200921100 九、發明說明： 【發明所屬之技術領域】 ^發，乃一種「改良式柱狀凝膠檢驗法（M〇dified c〇lumn Gel iest )」；該檢驗法是在柱狀管中利用凝膠介質(Sephadex)進行的一種 經過改良的血凝反應（紅細胞抗原與相應抗體在凝膠介質中可出現肉 眼可見的凝集反應）；特別之處在於： (一） 完全適合國内血庫檢測作業使用' (二） 該檢驗法能通過正反對照顯示出特異，因此克服了血庫學方法檢查 的不足。 一 (三） 可取5血庫檢驗傳統試管法的ΑΒ0血型檢驗、RH血型檢驗、不規則 抗體篩檢、不規則抗體鑑定、間接與直接庫姆氏AHG試驗、輸血交 叉试驗。 (四） 具有操作簡單、靈敏度和特異性高、標準化、結果清晰易判讀等優 點。 (五） 相較於傳統檢驗法，該檢驗法「無須使用微量吸管、專屬離心機及 丄專屬試劑產品，完全使用自製的設備和試劑」。 (六） 成本是目前相同產品的1/5至丨/1〇。 200921100 【先前技術】 } fIf技術—直依循美國血庫學會(aabb) «Si尨ϊ=Γΐ管法為主要方以 polyene (-) (ussmm，、傳統的間接及直接ahg檢驗，有步驟繁瑣 的血庫檢驗令傳統試管法對ABO血型鑑定的錯誤$約月匕 ϋί、Ϊ規則抗體篩檢約3. 8〜13. 9%與不規則抗體ίίί =====美國有關輪二^ 200921100 【發明内容】 (一）所欲解決之問題： 1. 傳統的間接及直接AHG檢驗，有步驟繁瑣（如洗滌期間不正確程 序）、敏感度低、人為技術、訓練差異等不利因素困擾，影響其再 現性和可靠性。94年中華民國醫檢學會血庫能力試驗的血^驗 中傳統試管法對ΑΒ0血型鑑定的錯誤率約〇〜2.1%、不規則抗體篩 檢約3. 8〜13. 9%與不規則抗體鑑定約〇〜4.1%、輸血交又試驗約 8.1~36. 8%。在美國有關輸血錯誤的文獻中，由血庫實驗室錯誤造 成的約佔5~29%。 、 2. 將先前技術所造成「偽陰性」問題詳述如后： (1) 紅血球洗滌不全，殘存有2wi/mi以上的血清時，就會中和 Anti Human Globulin’因此洗務紅血球時須完全洗淨。 (2) 實驗延遲或中斷，AHG試劑沒有在紅血球洗滌後馬上加入，會 造成紅血球上附著的抗體與其分開，形成偽陰性反應。 (3) 加入抗血清後未立即判讀結果。 (4) 忘記加 Anti Human Globulin。 (5) 離心不完全或過度離心造成溶血反應。 P)未在顯微鏡下觀察’忽略掉弱反應或Mixd Field。 (Ό沒有於室溫下再放置5分鐘，會漏掉補體的反應。如果放置 超過10分鐘，反應也會變弱。 (8) 紅血球濃度也會影響判讀，濃度太濃會使反應變弱，濃 淡則凝集反應不易判讀。 (9) 洗滌紅血球的Saline若pH值太低則會降低AHG的敏感性， Saline的pH值應在7. 0-7. 2之間。 _ λ 4( 1〇)實驗結果判讀錯誤，將弱反應判讀成陰性反應。 ―)解決問題之技術手段： 1. ΐΐ狀管中利用凝膠介質（Sephadex)進行的一種經過改良的血凝 應（紅細胞抗原與相應抗體在凝膠介質中可出現肉眼可見的凝 集反應）。 t通，正反對照顯示出特異，因此克服了血庫學方法檢查的不足。 ^無須使用微量吸管、專屬離心機及專屬試劑產品，完全使用 一 自製的設備和試劑」之方法，從而使操作簡單化。 二）對照先前技術之功效·· 1.解決「傳統檢驗法之『偽陰性』問題」 (1) 根據國外文獻報告數據顯示’改良式柱狀凝膠檢驗法 (Modified Column Gel Test)，經與傳統 AHG 試管法比較， =用傳統AHG試管法結果為陰性反應的檢體，如改用微柱凝 膠AHG試驗其中約有28%被偵測為陽性反應。 (2) 於本院，傳統dAT檢驗在8〇年度的陽性率為16%(25/15幻， 200921100 經使用改良式Gel Test,在84年8月至86年7月期間765 件檢體中，有陽性反應者計有152例，陽性率為19. 9%，陽性率 提昇了約25%，與國外使用微量Gel Test的文獻報告數據相 近。 (3)換言之，改良式柱狀凝膠檢驗法（Modified Column Gel Test) 能有效解決「傳統檢驗法之『偽陰性』問題」，具有高度之進 步性、新穎性。 2.成本低廉： 目前國内除財團法人國泰综合醫院外之所有醫學中心及部分區域 醫院’皆有引進瑞士 DiaMed公司的The ID Micro Typing System (Gel Test)產品或美國 〇rth〇-Clinical Diagnostics 公司的 (ID-MTS) Gel Test ;惟因成本過高和使用時效上無法滿足血庫實 驗室實際作業的需求’目前只能取代部分的血庫檢驗作業。改良 式柱狀凝膠檢驗法（Modified Column Gel Test)成本乃目前相 同產品的1/5至1/10’成本低廉’於產業之利用上有高度之優勢。 3. 操作簡便： (1) 改良式柱狀凝膝檢驗法（Modified Column Gel Test)乃以 實驗室内現有的血庫離心機(](111)€^861'011^1：1<：11)中加設一 台控制副機，使用不是微量設計的HLA離心試管自製成Gel Tube，以傳統塑膠吸管操作實驗；其方法乃參考The ID_Micr〇200921100 IX. Description of the invention: [Technical field to which the invention pertains] ^ Hair is a "M〇dified c〇lumn Gel iest"; this test is used in the columnar tube A modified hemagglutination reaction by the gel medium (Sephadex) (the red blood cell antigen and the corresponding antibody can be macroscopically agglutinated in the gel medium); the special features are: (1) It is completely suitable for domestic blood bank testing operations. ' (2) The test can show specificity through positive and negative control, thus overcoming the insufficiency of blood bank examination. One (3) It is desirable to test the ΑΒ0 blood type test, RH blood type test, irregular antibody screening, irregular antibody identification, indirect and direct Comm's AHG test, and blood transfusion test in the traditional test tube method. (4) It has the advantages of simple operation, high sensitivity and specificity, standardization, clear results and easy interpretation. (v) Compared with the traditional test method, the test method “does not use micropipettes, exclusive centrifuges and exclusive reagent products, and completely uses homemade equipment and reagents”. (vi) The cost is currently 1/5 to 丨/1〇 of the same product. 200921100 [Previous technology] } fIf technology—following the American Society of Blood Banks (aabb) «Si尨ϊ=Γΐ管法 as the main party with polyene (-) (ussmm, traditional indirect and direct ahg test, step-by-step blood bank The inspection method makes the error of the traditional test tube method for ABO blood group identification: about 匕ϋ月匕ϋί, Ϊ regular antibody screening about 3. 8~13. 9% with irregular antibody ί ίί ===== US related round 2 ^ 200921100 】 (1) Problems to be solved: 1. Traditional indirect and direct AHG tests, which have cumbersome steps (such as incorrect procedures during washing), low sensitivity, artificial techniques, training differences and other unfavorable factors, affecting their reproducibility. And the reliability. The error rate of the traditional test tube method for the blood test of the blood test in the blood test of the Chinese Medical Association in the past 94 years was about 2.1%, and the irregular antibody screening was about 3. 8~13. 9% with Irregular antibody identification was about 4.1~4.1%, and blood transfusion was tested about 8.1~36.8%. In the US literature on blood transfusion errors, about 5~29% caused by blood bank laboratory errors. 2. The "false negative" problem caused by technology is detailed as follows : (1) Red blood cells are incompletely washed, and when there is serum above 2wi/mi, it will neutralize Anti Human Globulin', so it must be completely washed when washing red blood cells. (2) Delay or interruption of experiment, AHG reagent is not washed in red blood cells Immediately after the addition, the antibodies attached to the red blood cells are separated from each other to form a pseudo-negative reaction. (3) The results are not immediately interpreted after the addition of the antiserum. (4) Forgetting to add Anti Human Globulin. (5) Incomplete centrifugation or excessive centrifugation Hemolysis reaction P) Not observed under the microscope 'ignoring weak reaction or Mixd Field. (ΌThere is no room temperature for 5 minutes, and the reaction of the complement will be missed. If it is left for more than 10 minutes, the reaction will be weak. (8) The concentration of red blood cells will also affect the interpretation. If the concentration is too strong, the reaction will be weak. The agglutination reaction is not easy to interpret. (9) If the pH of the Saline for washing red blood cells is too low, the sensitivity of the AHG is lowered, and the pH of the Saline should be between 7. 0 and 7. 2. _ λ 4 (1〇) The experimental results were misinterpreted and the weak response was interpreted as a negative reaction. ―) Technical means to solve the problem: 1. A modified hemagglutination using a gel medium (Sephadex) in the fistula (red blood cell antigen and corresponding antibody A macroscopic agglutination reaction can occur in the gel medium). T-pass, positive and negative control showed specificity, thus overcoming the lack of blood bank method examination. ^Since the use of micropipettes, proprietary centrifuges and proprietary reagents, complete use of a proprietary device and reagents, the operation is simplified. 2) Controlling the efficacy of the prior art·· 1. Solving the “false negative” problem of the traditional test method (1) According to the foreign literature report data, “Modified Column Gel Test” Compared with the traditional AHG test tube method, the sample with the negative result of the traditional AHG test tube method, such as the micro-column gel AHG test, about 28% of them were detected as positive. (2) In this hospital, the traditional dAT test has a positive rate of 16% in the 8th year (25/15 illusion, 200921100 using the modified Gel Test, 765 samples from August 84 to July 86) There were 152 positive responders, the positive rate was 19.9%, and the positive rate increased by about 25%, which is similar to the data reported in the foreign literature using the micro-Gel Test. (3) In other words, the improved columnar gel The Modified Column Gel Test can effectively solve the "false negative" problem of the traditional inspection method, which is highly progressive and novel. 2. Low cost: At present, all medicines except the Guotai General Hospital in China The Center and some regional hospitals have introduced the ID Micro Typing System (Gel Test) from DiaMed, Switzerland or the ID-MTS Gel Test from the United States 〇rth〇-Clinical Diagnostics; but because of the high cost and aging Unable to meet the needs of the actual operation of the blood bank laboratory' currently only replaces part of the blood bank inspection operation. The cost of the Modified Column Gel Test is currently 1/5 to 1/10' of the same product. 'to The use of the industry has a high degree of advantage. 3. Easy to operate: (1) The modified Column Gel Test is based on the existing blood bank centrifuge in the laboratory (] (111) € 861 ' Add a control auxiliary machine to 011^1:1<:11), use the HLA centrifuge tube which is not micro-design to make the Gel Tube, and experiment with the traditional plastic pipette; the method is based on The ID_Micr〇
Typing System™ (ID-MTS) Gel Test 使用的美國 〇rtho -Typing SystemTM (ID-MTS) Gel Test used in the United States 〇rtho -
Clinical Diagnostics公司離心機產品，設計製造一台 KUBOTA KA2200血庫離心機的控制副機及改良離心試管（圖 1)(註：因KUBOTA KA2200血庫離心機該機型國内血庫實驗 室佔有率九成以上）。 (2) 改良式柱狀凝膠檢驗法（Modified Column Gel Test)「無須 使用微量吸管、專屬離心機及專屬試劑產品，完全使用自…製 的設備和試劑」；相較於傳統檢驗法甚為簡便，於產業之利用 更有高度助益。 ' 4. 產業可利用性： 改良式柱狀凝膠檢驗法（Modified Column Gel Test)完全適合 國内血庫作業使用，可完全取代傳統的庫氏試驗 0 (Anti-Human-Globulin Test，Coombs，AHG Test)外，在血庫作 業令對ΑΒ0血型鑑定、RH血型鑑定、不規則抗體篩檢與鑑定、輪 士交又試驗、血小板抗體篩檢與交又試驗，提供另一種血庫技術 應用。除了可提昇血庫學試劑國產化的能力，同時對柱狀 檢測）提供基礎。 8 200921100 【實施方式】 1. 使用低離子強度緩衝液加入Dextran gel，再加入AHG，裝入「改 良離心試管」。 2. 再將欲受檢的紅血球懸浮液加入裝有Dextran gei的「改良離心 試管」内（圖1)。 3. 離心（70g) 10分鐘後判讀。 4. 當紅血球凝集或其與gei内含之抗體反應時，若紅血球經離心後 ，法下降至管底則為陽性反應，依其下降與否之程度可分為 ^+』'『3+』'『2+』、『1+』、1』、『陰性反應』」（利用661卩虹1；化163 Umolecula^ sieving作用，經過離心後，紅血球凝集依其分 小通過篩子的速度就不同，因此可將其區分。沒有凝集的 球ί部通過試管到達最底端；而凝集的紅血球則停留在試管 上鳊，越大的凝集則在越上端）（圖2〜6)。 200921100 【圖式簡單說明】 1. 圖1 :「改良離心試管」之示意圖。 2. 圖2圖改良式柱狀凝膠檢驗法（Modified Column Gel Test)」之示意 5. 圖程度分為『4+』、『3+』、『2+』、『1+』、 6, 200921100 【主要元件符號說明】Clinical Diagnostics centrifuge products, design and manufacture of a KUBOTA KA2200 blood bank centrifuge control auxiliary machine and improved centrifuge tube (Figure 1) (Note: due to KUBOTA KA2200 blood bank centrifuge, the domestic blood bank laboratory occupancy rate of this model is over 90% ). (2) Modified Column Gel Test "There is no need to use micropipettes, exclusive centrifuges and exclusive reagent products, completely using equipment and reagents made by..." compared to traditional inspection methods. It is simple and easy to use in the industry. 4. 4. Industrial Applicability: The Modified Column Gel Test is fully suitable for domestic blood bank operations and can completely replace the traditional Anti-Human-Globulin Test (Coombs, AHG). In addition, in the blood bank operation order, 血0 blood type identification, RH blood type identification, irregular antibody screening and identification, rotation test, platelet antibody screening and cross-test, provide another blood bank technology application. In addition to improving the ability to localize blood bank reagents, it also provides a basis for columnar detection. 8 200921100 [Embodiment] 1. Add Dextran gel using low ionic strength buffer, add AHG, and put in "modified centrifuge tube". 2. Add the red blood cell suspension to be tested to the modified centrifuge tube containing Dextran gei (Figure 1). 3. Centrifuge (70g) and read after 10 minutes. 4. When the red blood cells agglutinate or react with the antibodies contained in the gei, if the red blood cells are centrifuged, the method is down to the bottom of the tube, which is a positive reaction. According to the degree of decline or not, it can be divided into ^+』'『3+』 '『2+』,『1+』,1』,“negative reaction』” (using 661卩虹1; 163 Umolecula^ sieving effect, after centrifugation, the speed at which the red blood cells agglutinate through the sieve is different. Therefore, it can be distinguished. The ball ί without agglutination reaches the bottom end through the test tube; the agglutinated red blood cells stay on the test tube, and the larger the agglutination is at the upper end) (Figs. 2 to 6). 200921100 [Simple description of the diagram] 1. Figure 1: Schematic diagram of "modified centrifuge tube". 2. Figure 2 shows the schematic of the Modified Column Gel Test. 5. The degree of the graph is divided into "4+", "3+", "2+", "1+", 6, 200921100 [Main component symbol description]