Evaluation of different methods for antibody titre determination in ABO-incompatible kidney transplantation
Highlight box
Key findings
• Our findings demonstrate the benefits of the microcolumn gel card test (MGT) method (i.e., reduced inter-laboratory differences and increased repeatability) has important clinical implications for the selection of appropriate methods to detect antibodies in ABO-I kidney transplantation.
What is known and what is new?
• Previously, studies on ABO-I kidney transplant antibody titer were more simply focused on laboratory results, while rarely referred to the clinical situation. Moreover, the experimental methods are diverse, and the results vary significantly, lacking a unified experimental approach.
• Highlighting the importance of using a unified method to detect antibody titer in clinical practice,
What is the implication, and what should change now?
• It is important that clinical laboratories employ a consistent method of sample testing for a particular patient, and indicate the approach used when reporting results. the MGT could effectively monitor titer changes following surgery.
Introduction
Kidney transplantation is a feasible treatment for most patients with end-stage renal diseases. However, the shortage of donor organs greatly hampers the clinical practice of kidney transplantation. Using kidneys from ABO blood type-incompatible (ABO-I) living donors has previously been considered to be contraindicated for end-stage renal disease patients. The high titre of antibodies in recipient serum may react with blood type antigen attached to the allograft, and induce extensive microvascular thrombosis and renal cortical necrosis, leading to rejection responses including hyperacute rejection, acute rejection, and chronic rejection (1-4). If not treated promptly, anti-ABO antibody-mediated rejection usually causes graft loss.
In recent years, the inherent obstacles in ABO-I grafts have been largely overcome by the development of novel immunosuppressors (such as mycophenolate mofetil) (5) and advanced plasma exchange technology (6,7). However, there is still a certain incidence of antibody-mediated rejection induced by blood type antibody in ABO-I kidney transplantation, despite the implementation of reasonable treatments prior to surgery. The reason for this is a lack of not only a reliable ABO-I antibody baseline set for clinical intervention but also of reproducibility and precision in ABO antibody testing. There is a consensus among clinicians that interlaboratory repeatability variations the titres of anti-A and/or anti-B antibody should be reduced to 1:16 or lower before ABO-I transplantation surgery (8,9), which can be achieved by plasma exchange or double filtration plasmapheresis. It is also considered important to monitor post-operative antibody titres and maintain them at a safe level. Therefore, the basic principle for blood-type antibody treatment is the timely and accurate surveillance of antibody titres (10). However, because antibody detection methods differ between laboratories, there is no unified baseline to confirm the real titre. Therefore, it is imperative to choose a universally appropriate method to determine the anti-ABO antibody levels prior to surgery, and to set practical cut-off titre levels accordingly.
The most common method for detecting antibody levels in laboratories is the tube test (TT), which is simple and low-cost, but time-consuming and relies on naked-eye interpretation (11). The microcolumn gel card test (MGT) and glass bead card test (GBT) methods, which are based on column agglutination technology, were developed in the 1990s (12,13). These methods involve centrifuging a column on a reagent card, with agglutinated red blood cells (RBCs) becoming trapped in the gel or glass beads and unagglutinated RBCs accumulating at the bottom of the column. These methods are simple to conduct, with good repeatability, and offer interpretable results, and card tests have therefore been widely employed for antibody detection in many laboratories (14,15). However, because of the differences in detection principles between the tests, the results obtained with the same blood sample can vary.
The threshold for the antibody titre before renal transplantation has always been a matter of debate. For example, in one study in Melbourne, Australia, the preoperative A/B antibody titre was set to <1:32 (tube method) or <1:8 [ortho method: using Column Agglutination Technique (CAT) with the Ortho Clinical Diagnostics AutoVue Innova automated method] (16), while in another study at John Hopkins University in the USA, the preoperative A/B antibody titre was set to <1:16 (tube method) (17). Furthermore, a study based on 60 consecutive ABO-incompatible kidney transplantations collected from different centres showed considerable differences in preoperative antibody titres; these centres used distinct hemagglutination strategies with preoperative A/B antibody titres of 1:8 in Uppsala, Sweden, 1:32 in Stockholm, Sweden, and 1:128 in Freiburg, Germany (18,19). As demonstrated by these reports, there is a lack of systematic evaluation of the deviation in titre resulting from these different methods, and how this deviation impacts the blood type antibody removal treatment (such as antibody reduction and immunomodulatory therapies, plasma exchange, double-filtration plasmapheresis, etc.) during the perioperative period of kidney transplantation. In this study, we tested the blood type antibody levels of 210 blood samples collected from 44 patients before/after ABO-I kidney allograft surgery from August 2020 to May 2022. We evaluated three antibody detection methods (TT, MGT, and GBT) systematically, with the aim of providing a practical reference for ABO-I kidney transplantation. We present this article in accordance with the MDAR reporting checklist (available at https://tau.amegroups.com/article/view/10.21037/tau-24-617/rc).
Methods
Reagents
Gel cards (Neutral cards, Beijing Strong Biotechnologies, China) and glass cards (Tianjing Dexiang Biotechnology Co., Ltd., China) were used for anti-A/B IgM antibody detection. Gel cards (utilize a column filled with a gel and AHG, Bio-Rad DiaMed, Cressier-FR-Switzerland) and glass cards (utilize a column containing minute glass beads and AHG, Ortho BioVue System Poly Cassette, Raritan, USA) were used for anti-A/B IgG antibody detection, in accordance with the manufacturer’s instructions. Anti-human globulin (AHG) reagent kits and 2-mercaptoethanol (2-ME) were obtained from Shanghai Hemo Pharmaceutical & Biological, Co., Ltd., China. 5% RBC suspensions of Group A and Group B were obtained from Shanghai Hemo Pharmaceutical & Biological, Co., Ltd., China. This study was performed using reagents authorised by the manufacturer of the original Conformité Européene (CE) marked product.
Sample
EDTA-anticoagulated samples were obtained from patients before or after ABO-I kidney transplantation in our centre (The First Affiliated Hospital of University of Science and Technology of China). Of the 210 samples included in this study, 70 were from patients with blood type A, 70 from type B, and 70 from type O (Table 1). Samples with an IgM titre of >512 were excluded because incomplete degradation of IgM could interfere with the IgG analysis. Characteristics of the patients are shown in Table 2. All samples were assessed by a single technician to reduce variance.
Table 1
| Blood type | All, n | Anti-A, n | Anti-B, n |
|---|---|---|---|
| A | 70 | 70 | |
| B | 70 | 70 | |
| O | 70 | 70 | 70 |
| Total | 210 | 140 | 140 |
Table 2
| Characteristics | Value |
|---|---|
| Age at transplantation (years) | 33.6±7.84* |
| Gender, n | |
| Male | 40 |
| Female | 4 |
| Liver doner | 100% |
| HLA mismatches | 2.3±1.5* |
| Recipient and donor blood group, n | |
| O (donor A/B/AB) | 18 |
| A (donor B/AB) | 14 |
| B (donor A/AB) | 12 |
*, mean ± standard deviation. HLA, human leukocyte antigens.
Sample preparation
Samples were first centrifuged to separate the blood cells and serum. Each serum sample was divided into two parts, one for anti-A/B IgM antibody detection and one for anti-A/B IgG antibody detection. The 0.3 mL of serum was serially two-fold diluted with 0.3 mL of 0.9% normal saline. The reaction for anti-A/B IgM antibody detection was carried out at room temperature, whereas that for anti-A/B IgG antibody detection was performed at 37 ℃. To ensure that the resulting titres corresponded to only the level of IgG antibodies present in the sample for the IgG test, IgM antibodies in the sample were inactivated by adding an equal volume 2-ME and incubation for 2 h at 37 ℃.
This study was approved by the Ethics Committee of The First Affiliated Hospital of University of Science and Technology of China (No. 202207281032000186014). Our hospital is a clinically research-oriented institution. Our research has obtained informed consent from all patients, and the study was conducted in accordance with the Declaration of Helsinki (as revised in 2013).
Titre determination via the TT
Anti-ABO IgM titres
Serial dilutions of each sample were prepared as described above, and 50 µL of 5% Group A or B RBC suspension was pipetted into the diluted serum (100 µL) in a glass tube, and then centrifuged for 15 s. After shaking the tube gently, the resulting agglutination was judged by observation within two minutes. The titre was set as the highest dilution showing 1+ agglutination.
Anti-ABO IgG titres
Serial dilutions of 2-ME pre-treated samples were prepared and mixed with RBC suspension as described above. The tubes were then incubated at 37 ℃ for 30 min. After antibody-bound RBCs in tubes were washed and centrifuged three times with 0.9% normal saline, 0.1 mL antiglobulin was then added into the tubes, and centrifuged for 15 s, observe and judge the generated agglutination within two minutes. The titre was set as the highest dilution showing 1+ agglutination.
Titre determination via the MGT and GBT
Anti-ABO IgM titres and IgG titres
A mixture of 100 µL of plasma dilution and 20 µL of 5% RBC suspension was pipetted into the card. The card was centrifuged for 15 s, after which the resulting agglutination was observed. Agglutination was graded from 0 to 4+ in accordance with the manufacturer’s guidelines. The titre was set as the highest dilution showing 1+ agglutination. For IgG detection, the samples were incubated at 37 ℃ for 20 min before centrifugation.
Interpretation of test results
For positive samples with detectable anti-A/B antibody levels, the agglutinated RBCs were trapped in or above the microtube gel or glass bead card after being bound by antibody. For negative samples lacking anti-A/B antibodies, the RBCs remained unagglutinated and moved through the microtube gel or glass beads to form a pellet at the bottom of the tube. The determination of red blood cell agglutination strength is shown in Table S1.
Statistical analysis
Mean anti-A/B antibody titres and their deviations according to each of the three detection methods were analysed by Kruskal-Wallis tests, and differences with a P value of <0.05 were considered statistically significant. Statistical tests were conducted using Graph Pad Prism 5.0.
Results
Titre distribution in the non-O blood group
Both IgG and IgM play important roles in the process of ABO-I kidney transplantation because both can activate complement and cause platelet aggregation and inflammation. Therefore, both antibody types are taken into consideration throughout a patient’s treatment. The distributions of the measured titres in the non-O blood group are shown in Figure 1. For the non-O blood group, the anti-A IgM titres in blood group B, as determined by the TT, ranged from 2 to 64, similar to the corresponding results for the MGT. Unexpectedly, the anti-A IgM titres determined by the GBT ranged from 8 to 512 (Figure 1A). A similar pattern of results was observed for blood type A, in which the anti-B IgM titres ranged from 1 to 64 according to the TT and MGT results but ranged from 4 to 512 according to the GBT results (Figure 1B).
To assess whether the pattern of IgM titres determined by the three methods is also applicable to the measurement of IgG titres, we performed a comparative analysis of the IgG titre results (Figure 1C,1D). The titres for IgG antibodies ranged from 1 to 32 for anti-A IgG in blood group B and for anti-B IgG in blood group A according to the TT. The titres measured by the MGT ranged from 2 to 64 in both blood groups A and B. In contrast with the IgM titre distributions, the IgG antibody titres according to the MGT were always higher than those according to the TT. All samples had higher titres according to the GBT compared with the other two methods.
Titre distribution in the O blood group
The main antibody type produced by individuals in group B or A was mainly IgM, while the type of anti-A and anti-B antibody in group O was mainly IgG. In our study, some of the specimens were from patients after transplantation. Therefore, low IgG antibodies were detected in O type specimens, while low levels of IgM antibodies were also detected in the non-O blood group. There were no differences in the titre steps for the different blood groups. For group O serum, the anti-A IgM titres ranged from 2 to 32, and the anti-B IgM titres ranged from 1 to 32 according to the TT results, thus comparable results were observed by the MGT. The IgM titres, as measured by the GBT, were significantly different from those measured by the TT and MGT for both anti-A antibody [4–256] (Figure 2A) and anti-B antibody [4–256] (Figure 2B). By contrast, the distributions of the IgG antibody titre obtained by different methods in the O blood group (Figure 2C,2D) were the same as those in the non-O blood group.
Overall, the IgM titre distributions measured by the TT and MGT were similar for all blood groups, while the IgM titres measured by the GBT were always higher than those determined by the other two methods. Different from the IgM titre distributions, the IgG titres measured by the MGT were higher than those measured by the TT. The GBT appears to overestimate anti-A/B antibody levels.
Deviation among different anti-A/B antibody detection methods according to patient blood group
To evaluate the differences among the three antibody detection methods, we conducted a statistical analysis of their results. Kruskal-Wallis tests were employed to derive a score measuring the deviation among these methods. For statistical comparisons, we converted all titre values to a log value (logarithm base 2 of the titre value). Table 3 summarises the mean score of the antibody titrations measured with the TT, MGT, and GBT. The mean antibody titration score of IgM titres in the non-O blood groups for the GBT was notably higher than those obtained with the TT and MGT [6.37±1.73 vs. 3.81±1.50 and 4.17±1.39 (P<0.001 for both) in blood group A, 6.24±1.47 vs. 3.77±1.22 and 4.04±1.39 (P<0.001 for both) in blood group B]. The same pattern was observed regarding IgG titres. The results obtained by the GBT were significantly higher than those obtained with the other two methods [5.24±1.63 vs. 2.86±1.29 for the TT and 3.56±1.46 for the MGT (P<0.001 for both) in blood group A, 5.67±1.41 vs. 3.06±1.33 for the TT and 4.11±1.31 for the MGT (P<0.001 for both) in blood group B]. No significant differences were found between IgM antibody titres as measured by the TT and MGT. However, a significant IgG titre disparity was observed between the TT and MGT results. Similarly, poor agreement in IgG antibody titre was found between the TT and GBT results, whereas there was a higher degree of agreement between IgM antibody titres from these two methods. The same pattern was also observed for the O blood group, as shown in Table 3. The statistical analysis results of three methods for detecting IgM and IgG are shown in Table 4.
Table 3
| Blood type | Methods | Anti-A | Anti-B | |||
|---|---|---|---|---|---|---|
| IgM | IgG | IgM | IgG | |||
| O | TT | 2.97±1.09 | 4.14±1.27 | 2.66±1.43 | 3.67±1.63 | |
| MGT | 3.30±1.21 | 5.15±1.31 | 2.93±1.60 | 4.60±1.68 | ||
| GBT | 5.39±1.45 | 6.91±1.22 | 5.16±1.63 | 6.36±1.86 | ||
| A | TT | 3.81±1.50 | 2.86±1.29 | |||
| MGT | 4.17±1.39 | 3.56±1.46 | ||||
| GBT | 6.37±1.73 | 5.24±1.63 | ||||
| B | TT | 3.77±1.22 | 3.06±1.33 | |||
| MGT | 4.04±1.39 | 4.11±1.31 | ||||
| GBT | 6.24±1.47 | 5.67±1.41 | ||||
The mean anti-A and anti-B antibody titres determined using the three methods according to the patient’s blood group. GBT, glass bead card test; MGT, microcolumn gel card test; IgG, immunoglobulin G; IgM, immunoglobulin M; SD, standard deviation; TT, tube test.
Table 4
| Antibody | Comparing methods | P | 95% CI |
|---|---|---|---|
| IgM | TT-MGT | 0.01 | −0.541 to 0.059 |
| TT-GBT | <0.001 | −2.759 to −2.241 | |
| MGT-GBT | <0.001 | −2.466 to −1.934 | |
| IgG | TT-MGT | <0.001 | −1.243 to −0.737 |
| TT-GBT | <0.001 | −2.923 to −2.397 | |
| MGT-GBT | <0.001 | −1.937 to −1.403 |
CI, confidence interval; GBT, glass bead card test; IgG, immunoglobulin G; IgM, immunoglobulin M; MGT, microcolumn gel card test; TT, tube test.
Comparison of anti-A/B antibody titres among the three detection methods
An acceptable blood type antibody baseline is the basis of successful ABO-I kidney transplantation; however, it is important to choose an appropriate method for antibody titre determination when setting this baseline. To further evaluate the variance between the antibody titre results obtained by the three methods, we compared all log values between methods. A comparison of the variance of the MGT results with those of the TT results for IgM titres in the non-O blood group showed that almost all the variance among the results was within ±1 dilution; furthermore, 41 (58.6%) of the 70 samples in blood group A and 45 (64.2%) of the 70 samples in blood group B had identical titres (Figure 3A,3B).
However, for IgG antibody, we found that about 87.1% and 97.1% of the titre determined by the MGT was higher than that determined by the TT in blood group A and blood group B, respectively; among these samples, 58 (82.8%) in blood group A and 62 (88.5%) in group B had a titre of one dilution higher, and a few samples had a titre of two or three dilutions higher than the TT titres (Figure 3C,3D). Regarding O blood group, the same conclusion was drawn. More than half of the samples had identical titres according to the MGT and TT (Figure 4A,4B). The difference between the two detection methods was also applicable to type O blood samples (Figure 4C,4D). Therefore, we can conclude that the IgM titres determined by these two methods show high consistency regardless of blood type.
We also compared the results obtained by the GBT and TT, and found that there was a 1–4 serial dilution step difference between the IgM titres obtained by the GBT and TT in all blood groups, and almost half of the samples differed by three dilution steps. A similar pattern was observed regarding the IgG titres. All titres determined using the GBT varied more than one standard dilution compared with those determined using the TT. In summary, our results emphasise the importance of the choice of method for titre determination.
Clinical application of different detection methods
Continuous monitoring of preoperative antibodies can assess the efficacy of the pretreatment regimen while also providing a safe antibody titre for surgery. In our centre, the TT was initially used for titre analysis and the results were reported to clinicians, while the GBT results were used as a laboratory reference. In this context, we reviewed two cases of delayed graft function and increased creatinine level after renal transplantation, which were both reported with safe anti-A/B antibody levels before surgery based on the TT (Table 5). Case 1 was a 23-year-old male with the O blood type, and the donor was of blood type A. The post-operation anti-A IgM and IgG titres were 1:8 and 1:4 (TT), respectively. However, the creatinine level increased from 204 to 378. The titre was thus immediately analysed by the TT and MGT, and the results showed that although the anti-A IgM titre determined by the TT remained at a safe threshold (1:16), the MGT revealed that the titre was elevated to 1:32 (Figure 5A). Three rounds of therapeutic plasmapheresis were subsequently implemented, and the anti-A IgM titres, as measured by both the TT and MGT, dropped down to a safe level (1:4 and 1:8); as expected, the creatinine level decreased to 204. During this whole process, the anti-A IgG level was maintained at a stable level (<1:16). A similar situation occurred with case 2. The recipient was a 32-year-old male of blood type B and the donor was blood type A. The operation was performed for these recipients after blood type antibodies reached a safe range, with a titre less than 1:16. Figure 5B shows the creatinine and titre monitoring data. It was observed that the IgM antibody titre obtained by the manual method remained within the safe range (≤16), while the titre obtained by the MGT exceeded the safe value (1:16). After therapeutic plasmapheresis, the titre returned to a safe level and the patient showed a greater reduction in their creatinine level. Our case demonstrates the limitations of traditional manual methods, which may be less sensitive than the MGT method in monitoring postoperative titre changes. This also indicates that the MGT method is more reliable and could replace, or at least be used as a reference for, the traditional TT method of titre measurement, to prevent graft rejection and delayed graft function caused by postoperative titre rebound.
Table 5
| Characteristics | Patient 1 | Patient 2 |
|---|---|---|
| Age (years) | 23 | 33 |
| Gender | Male | Male |
| Recipient and donor blood group | O(A) | A(B) |
| HLA mismatches | 3 | 3 |
| DSA | Negative | Negative |
| PE times after surgery | 3 | 2 |
DSA, donor specific antibody; HLA, human leukocyte antigens; PE, plasma exchange.
Discussion
The answer to the question of whether IgM or IgG plays critical in the rejection of an ABO-I kidney transplantation remains controversial. Some reports documented that anti-ABO IgM binds more avidly compared with IgG, and that patients with high anti-ABO IgG titres have similarly outcomes comparable to those who have low anti-ABO IgG titres. Furthermore, ABO-I kidney transplantation surgery can be successfully performed by reducing IgM titre to 4 or less, regardless of IgG titre (16). However, this view was opposed by some other researchers who state that IgG titre play a significant role in immunoreaction after ABO-I kidney transplantation, while IgM titres do not affect clinical decisions (17). Therefore, IgG titres are thought to be critical for assessing patients’ condition before and after organ transplantation, and plasma exchange treatment should be performed when anti-ABO antibody titre at the AHG phase is higher than 16 before surgery (8). In spite of these point of view, we consider both anti-ABO IgM and IgG titres are important throughout a patient’s treatment, and surgery will not be performed till both titres are less than 16.
The TT is a long-established method to determine anti-ABO antibody titres; however, several experimental factors, including temperature, equipment, and reagents such as antihuman globulin, may influence the outcome accuracy. The results obtained by the TT usually vary according to the operator; for example, a report indicated that the titre may vary from 8- to 64-fold, depending on differences in the preparation procedure and data interpretation across laboratories (20). Despite this, the TT method is still widely used because of its relatively acceptable speed and low cost (21). Here, we compared the anti-A/B antibody titres determined by three different methods and found a good correlation between the IgM titres assessed by the MGT and TT. However, our data indicated that the IgG titres determined by the MGT were always one standard dilution higher than those determined by the TT across all ABO blood groups; this finding is similar to those reported by other institutions (22,23). One possible reason for this difference in outcomes regarding IgG titre detection between the two methods may be the elimination of a wash step in the MGT.
A low level of anti-ABO antibody is the established prerequisite for ABO-I kidney transplantation (24-26). The acceptable antibody level prior to surgery is inconsistent between transplantation centres. Our previous retrospective analysis suggested that patients with a TT titration less than 16 were suitable for ABO-I kidney transplantation. The present work indicates that the MGT is an acceptable method for monitoring the anti-A/B antibody titre due to its reliability, reproducibility, and stability. Notably, the MGT method offers the advantages of ease of use, high reproducibility, and simple reporting. Based on these advantages, the use of the MGT may reduce intra- and inter-laboratory variations in anti-A/B titre measurements. In addition, the test period can be shortened using the MGT method.
The anti-A/B titres in samples taken at the room temperature phase (IgM) and the AHG phase (IgG) were always higher when measured by the GBT method compared with the other two methods, being 2–4 serial dilution steps higher than those measured by the TT. GBT is a method based on column agglutination technology, and the high sensitivity of the card method often leads to a high experimental titre (27,28).
The detection of antibody titre is difficult to standardise and it is therefore challenging to determine an optimum antibody titre for ABO-I kidney transplantation. However, choosing reliable and standardised testing methods is the goal of transplantation centres. A safe and reliable titre of antibody is closely associated with the outcome and prognosis of clinical transplantation. Our study highlights several limitations that should be noted. First, the interpretation of the TT method was mainly based on human visual observations, with the degree of agglutination being judged by the naked eye. The TT method involves more experimental steps compared with the card test, particularly in the identification of IgG antibodies, which also results in the TT detecting the lowest titres among the three methods analysed. In the absence of any reagents that may improve the sensitivity of the MGT for IgM detection, we found that the results obtained by the MGT did not differ significantly from those obtained by the manual TT method. The addition of a reaction enhancer in the GBT increased the reactivity between ABO blood type antigen and antibody, thus elevating the sensitivity. We propose using MGT to check the patient’s titre after surgery because it is more sensitive than the usual manual method and can detect titre changes more accurately. We compared the results obtained from another kidney transplantation centre using different methods, and found that the titres in our laboratory differed at no more than one stage for the same samples and titration procedure, indicating that the same trends were observed among the three methods.
Previously, studies on ABO-I kidney transplant antibody titre were more simply focusing on laboratory results, while rarely referred to the clinical situation; in our study, we found that MGT method could reduce the inter-laboratory difference, especially in detecting IgG antibody titres. Importantly, there is very limited literature in China on the study of antibody titre of kidney transplantation. Our hospital, a pioneer in ABO incompatible kidney transplantation in China, tops the nation in cross-blood living donor kidney transplants amongst family members. Our institution has the third-highest rate of living donor kidney transplantation in China, making the antibody detection techniques used by our institution a valuable domestic and even international resource. However, our study did not conduct experiments on samples with high IgM titres. Such samples were excluded because any remaining IgM that was not inactivated by 2-ME treatment would greatly affect the accuracy of the IgG titre. This is especially important for samples from patients in blood groups A and B, which mostly consist of IgM antibodies. Dithiothreitol is also used to inactivate IgM in some laboratories; however, it is reported that it may destroy IgG (29). Therefore, further research is still needed for those samples with ultra-high anti-A/B antibody titres.
Conclusions
In conclusion, our study indicates that titre measurements are mainly dependent upon the technology employed during the testing procedure. Among the three methods reported here, the GBT had the highest sensitivity, caution should be exercised when using GBT to determine transplant eligibility criteria, which may allow the cut-off titre to be set to a more secure level for transplantation, but at the same time, it will prolong the elimination time of patient antibody titres. Our results demonstrate that it is practical to adopt the MGT for anti-A/B titre testing. As clinicians usually focus only on the target titre without taking into account the approach used for the measurement, it is important for clinical staff and laboratory staff to determine the safe limits of the blood-type antibody titres according to the technology used throughout the whole perioperative period.
Acknowledgments
The authors acknowledge financial support from the China Postdoctoral Science Foundation (No. 2020M671889) and the Natural Science Foundation of Anhui Province of China (No. 1908085MH247).
Footnote
Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://tau.amegroups.com/article/view/10.21037/tau-24-617/rc
Data Sharing Statement: Available at https://tau.amegroups.com/article/view/10.21037/tau-24-617/dss
Peer Review File: Available at https://tau.amegroups.com/article/view/10.21037/tau-24-617/prf
Funding: This work was supported by
Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tau.amegroups.com/article/view/10.21037/tau-24-617/coif). The authors have no conflicts of interest to declare.
Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This study was approved by the Ethics Committee of The First Affiliated Hospital of University of Science and Technology of China (No. 202207281032000186014). Our research has obtained informed consent from all patients, and the study was conducted in accordance with the Declaration of Helsinki (as revised in 2013).
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References
- Ishida H, Kondo T, Shimizu T, et al. Postoperative rebound of antiblood type antibodies and antibody-mediated rejection after ABO-incompatible living-related kidney transplantation. Transpl Int 2015;28:286-96. [Crossref] [PubMed]
- Morozumi K, Takeda A, Otsuka Y, et al. Reviewing the pathogenesis of antibody-mediated rejection and renal graft pathology after kidney transplantation. Nephrology (Carlton) 2016;21:4-8. [Crossref] [PubMed]
- Naik RH, Shawar SH. Renal Transplantation Rejection. Treasure Island (FL): StatPearls Publishing; 2025.
- Justiz Vaillant AA, Mohseni M. Chronic Transplantation Rejection. Treasure Island (FL): StatPearls Publishing; 2025.
- Kahwaji J, Vo AA, Jordan SC. ABO blood group incompatibility: a diminishing barrier to successful kidney transplantation? Expert Rev Clin Immunol 2010;6:893-900. [Crossref] [PubMed]
- Cervantes CE, Bloch EM, Sperati CJ. Therapeutic Plasma Exchange: Core Curriculum 2023. Am J Kidney Dis 2023;81:475-92. [Crossref] [PubMed]
- Chen YY, Sun X, Huang W, et al. Therapeutic apheresis in kidney diseases: an updated review. Ren Fail 2022;44:842-57. [Crossref] [PubMed]
- Tobian AA, Shirey RS, Montgomery RA, et al. ABO antibody titer and risk of antibody-mediated rejection in ABO-incompatible renal transplantation. Am J Transplant 2010;10:1247-53. [Crossref] [PubMed]
- Cen M, Wang R, Kong W, et al. ABO-incompatible living kidney transplantation. Clin Transplant 2020;34:e14050. [Crossref] [PubMed]
- Bentall A, R, Barnett AN, Braitch M, et al. Clinical outcomes with ABO antibody titer variability in a multicenter study of ABO-incompatible kidney transplantation in the United Kingdom. Transfusion 2016;56:2668-79. [Crossref] [PubMed]
- Kang SJ, Lim YA, Baik SY. Comparison of ABO antibody titers on the basis of the antibody detection method used. Ann Lab Med 2014;34:300-6. [Crossref] [PubMed]
- Lapierre Y, Rigal D, Adam J, et al. The gel test: a new way to detect red cell antigen-antibody reactions. Transfusion 1990;30:109-13. [Crossref] [PubMed]
- Schneider D, Vicarioto M, Coluzzi S, et al. ABO antibody titres: a multisite comparative study of equivalency and reproducibility for automated solid-phase and haemagglutination titration, and manual dilution with gel column agglutination technology. Blood Transfus 2022;20:329-37. [PubMed]
- Datta SS, Basu S, Reddy M, et al. Comparative evaluation of the conventional tube test and column agglutination technology for ABO antibody titration in healthy individuals: a report from India. Immunohematology 2021;37:25-32. [Crossref] [PubMed]
- Wu S, Wu Y, Guo G, et al. Comparison of the Detection Rate and Specificity of Irregular Red Blood Cell Antibodies Between First-Time Pregnant Women and Women With a History of Multiple Pregnancies Among 18,010 Chinese Women. J Pregnancy 2024;2024:5539776. [Crossref] [PubMed]
- Flint SM, Walker RG, Hogan C, et al. Successful ABO-incompatible kidney transplantation with antibody removal and standard immunosuppression. Am J Transplant 2011;11:1016-24. [Crossref] [PubMed]
- Lonze BE, Bae S, Kraus ES, et al. Outcomes and risk stratification for late antibody-mediated rejection in recipients of ABO-incompatible kidney transplants: a retrospective study. Transpl Int 2017;30:874-83. [Crossref] [PubMed]
- Kumlien G, Wilpert J, Säfwenberg J, et al. Comparing the tube and gel techniques for ABO antibody titration, as performed in three European centers. Transplantation 2007;84:S17-9. [Crossref] [PubMed]
- Tydén G, Donauer J, Wadström J, et al. Implementation of a Protocol for ABO-incompatible kidney transplantation--a three-center experience with 60 consecutive transplantations. Transplantation 2007;83:1153-5. [Crossref] [PubMed]
- AuBuchon JP, de Wildt-Eggen J, Dumont LJ, et al. Reducing the variation in performance of antibody titrations. Arch Pathol Lab Med 2008;132:1194-201. [Crossref] [PubMed]
- Nayak S, Makroo RN, Prakash B, et al. Comparative Evaluation of Five Different Methods of Anti-ABO Antibody Titration: An Aid for ABO-Incompatible Organ Transplants. Ther Apher Dial 2019;23:86-91. [Crossref] [PubMed]
- Flesiopoulou I, Pouliakis A, Politou M, et al. Red Blood Cell Alloantibody Titration - Does the Titration Method Matter? Clin Lab 2020; [Crossref] [PubMed]
- Shirey RS, Cai W, Montgomery RA, et al. Streamlining ABO antibody titrations for monitoring ABO-incompatible kidney transplants. Transfusion 2010;50:631-4. [Crossref] [PubMed]
- Mohamed M, Sweeney T, Alkhader D, et al. ABO incompatibility in renal transplantation. World J Transplant 2021;11:388-99. [Crossref] [PubMed]
- Xu P, Zhao N, Wang J. Success rate and safety of living donor kidney transplantation in ABO blood group incompatible relatives: A systematic review and meta-analysis. Transpl Immunol 2023;81:101921. [Crossref] [PubMed]
- Kim H, Choe W, Shin S, et al. ABO-incompatible kidney transplantation can be successfully conducted by monitoring IgM isoagglutinin titers during desensitization. Transfusion 2020;60:598-606. [Crossref] [PubMed]
- Charong N, Kooltheat N, Plyduang T. High-sensitivity detection of clinically significant red blood cell antibodies by the column agglutination technique. Adv Clin Exp Med 2021;30:1205-14. [Crossref] [PubMed]
- Sigdel A, Chalise G, Bolideei M, et al. Comparison between the Manual Method of Indirect Coombs via Gel Technology and Solid Phase Red Cell Adherence. Maedica (Bucur) 2021;16:200-6. [Crossref] [PubMed]
- Knight RC. Measuring IgG anti-A/B titres using dithiothreitol (DTT). J Clin Pathol 1978;31:283-7. [Crossref] [PubMed]
