Comparing the Tube and Gel Techniques for ABO Antibody Titration, as Performed in Three European Centers 林 良 豐

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1 Comparing the Tube and Gel Techniques for ABO Antibody Titration, as Performed in Three European Centers 林 良 豐

2 Introduction Data from 60 consecutive ABO-incompatible kidney transplantations performed in Stockholm, Sweden;Freiburg, Germany; and Uppsala, Sweden, revealed different A/B antibody titer levels in the three patient populations, with median titers of 1:32, 1:128, and 1:8, respectively. Because it is unlikely that the different populations would differ significantly with regard to A/B antibody levels, we wanted to investigate whether these differences were method-related.

3 All German transplantation patients undergo a vaccination program pretransplant, and because it is well known that vaccination can booster A/B antibody titers, this could be one explanation for intercenter differences. However,tube hemagglutination technique is, at the very best, a semiquantitative technique with a several titer steps difference needed for a significant intracenter change.

4 Previous studies have also shown substantial intercenter variation regarding analysis of A/B antibodies. In our previous study, patient samples were analyzed using three different tube agglutination techniques. The centers differed in almost every detail, such as use of patient plasma or serum, the medium used for dilution of patient samples, incubation time, centrifugation time and speed, and use of polyclonal vs. monoclonal secondary step antibody for indirect agglutination. All these details can affect titer results.

5 In February 2006, Stockholm switched to a gel microcolumn hemagglutination technique using Micro Typing Cards and reagents from DiaMed AG Plasma from the patient is stepwise diluted 1:2 with PBS from the intended kidney donor, or from a single blood donor with the same ABO blood group, including A1/A2 subtype, as the kidney donor, are used to make a 0.8% suspension

6 In each incubation well, 50μL of cell suspension is mixed with 25 μl of plasma dilution. Micro Typing Cards with NaCl (DiaMed- ID,NaCl/enzyme test and cold agglutinins) are used for direct agglutinationwith incubation for 10 min at room temperature (RT) and centrifugation for 10 min at 85g.

7 For indirect agglutination,micro Typing Cards with anti-immunoglobuling(igg) monoclonal antibody (DiaMed-ID, Coombs anti-igg) are used for incubation for 15 min at 37 C and centrifugation for 10 min at 85g. The inverted value of the highest plasma dilution that gives a 1agglutination reaction is interpreted as the titer.

8 As internal reference, DiaMed Anti-D reference reagent is titrated according to the manufacturer s instructions using Micro Typing Cards with anti-igg monoclonal antibody. A weak reaction is found with a 1:16 dilution and anr0r RBC. In hemagglutination technique, direct agglutination in saline (NaCl) and indirect agglutination (using the indirect antiglobulin test, IAT) are arbitrarily interpreted as immunoglobulin M (IgM) and IgG, respectively.

9 When plasma is not treated with dithiothreitol to inactivate IgM molecules, indirect agglutination titers may be a mix of IgM antibodies reacting at 37 C, and IgG antibodies. However, both IgG and IgM antibodies are likely to be active in humoral rejection, and coldreacting (30 C) antibodies are usually not of clinical significance. Also, anti-a/b of IgG class may affect direct agglutination

10 Material And Method As a first step, current local methods for A/B antibody titration, that is, two different tube techniques in Freiburg and Uppsala and gel technique in Stockholm, were compared. Three samples were drawn from each of 21 healthy blood donors with blood group O and analyzed in the three centers within 1 week using current local methods and local test RBCs.

11 All three centers used single-blood donor A1, Leab+, and B, Lea-b+, RBCs. In Stockholm and Uppsala, plasma was used, whereas in Freiburg, serum was used. For each sample, anti-a and anti-b antibody was analyzedusing direct (NaCl) and indirect agglutination technique(using the IAT) (n84 titrations).

12 The results confirmed method-related differences (Fig. 1), with indirect (IAT) titers being a median of one titer step higher in Freiburg and a median of one titer step lower in Uppsala compared with titer results obtained in Stockholm.

13

14 the results in Freiburg and Uppsala varied from five stepslower to three steps higher compared with Stockholm. Results in Freiburg varied from one step lower to five steps higher compared with Stockholm (Fig. 2A).

15

16

17 As a second step,21 healthy blood donors blood donor A1, Lea-b, and B, Lea-b, RBCs that were also sent from Stockholm. Plasma dilutions and RBC suspensions were prepared locally according to the common instruction.

18 Median IAT titer levels were 1:64, 1:128, and 1:128 in Stockholm, Freiburg, and Uppsala, respectively. For each sample and titration procedure, the results in Freiburg and Uppsala were compared with results in Stockholm and expressed as number of titer steps difference higher or lower.

19 For the same sample and titration procedure, both for direct and for indirect agglutination, the results in Uppsala varied from the same titer to 4 titer steps higher compared with Stockholm. In Freiburg, they varied from 1 to 3 steps higher for IAT and from 1 step lower to 2 steps higher for direct agglutination compared with Stockholm

20

21

22 Conclusion In conclusion, intercenter variation in A/B antibody titration was reduced from amedian of three (range 0 to 6) titer steps using different hemagglutination techniques, to amedian of one (range 0 to 4) titer step using the same gel hemagglutination technique and the same test RBCs. Considering the inherent variability in hemagglutination technique, it is probably necessary to use exactly the samemethod to achieve similar results in different centers.

23 Conclusion Because it is impossible to use the same test RBCs worldwide, kidney donor RBCs are suggested for test RBCs for A/B antibody titration in ABOincompatible kidney transplantation patients. A more reproducible method for A/B antibody quantification than hemagglutination is needed to develop the protocol for ABO-incompatible kidney transplantation.

24 Enzyme-linked immunosorbent assay technique, flow cytometry, surface plasmon resonance, and KODE technology are candidates, but these techniques are not available in all laboratories. A change from tube to gel hemagglutination technique offers a simple way to some improvement.

25 THE END

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