Int J Blood Transfus Immunohematol 2017;7:15–25. Ono et al. 15 www.ijbti.com ORIGINAL ARTICLE PEER REVIEWED OPEN| OPEN ACCESS ACCESS Comparative study of two automated pre-transfusion testing systems (microplate and gel column methods) with standard tube technique Takako Ono, Hitoshi Ohto, Hiroyasu Yasuda, Rie Hikichi, Kinuyo Kawabata, Keiji Minakawa, Satoshi Ono, Masami Kikuchi, Akiko Sugawara, Shunnichi Saito, Nozomi Takano, Kenneth E. Nollet ABSTRACT MP and 1 of 272 with GC). MP missed 2 (anti-E and –Fyb) of 8 antibodies and GC missed 5 (2 Aims: Clinically significant antibodies may anti-E and 1 each of -Fyb, -Jka and –Lea) of 10 become undetectable and still provoke antibodies, which manual PEG-IAT detected. transfusion reactions. Hemolysis has been Among 11 known alloantibodies, MP detected 7 reported among transfusion recipients with antibodies at higher dilution than tube PEG-IAT, anti-Kidd that was undetectable by gel column whereas GC showed lower scores in 7 samples but detectable by other methods. Methods: than tube PEG-IAT and missed 2 clinically We compared two automated technologies - significant antibodies (anti-C and anti-Fyb). microplate (MP) and gel column (GC) methods Conclusion: Automated systems, comparable - with manual methods in ABO/RhD typing, to manual tube technique for forward grouping irregular antibody screening, identification, of ABO and RhD, are less sensitive in ABO titration, and detection threshold of mixed- reverse testing. As GC more often failed to detect field agglutination. Results: Automated systems clinically significant antibodies of low-titer, agreed generally with tube results in 98% or users should be especially mindful of possible more of ABO forward and RhD groupings, but post-transfusion hemolysis. showed weaker reactions in ABO reverse testing against A1 (K=0.88 with MP and K=0.77 with Keywords: ABO grouping, Gel column method, GC) and B cells (K=0.66 with MP and K=0.68 Irregular antibody, Microplate method, Tube with GC) and failed to detect some anti-A (2 of technique 273 samples with MP) and anti-B (2 of 273 with How to cite this article Takako Ono1, Hitoshi Ohto1, Hiroyasu Yasuda1, Rie Hikichi1, Ono T, Ohto H, Yasuda H, Hikichi R, Kawabata Kinuyo Kawabata1, Keiji Minakawa1, Satoshi Ono1, Masami K, Minakawa K, Ono S, Kikuchi M, Sugawara A, Kikuchi1, Akiko Sugawara1, Shunnichi Saito1, Nozomi Takano1, Saito S, Takano N, Nollet KE. Comparative study Kenneth E. Nollet1 of two automated pre-transfusion testing systems Affiliations: 1Department of Blood Transfusion and Trans- (microplate and gel column methods) with standard plantation Immunology, Fukushima Medical University Hos- tube technique. Int J Blood Transfus Immunohematol pital, Fukushima, Japan. 2017;7:15–25. Corresponding Author: Hitoshi Ohto, MD, PhD, Depart- ment of Blood Transfusion and Transplantation Immunology, Fukushima Medical University Hospital, Hikariga-oka, Fuku- shima City, Fukushima 960-1295, Japan; Email: hit-ohto@ Article ID: 100030IJBTITO2017 fmu.ac.jp ********* Received: 30 January 2017 Accepted: 20 April 2017 doi:10.5348/ijbti-2017-30-OA-3 Published: 16 May 2017 International Journal of Blood transfusion and Immunohematology, Vol. 7; 2017. ISSN – [2230-9020] Int J Blood Transfus Immunohematol 2017;7:15–25. Ono et al. 16 www.ijbti.com INTRODUCTION an indirect antiglobulin (IAT) procedure. The RBCs were incubated at 37oC for 30 minutes, then washed 3 Automated pre-transfusion testing has gained times with saline. A rabbit-source antihuman reagent favor to overcome drawbacks of tube technique, which (Ortho Anti-IgG (Rabbit), Ortho-Clinical Diagnostics), is subject to human error even in laboratories well- was added to each tube, centrifuged, resuspended and staffed with dedicated transfusion specialists. Studies examined for agglutination. IAT Control cells coated with comparing standard tube technique with automated IgG (prepared in-house) were added to each negative test pre-transfusion testing have focused on the sensitivity to confirm antihuman reactivity. of irregular antibody detection [1–5]. Automated The antibody screen test was performed using a, microplate (MP) systems have been reported to be more polyethylene-glycol (PEG)-IAT method. Two drops of sensitive than standard tube technique [4]. Conversely, patient’s plasma and 1 drop each of a 3-cell screen set a collaborative cohort found gel column (GC) systems (SURGISCREEN, Ortho-Clinical Diagnostics) and a to be less sensitive than low-ionic strength solution- special screen cell (Di(a+), Ortho-Clinical Diagnostics) enhanced indirect antiglobulin tests in detecting were mixed, centrifuged, resuspended and examined for clinically significant irregular antibodies [6]. In a recent direct agglutination. To each tube, 2 drops of 20% PEG series of 17 patients with post-transfusion hemolysis, (prepared in-house)[8] were added as a enhancer and anti-Jka was detectable by MP but not by GC [7]. In this incubated at 37oC for 15 minutes, washed 4 times with study, we compare two desk-top automated systems, MP saline, and 2 drops of antihuman reagent (Ortho Anti-IgG (ECHO, Immucor) and GC (WADiana Compact, Grifols), (Rabbit), Ortho-Clinical Diagnostic) were added. After with standard tube technique for ABO grouping, RhD incubation, and centrifugation, the tubes were examined typing, irregular antibody screening, titration of known for agglutination. The in-house IgG-coated RBCs were irregular antibodies, and detection threshold of mixed- added to each negative test [7]. field agglutination. If any patient sample reacted with any screen cell, antibody identification was performed using an eleven- cell panel (RESOLVE Panel A and B, Ortho-Clinical MATERIALS AND METHODS Diagnostics) and/or sixteen-cell panel (PANOCELL-16, Immucor K.K., Tokyo, Japan) with PEG-IAT. Samples Solid phase method using an automated To compare ABO/D blood grouping between a direct agglutination method on a microplate and a standard tube instrument method, and to antibody screening between a solid phase For ABO/D grouping, performed by direct (SP) method and a standard tube method, we processed agglutination method on a microplate 20 µL of patient’s 273 consecutive routine samples during October 2012. red cell suspension and 40 µL of murine monoclonal Then, to compare blood grouping and antibody screening ABO reagents (anti-A, anti-B, Immucor K.K.,) and between a gel column (GC) method and standard tube monoclonal Rh reagent and control (Gamma-clone method, we processed 272 consecutive routine samples anti-D and Monoclonal Control, Immucor K.K.) were during December 2012. Samples of newborns were pipetted onto a microwell plate (Galileo Echo, Immucor excluded from this study. K.K.), centrifuged, resuspended and agglutination was interpreted by the automated algorithm and camera. For Standard tube method ABO reverse typing, 15 µL of each of the reverse grouping cells (Referencell, Immucor K.K.) and 30 µL of patient’s For determining the ABO group of red blood cells plasma were mixed, centrifuged, resuspended and (RBC), 1 drop of each reagent (Bioclone anti-A, Bioclone examined for agglutination. anti-B, Ortho-Clinical Diagnostic, Tokyo, Japan), and 1 For the solid phase antibody screening, 25 µL of drop of 3–5% patient’s RBCs were added to appropriately- patient’s plasma and 50 µL of additive (Capture LISS, labeled tubes, mixed, centrifuged, resuspended and Immucor K.K.) were added to the plate wells containing examined for agglutination. For the ABO reverse test, 2 the RBC monolayers (Capture-R Ready-Screen(3), drops each of patient’s plasma and 1 drop of reagent cells Immucor K.K.), The automated analyser (Galileo Echo, (AFFIRMAGEN A or B cells, Ortho-Clinical Diagnostics) 1 Immucor K.K.) completed the testing, added the indicator were mixed, centrifuged, resuspended and examined for cells (Capture-R Ready Indicator Cells) and interpreted agglutination. the results. All negative reactions, were checked visually For D typing, 1 drop of reagent (Bioclone anti-D, Ortho- for confirmation per the Manufacturer’s Operators’ Clinical Diagnostics) and 1 drop of the corresponding Manual. control (Rh-hr Control, Ortho-Clinical Diagnostics) When any potential antibody was suspected, and 3–5% patient RBCs were mixed, centrifuged, identification was performed using the manual PEG-IAT resuspended and compared for agglutination. If there method. was no agglutination of the RBCs in both the reagent and control tubes, the weak D test was performed using International Journal of Blood transfusion and Immunohematology, Vol. 7; 2017. ISSN – [2230-9020] Int J Blood Transfus Immunohematol 2017;7:15–25. Ono et al. 17 www.ijbti.com Gel column method using an automated 0.6), moderate agreement; K(0.21–0.4), fair agreement; instrument and K(0.0–0.2), slight agreement. Titration score was analyzed by the Friedman test and the nonparametric For ABO/D grouping, 10 µL of patient’s RBC Wilcoxson’s signed rank test. Differences with a p value of suspension was added to the appropriate column on the 0.05 or less were considered statistically significant. All gel card. The gel cards are prepared with antisera (DG Gel statistical analyses were performed with SPSS Statistics ABO/Rh (2D) Card, Kainos Laboratories, Tokyo, Japan) version 24 (IBM Corporation). already added. The ABO reverse test was performed using 50 µL of 0.8% RBC (DG Reverse-Cyte A1,B 0.8%, Kainos Laboratories) All pipetting and processing is performed RESULTS by an automated analyzer (WADiana Compact, Kainos Laboratories). After centrifugation, the cells pellet ABO grouping and RhD typing either at the top (positive reaction) or bottom (negative reaction) of the matrix. The results are read automatically In ABO forward grouping of 273 samples, MP by the instrument and can be confirmed visually concordance with standard tube technique was 99.3% For the antibody screen, 50 µL of patient’s plasma (K = 0.985) for A and 98.9% (K = 0.976) for B in were mixed with 25 µL each of 3 screen cells (Search- agglutination strength (Table 1A and Table 1B).