준편차, 즉 USD (usual SD) 를구하여설정하는데 [2] 이를벗어날경우임상적결정에영향을줄수있는의미있는차이로보았다. 본연구에서는이같은실험실자체의기준을정하지못하였으나같은제조사의장비를사용한 Proctor 등 [3] 의기준을따라나트륨은 2%, 칼슘, 염소, 포도당및칼륨은 5%, 다른화학물질들은 10% 를허용기준으로보았고, 두검체간통계적차이가있었던 20종물질중칼륨만이이를벗어나임상적결정에영향을줄수있는오차를보였다. 허용기준내에속하지만 ALT, 인, 총빌리루빈, 직접빌리루빈, 크레아티닌은통계적관련있는차이가 5% 이상으로상대적높았는데이들물질이외부정도관리에대한정밀도나정확도의허용범위가넓은물질이라는점을볼때혈장과혈청사이의오류즉, 검체형태에의한오류로만볼수는없을것으로생각되었다. 일반화학종목에대해혈장과혈청간검체를비교한다른연구를살펴보면칼륨외에는연구마다같은물질에도이견을보인다 [4-12]. 총빌리루빈은한연구에서혈장과혈청간통계적으로차이가있었으나 SCL 을벗어나지않아 [4] 본
일반임상화학검사에서의진공겔혈장시험관의평가 207 연구와동일한결과를보였고, 또다른연구자들은통계학적으로유의한차이가없었다고보고하고있다 [5,6]. 인역시본연구와동일하게통계적관련있는차이가있었지만 [7,8] 모두 SCL 범위내에속해임상적인영향은없다고평가되었다 [8]. 심장표지자 cardiac troponin I (ctni) 는본연구에서는리튬-헤파린혈장과혈청의비교와리튬-헤파린과나트륨- 헤파린혈장간비교모두에서통계적차이가나타나지않았으나, Miles 등 [11] 은본연구와동일한장비로측정했을때혈장과혈청간의통계적의의가있으면서 11% 차이가있다 Table 1. Comparison between test results of plasma and serum Analyte N Mean±SD Mean bias (95% Confidence interval) Unit Plasma-LH Serum P value Serum-Plasma Differences(%)* AST 45 U/L 22.24 ± 5.45 22.31 ± 5.17 0.769 - - ALT 45 U/L 20.91 ± 8.77 22.09 ± 8.96 < 0.05 1.178 (0.766, 1.589) - 5.52 Glucose 45 mg/dl 91.62 ± 14.24 93.71 ± 13.64 < 0.05 2.089 (1.029, 3.149) - 2.30 Calcium 45 mg/dl 9.15 ± 0.35 9.22 ± 0.35 < 0.05 0.711 (0.026, 0.115) - 0.76 Phosphate 45 mg/dl 3.26 ± 0.45 3.48 ± 0.43 < 0.05 0.222 (0.158, 0.285) - 6.40 Uric acid 45 mg/dl 5.1 ± 1.32 5.2 ± 1.34 < 0.05 0.035 (0.012, 0.058) -0.66 GGT 45 U/L 27.4 ± 22.69 27.96 ± 22.87 < 0.05 0.556 (0.163, 0.948) - 1.49 Alkaline phosphatase 44 IU/L 63.55 ± 15.03 66.18 ± 15.55 < 0.05 2.636 (2.232, 3.041) - 4.01 Total protein 45 g/dl 7.95 ± 0.38 7.74 ± 0.41 < 0.05-0.213 (-0.256,-0.170) 2.80 Albumin 45 g/dl 4.61 ± 0.29 4.66 ± 0.28 < 0.05 0.048 (0.019, 0.077) - 1.05 Total bilirubin 45 mg/dl 0.58 ± 0.26 0.62 ± 0.26 < 0.05 0.037 (0.026, 0.048) - 6.80 Direct bilirubin 45 mg/dl 0.19 ± 0.07 0.2 ± 0.06 < 0.05 0.011 (0.004, 0.018) - 5.53 BUN 45 mg/dl 15.46 ± 3.83 15.36 ± 3.89 0.143 - - Creatinine 45 mg/dl 0.85 ± 0.16 0.81 ± 0.16 < 0.05-0.042 (-0.051, -0.032) 5.31 Triglyceride 45 mg/dl 116.53 ± 72.29 120.84 ± 73.61 < 0.05 4.311 (2.83, 5.79) - 3.38 Cholesterol 45 mg/dl 185.2 ± 38.05 191.84 ± 39.66 < 0.05 6.644 (5.015, 8.274) - 3.39 LDL 45 mg/dl 129.58 ± 34.49 130.84 ± 35.18 < 0.05 1.267 (0.016, 2.518) - 0.94 HDL 45 mg/dl 55.64 ± 16.06 55.73 ± 15.44 0.712 - - Ferritin 45 ng/ml 116.37 ± 43.27 117.76 ± 43.91 < 0.05 1.406 (0.895, 1.918) - 1.17 UIBC 45 µg/dl 206.4 ± 68.58 199.03 ± 68.15 < 0.05-7.370 (-12.739,-2.002) 4.07 CK 45 U/L 107.36 ± 61.12 110.13 ± 62.63 < 0.05 2.778 (1.421, 4.135) - 2.77 LD 45 mg/dl 326.19 ± 59.73 328.77 ± 58.77 0.296 - - CRP 35 mg/dl 0.17 ± 0.36 0.17 ± 0.36 0.337 - - Magnesium 45 mg/dl 2.24 ± 0.17 2.26 ± 0.18 < 0.05 0.013 (0.002, 0.024) - 0.56 Total CO 2 45 mmol/l 22.74 ± 2.78 25.71 ± 2.43 0.444 - - Sodium 35 mmol/l 141.09 ± 2.74 141.63 ± 2.73 < 0.05 0.40 (0.035,0.765) - 0.28 Potassium 35 mmol/l 3.88 ± 0.27 4.18 ± 0.26 < 0.05 0.304 (0.261, 0.347) - 7.32 Chloride 35 mmol/l 104.49 ± 2.06 104.46 ± 2.25 0.462 - - Cardiac TnI 42 ng/ml 0.001 ± 0.003 0.002 ± 0.007 0.279 - - CK-MB 42 ng/ml 1.05 ± 0.60 1.01 ± 0.57 0.365 - - Myoglobin 42 ng/ml 42.43 ± 15.63 42.79 ± 15.67 0.426 - - Amylase 32 U/L 62.59 ± 21.15 62.91 ± 21.59 0.134 - - Lipase 32 U/L 228.81 ± 40.37 233.38 ± 46.19 0.152 - - *Difference%: the average of [(test value of plasma-test value of serum)/ test value of serum] 100. Abbreviations: LH, lithium heparin; GGT, γ-glutamyl transferase; BUN, blood urea nitrogen; UIBC, unsaturated iron binding capacity; CK, creatine kinase; LD, lactate dehydrogenase; CRP, C-reactive protein TnI, troponin I.
208 서관용 이현철 김유경 김지혜 Table 2. The effect of storage for 24 hours in serum and plasma Analytes N Mean bias (95% Confidence interval) Serum T 0 - T 24 Difference(%) * Plasma T 0 - T 24 Difference(%) * AST 45 0.778 (0.330, 1.226) - 3.83-0.333 (-0.854, 0.187) 1.81 ALT 45 0.867 (0.352, 1.381) - 5.04 1.6 (0.894, 2.306) - 9.41 Glucose 45 0.756 (-0.363, 1.874) - 0.92 37.4 (32.821, 41.979) -41.29 Calcium 45 0.008 (-0.069, 0.086) - 0.06-0.080 (-0.141, -0.019) 0.88 Phosphate 45-0.080 (-0.131, -0.029) 2.5-0.226 (-0.269, -0.184) 7.27 Uric acid 45-0.026 (-0.047, -0.058) 0.54-0.033 (-0.058, -0.009) 0.61 GGT 45-0.356 (-0.856, 0.145) 1.53-0.800 (-1.188, -0.412) - 3.19 Alkaline phosphatase 44-1.023 (-1.396, -0.649) 1.73 1.955 (1.473, 2.436) 3.36 Total protein 45-0.037 (-0.086, 0.010) 0.52-0.051 (-0.097, -0.006) 0.66 Albumin 45-0.017 (-0.049, 0.013) 0.44 0.029 (-0.009, 0.067) - 0.53 Total bilirubin 45 0.06 (0.043, -0.077) -10.47 0.007 (-0.004, 0.017) - 0.94 Direct bilirubin 45 0 (-0.008, 0.008) 0.65 0.005 (-0.003, 0.014) - 1.28 BUN 45-0.087 (-0.228, 0.054) 0.6-0.131 (-0.259, -0.003) 0.81 Creatinine 45-0.022 (-0.032, -0.013) 2.76-0.029 (-0.037, -0.021) 3.51 Triglyceride 45-6.711 (-10.043, -3.379) 6.54-1.311 (-4.535, 1.913) - 0.16 Cholesterol 45 2.089 (-1.185, 5.363) - 0.68 2.467 (-0.761, 5.695) - 0.95 LDL 45 4.222 (2.480, 5.965) - 3.28 5.911 (3.811, 8.011) - 4.31 HDL 45 1.289 (0.751, 1.827) - 2.36 0.044 (-0.633, 0.722) 0.32 Ferritin 45-0.493 (-0.800, -0.187) 0.52-0.804 (-1.125, -0.484) 0.77 UIBC 45 1.757 (-6.128, 9.642) - 0.4 7.251 (0.395, 14.107) - 3.39 CK 45 0.044 (-1.023, 1.112) 0.34-2.444 (-3.862, -1.027) 2.75 LDH 45-8.871 (-11.349, -6.393) - 2.66-43.273 (-49.638, -36.909) 13.32 CRP 35 0.005 (-001, 0.010) - 1.16-0.01 (-0.008, 0.005) 7.5 Magnesium 45-0.035 (-0.058, -0.012) 1.54-0.051 (-0.70, -0.032) 2.3 Total CO 2 45 0.54 (0.042, 1.038) - 1.75 3.402 (3.028, 3.777) -13.14 Sodium 35-1.486 (-2.164, -0.807) 1.07-1.086 (-1.837, -0.334) 0.79 Potassium 35-0.046 (-0.071, -0.020) 1.1-0.251 (-0.295, -0.208) 6.61 Chloride 35-1.029 (-1.545, -0.512) 0.99-0.514 (-0.996, -0.033) 0.5 * %Difference=average of [(T 24-T 0)/T 0 100]. Abbreviations: T 0, test value of initial time; T 24, test value of storage for 24 hours; see Table 1. Table 3. Comparison of lithium heparin and sodium heparin Analyte Unit N Mean value of initial time Mean value of 24h Plasma-LH Plasma-SH P value Plasma-LH Plasma-SH P value TnI ng/ml 50 1.69 1.63 0.109 1.55 1.29 0.996 CK-MB ng/ml 52 1.22 1.16 0.317 1.08 1.10 0.998 Myoglobin ng/ml 50 82.30 82.30 0.978 83.80 87.70 1 Ammonia µmol/l 40 17.53 18.20 0.628 NA NA - Abbreviations: LH, lithium heparin; SH, sodium heparin; TnI, troponin I; CK, creatine kinase; NA, not applicable.
일반임상화학검사에서의진공겔혈장시험관의평가 209 고보고한바있다. 그러나두연구간대상환자군의차이가있어, 본연구에서는포함되지않은고농도의검체결과로인해이러한차이가기인했다고생각되었다. 이와같이대부분의항목이연구간차이를보이나칼륨의경우는많은연구들에서일치되게혈장에서낮은농도를보이고임상적인허용범위를넘는경우가많았다 [1,3,9,10]. 본연구에서도동일하게혈장농도가혈청보다낮았고차이는 7.32% 로다른연구와유사하게나타났다. 칼륨의혈장혈청간차이의원인은혈장의경우항응고제의사용으로응고형성에의한혈소판의파괴가적어칼륨의세포외유리가적기때문인것으로알려져있다 [10]. 혈장이응고시간이필요없는장점에도불구하고사용이제한되는것은혈장에대한평가가부족하다는점외에도세포와분리하지않고보관시혈장이더불안정하다고알려져있기때문이다. Boyanton 등 [2] 은혈장이나혈청을세포와즉시분리한경우와세포와접촉한상태의두조건으로나누어실온에서 56시간까지보관하며 4시간간격으로측정하였다. 세포와즉시분리가되었을때는 56시간까지혈장과혈청이동일한안정성를가지나, 상온에서세포와분리되지않을때임상에영향을줄수있는불안정성이혈장과혈청둘다에서나타나지만혈장의불안정성이정도가더크다고하며혈청을더적합한임상화학검체로선호하였다. 본연구에서리튬-헤파린혈장시험관과혈청시험관각각에서 24시간후의변화를보았는데리튬-헤파린혈장시험관에서포도당, 총이산화탄소, LD, 칼륨이혈청시험관에비해처음측정값보다의미있게증가하여불안정하였다. 이것은겔의세포와혈장을분리하는장벽역할이알려진것에반해충분하지못하다는사실을보여주고있고, 검사가지연될상황에검체의안정성을확보하기위해별도의자검체분리가필요하므로겔시험관의이점이줄어든다. 포도당, LD, 총이산화탄소는 4시간이상세포와접촉될때임상적영향을미칠만한변화가일어나는데 [2] 겔이포함된혈청시험관에서는 24시간경과한후에도이들물질이매우안정한것을확인할수있어알려진것처럼혈청시험관의겔이유용하였다. 혈장검체의경우도겔시험관의안정성이있다고알려져있지만 [1] 저자들은이에대한추후보충연구가필요하다고생각하였다. 혈청시험관은 24시간보관시대부분의물질이혈장보다안정하였으나 24시간후총빌리루빈감소가혈장에비해크게나타났는데혈청에서총빌리루빈의불안정성에대한보고가있으며 photodegradation 가능성을생각해볼수있다 [13,14]. 응급검사로시행되는심장표지자의경우, 본연구에서는리튬-헤파린혈장검체와나트륨-헤파린혈장검체간의차이가없는것으로나타나, 자검체분주가필요없는리튬-헤파린시험관의도입이유용할것으로생각되었다. 리튬-헤파린혈장검체는다양한임상화학검사에서혈청과비교하여결과가일치하여혈청을대신하여사용할수 있을것으로생각되어앞으로검사시간단축에기여할수있을것이다. 혈장의낮은칼륨농도는여러연구에서잘알려져있으므로새로운참고치의적용이필요할것이다. 그러나여전히검사가지연될때혈장의불안정성이겔시험관에서도나타나므로이점을반드시인지하고검사결과에영향을미치지않도록보완점을찾아야할것이다. 또한추가적으로임상화학외의다양한면역검사에도혈장시험관의평가가이루어질필요가있다. 요약배경 : 최근임상검사실에서는검사시간단축을목적으로혈장검체가혈청을대신하여사용되고있다. 저자들은일반적임상화학항목에대해리튬-헤파린진공겔혈장시험관을기존의진공겔혈청시험관과비교하였다. 방법 : 검진센터에내원한 52 명을대상으로하였다. 리튬 -헤파린혈장시험관과혈청시험관의비교를위해 30 종의일반화학검사항목과 3종의심장표지자를측정하였다. 결과는각시험관사이평균오차와백분율차이를계산하여비교하였다. 결과 : 리튬-헤파린혈장시험관과기존의혈청시험관사이의결과는칼륨을제외하고모두임상적으로일치하였다. 칼륨은혈장에서혈청에비해 7.32% 낮은농도를보였다. 24시간보관후리튬-헤파린혈장시험관에서포도당, lactate dehydrogenase (LD), 총이산화탄소, 칼륨의의미있는변화가관찰되었다. 결론 : 리튬-헤파린혈장시험관은기존의혈청시험관과비교하여임상화학검사에서임상적으로일치하는결과를보여검사시간단축을위해사용할수있을것이다. 그러나 24시간보관후에나타난혈장겔시험관의불안정성에대한주의가필요하다. 참고문헌 1. O'Keane MP and Cunningham SK. Evaluation of three different specimen types (serum, plasma lithium heparin and serum gel separator) for analysis of certain analytes: clinical significance of differences in results and efficiency in use. Clin Chem Lab Med 2006;44:662-8. 2. Boyanton BL Jr and Blick KE. Stability studies of twenty-four analytes in human plasma and serum. Clin Chem 2002;48:2242-7. 3. Proctor KA, Rao LV, Roberts WL. Gadolinium magnetic resonance contrast agents produce analytic interference in multiple serum assays. Am J Clin Pathol 2004;121:282-92. 4. Doumas BT, Hause LL, Simuncak DM, Breitenfeld D. Differences between values for plasma and serum in tests performed in the Ektachem 700 XR Analyzer, and
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