Materials and methods
Samples
Twenty seven serum samples were obtained from red blood cells. These samples contained antibodies which had been identified using the IAT technique. The serum samples were stored at a low temperature of -20oC and thawed immediately before the process of centrifugation was performed to avoid interference from fibrin clots.
Test RBCs
Samples of known antibodies were used which were homozygous and heterozygous for the genes controlling expression for the following antigens:
K, D, Jka, Jkb, C, Fya, Fyb, S, s, M, Kpb +c, E, K, c+E and CW.
The RBCs were suspended in the corresponding modified LISS provided by the manufacturers together with the instructions given in order for them to be used in the micro-tube systems. These instructions were as follows: (Lateral Grifols Gel: final concentration 0·8%, RBCs resuspended in diluents provided by Diagnostic Grifols; Ortho BioVue: final concentration 0·8%, RBCs resuspended in Ortho BLISS).
Antibody Identification
The sensitivity of the antibodies was determined using two systems. An 11-cell identification panel from the blood transfusion lab was used. During the experiment, some antibodies did not exist in the ortho panel i.e. CHa, Cob, Coa. Table 1 shows some of the cells from the panel of Ortho BioVue and Lateral Grifols.We were trained with the new gel since we had not interacted with the two systems before. Necessary materials for Lateral Grifols Gel and Ortho BioVue were obtained from the manufacture before all microtube tests were performed in parallel with strict adherence to the instructions given.
For Ortho BioVue, the test comprised of anti-human globulin anti-IgG, anti-C3d and poly-specific cassette with six micro-tubes. Each microtube had wide reaction vessel in the upper section while the lower part had AHG, a glass micro-bead matrix, dextran solution or PEG. The procedure involved mixing of 40 µL of sample with 50 µL of reagent RBCs before an incubation process was done at 37 °C for ten minutes. This was followed by a five minutes of centrifugation process using the preset cycle.
For Lateral Grifols system, the test consisted of a Coombs card which had eight micro-tubes. Each micro-tube had a wide reaction chamber region while the lower region had a gel and rabbit anti-human globulin reagent (AHG). The procedure used involved mixing of 25 µL of the sample with 50 µL of reagent RBCs. This was followed by fifteen minutes of incubation at 37oC and nine minutes of centrifugation using preset cycle of the centrifugation.
Antibody Titration
Four antibodies, anti-D, anti-JKa, anti-Fya, anti-K, were tested with known specificities of antibodies were tested using the two systems. Doubling of serial dilutions was performed using saline solution (9 g L−1 of sodium chloride) containing 6% of bovine serum albumin. RBC reagent was used with a single-dose expression of the required antigen for the titration process. The same RBC was used for titration with two microtube systems which was resuspended in the corresponding LISS as described in the above procedure. Results were obtained and recorded according to the system and quantified by adding the lengths of positive reactions.
Results
Concordant results were obtained for the 27 serum samples using Ortho BioVue and Lateral Grifocs column agglutination technology. For Ortho BioVue, homozygous cells got 44 while heterozygous cells got 33. On the other hand, Lateral Grifocs homozygous cells got 46 and heterozygous cells had 31. Table 2 gives a summary of the results after identification procedure for unknown antibodies using the two microtube column agglutination systems. Importantly, some antibodies did not show results because they lacked cells from the panels. Titration results for the four sera randomly investigated are given in table 3. There was a small variation between the BioVue and Lateral Grifocs microtube column systems with antibodies of doubtful clinical reliance showing highest score in the BioVue.