A comparison of ABO blood group antibody titration methodologies and ABO IgG subclass measurement against graft survival in ABO-incompatible kidney transplant patients at Hammersmith Hospital

  • Elias Bhonda

Student thesis: Doctoral Thesis


ABOiRTx has the potential to increase kidney donor availability and access to transplantation. However, precise monitoring of ABO antibodies is critical in guiding treatment and assessing patient suitability for ABOiRTx. Haemagglutination titration assay is a semi-quantitative methodology commonly used for measuring ABO antibodies and the amount of PE required pre- or posttransplantation. Due to the variation in titration assays across institutions, this study was conducted to investigate for agreement/disagreement between these methods and suggest proposals for standardisation. 50 plasma samples were tested against the titration methods, Tube IAT, Tube DRT, Bio-Rad IAT, Bio-Rad DRT, Bio-Vue IAT and Bio-Vue DRT. 30 of these samples were from ABOiRTx patients with graft outcome data available while 20 were from ESRD patients awaiting transplantation. An ELISA method for the measurement of ABO IgG antibody subclasses (IgG1, IgG2, IgG3 and IgG4) was also developed and used to measure subclass levels in 9 patients.
All titration results showed overall statistically significant difference using ANOVA multivariate analysis which meant that the methods cannot used interchangeably. However, upon pairwise analysis, association was found between the following pairs of methodologies: Tube IAT/Tube DRT, Tube DRT/Bio-Rad IAT and Bio-Rad DRT/Bio-Vue IAT. The number of PE was closely linked to baseline but not posttransplant ABO antibody titres. The use of one operator did not improve the variability between the different titration methods. This suggests that all ABO antibody titration methodologies in current use globally cannot be used interchangeably, thus making it difficult to objectively manage patients retransplantation. From the study results, a reasonable choice of methodology to propose for standardisation across transplant centres is the Bio-Rad IAT column method which can be developed over time and therefore provide the basis for all comparisons.
Using the Indirect ELISA technique, most of the cases that experienced graft rejection (5/9) had a combination of IgG1 and IgG2 subclasses. No association was found between ABO IgG2, IgG3 and IgG4 as individual entities against graft
outcome. IgG1 was the only antibody that demonstrated association with graft outcome. IgG2 was detected in 4/4 cases who did not experience graft rejection. IgG3 was detected in 4/5 graft rejection cases which supports its known feature of efficiency in activating complement. IgG4 was found in 1/9 cases, in a patient who experienced graft rejection. The sample size for this part of the study was too small, resulting in low statistical power. However, the Indirect ELISA technique developed in the study can be used to explore ABO IgG antibody subclass dynamics to further understand their role in ABOiRTx.
Date of AwardMar 2020
Original languageEnglish
SupervisorRoslyn Victoria Gibbs (Supervisor) & Fiona Regan (Supervisor)

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