1. Joan Cid, Núria Nogués, Rosa Montero, Marta Hurtado, Albert Briega and Rafael Parra. Blood and Tissue Bank. Barcelona, Spain. Comparison of three microtube column agglutination systems for antibody screening: DG Gel, DiaMed-ID and Ortho BioVue INTRODUCTION Antibody screening is performed to detect clinically relevant red blood cell (RBC) antibodies in transfusion recipients and blood donors. The classical low-ionic-strength-solution (LISS) tube indirect antiglobulin test (IAT) for detecting clinically significant antibodies has been replaced in many countries by microtube column agglutination systems, described for first time in 1988 and published in 1990 (1). The first microtube system was manufactured and distributed by DiaMed-ID (Switzerland), followed by Otho BioVue (Otho Diagnostic GmBH, Germany). Recently, a new microtube column agglutination system has been launched on the market by Diagnostic Grifols, S.A. (Barcelona, Spain) with the name of DG Gel. The aim of the present study was to evaluate the diagnostic accuracy of this new system, to analyze the antibody reactivity and to compare the data with the two cited well-established methods. METHODS Samples. We collected 3,024 specimens: 2,408 (79.6%) were plasma samples obtained from EDTA tubes and 616 (20.4%) were serum samples. We obtained 2,422 (80.1%) samples from blood donors in our blood bank; 382 (12.6%) samples from transfusion recipients and 220 (7.3%) from pregnant women in Hospital Vall d’Hebron (Barcelona, Spain). We stored all these samples in the refrigerator at 2-8 ºC and we processed them before 72 hours from extraction. We additionally obtained 100 samples containing antibodies of known specificity from the immunohematology laboratory of our blood bank. These samples had been stored in a frozen state at -30 ºC and were thawed immediately before processing. Test RBCs. The RBCs used for the antibody screening and for the panel identification were obtained from our selected blood donors. These RBCs were suspended to final concentration 0.8 % in the corresponding modified LISS provided by the manufacturers according to their instructions as follows. We calculated the conductivity of these solutions. DG Gel: 50 µL of RBCs in diluent provided by Diagnostic Grifols (Cond.: 5.09 mS/cm) + 25 µL of sample. Incubation: 15 min, 37 ºC. Centrifugation: 9 min. DiaMed-ID: 50 µL of RBCs in ID-CellStab (Cond.: 5.89 mS/cm) + 25 µL of sample. Incubation: 15 min, 37 ºC. Centrifugation: 10 min. Ortho BioVue: 50 µL of RBCs in Ortho BLISS (Cond.: 3.03 mS/cm) + 40 µL of sample. Incubation: 10 min, 37 ºC. Centrifugation: 5 min. RESULTS Antibody screening We performed the antibody screening of 3,024 fresh samples with the three microtube systems and we obtained concordant results in 3,019 (99.83%) and discrepant results in 5 (0.17%). Table 1 shows the specificities of the antibody after the identification steps using 11-cell panel and additional techniques, e.g. papain-treated reagent RBCs and IAT with polyethylenglycol (PEG) tube technique (2). Antibody titration We titrated 100 frozen samples containing antibodies with known specificities. The score mean and standard desviation of the antibody titration for each microtube system were the 34.31±26.7 for DG Gel, 30.3±27.76 for Diamed-ID, and 37.38±28.68 for Ortho BioVue (see Table 2). The antibody titration score was higher with the DG Gel when compared with DiaMed-ID in samples containing anti-K (46.23 vs. 44.38; p<0.05) and in samples containing anti-M (11.88 vs. 5.38; p<0.05). No significant differences were seen in other antibodies specificities. CONCLUSION All three microtube column agglutination systems work well showing a high estimated sensitivity and specificity. REFERENCES 1. Lapierre, Y. Rigal, D., Adam, J. Josef, D., Meyer, F., Greber, S. and Drot, C. (1990) Transfusion, 30, 109-113. 2. AABB (2002) Technical Manual, 14 th edition, Beshesda. Table 1. Antibody detected by the three systems using 2 screening cells. Mean±SD score of antibody titration SpecificitynDG GelDiaMed-IDOrtho BioVue Anti-D774.14±24.2374.43±23.8682.29±29.35 Anti-C463±16.4560.75±15.5470±19.71 Anti-c9 23.6719.442319.5528.2224.80 Anti-E10 37.4023.1433.3028.9834.3029.33 Anti-e2 20013016.5012.02 Anti-C w 3 15.339.614.679.50167.21 Anti-Ch2 38.5019.09248.483618.38 Anti-Jk a 7 20.5714.5015.8611.6522.4316.20 Anti-Jk b 2 194.248080210 Anti-Fy a 5 3424.8724.4024.7035.8026.01 Anti-Fy b 4 50.2514.6845.4511.145217.37 Anti-K13 46.2336.7344.3836.7250.3137.60 Anti-Kp a 2 17.506.3616.5012.022614.14 Anti-Kp b 112 19 Anti-M8 11.884.735.382.8216.887.69 Anti-N1392546 Anti-s3 2417.0824.6727.7929.6718.14 Anti-S5 3417.0130.4020.734121.15 Anti-Le a 2 808041.4187.07 Anti-Le b 2 11.502.125.503.53161.41 Anti-H/HI/I3 13.3313.7912.3310.6920.6714.18 Anti-PP 1 P K 2 48.5034.6441.5037.474829.69 Anti-Ge:2117 24 Anti-Vel1463343 Anti-Xg a 18674 Total10034.3130.337.38 Blood Group System Antibody specificity Number of Samples Detected DG GelDiaMed-IDOrtho BioVue Rh Anti-D666 Anti-c444 Anti-E313 Kell Anti-K111 Duffy Anti-Fy a 111 Kidd Anti-Jk a 101 MNS Anti-M111 Anti-S111 Lewis Anti-Le a 111 Mixed Anti-C/-D222 Anti-E/c/panaglutinin111 Anti-Jk b /-K111 NonspecificPanaglutinin002 False positive002 Negative300030032998 False negative030 Total3024 Table 2. Samples containing antibodies of known specificities used for antibody titration. DG GelDiaMed-IDOrtho BioVue Sensitivity 100% (97.07-100) 97.58% (93.09-99.50) 100% (97.07-100) Specificity 100% (99.88-100) 100% (99.88-100) 99.93% (99.76-99.99) PPV( a ) 100% (97.07-100) 100% (97-100) 98.41% (94.38-99.81) PNV( b ) 100% (99.88-100) 99.9% (99.71-99.98) 100% (99.88-100) Efficiency 100% (99.88-100) 99.9% (99.72-99.98) 99.94% (99.77-99.99) Table 3. Estimated diagnostic accuracy. In parenthesis the 95 % CI. (a) PPV: predictive positive value, (b) PNV: predictive negative value. Sensitivity and Specificity In order to obtain a comparative value between the new test and the other two we calculated the estimated diagnostic accuracy for each system (see Table 3). For calculating estimates of these statistical measures we include the 100 positive samples and we reported the confidence intervals (CI) for a sample size of 3,124.