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Comparative analysis of Luminex-based donor-specific antibody mean fluorescence intensity values with complement-dependent cytotoxicity & flow crossmatch results in live donor renal transplantation

Background & objectives: Antibodies specific to donor human leucocyte antigen (HLA) play a critical role in graft rejection and graft loss. In recent years, techniques for their detection have evolved significantly providing an ever-increasing degree of sensitivity and specificity, from the conventional cell-based assays to the advanced solid-phase system based on the Luminex platform. Consensus is still evolving on the routine employment of all these methods, either stand alone or in combination. The objective of this study was to explore the near-accurate mean fluorescence intensity (MFI) cut-off values detected on Luminex platform predicting the strength of cell-based crossmatch results.

Antibodies against the human leucocyte antigen (HLA) play a major role in the causation of antibody-mediated rejection and graft loss, thus making it a critical barrier for solid organ transplantation [1][2][3] . Almost 60 years have passed since the introduction of complement-dependent cytotoxicity (CDC) as the first technique for the detection of HLA antibodies in recipients before undergoing renal transplantation 4 . Since then, methodologies to detect HLA antibodies have progressed from purely target donor cell-based assays, such as CDC or flow cytometry to the more sensitive and specific HLA protein-based solidphase assay systems in the form of an enzyme-linked immunosorbent assay (ELISA) or HLA antigen-coated fluorescence bead assay based on a Luminex platform 5

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In the latter technique, the mean fluorescence intensity (MFI) is a measure of the degree of saturation of total antigens present on the beads by antibodies and is used as a surrogate marker for the level of antibody titres. Currently, there is a lack of consensus with regard to the optimum MFI cut-offs for classifying antibodies as positive or those that are significant.
Consensus is still evolving on the routine employment of all these methods either stand alone or together and the clinical relevance of antibodies detected by each of these. Several studies have focused on defining cut-off values for donor-specific antibodies (DSA) which can predict the CDC and flow crossmatch (FCXM) results to a reasonable degree of accuracy [6][7][8][9][10][11] . However, the MFI values of such a cut-off have differed in different studies and seem to be centre/laboratory specific.
The objective of this study was to actuate the MFI values of DSA detected by single antigen bead (SAB) assay to that of cell-based CDC and flow crossmatch results. This is perhaps the first Indian study to assess the MFI cut-off values of Luminex-based single antigen DSA that can accurately predict the strength of cell-based CDC and FCXM results.

Material & Methods
A total of 116 consecutive prospective ABO-compatible primary renal transplant recipients who reported for pre-transplant workup at the department of Transplant Immunology and Immunogenetics, All India Institute of Medical Sciences (AIIMS), New Delhi, between May 2012 and July 2014, were included in the study. All patients underwent antibody detection by three methods: (i) CDC crossmatch, (ii) flow cytometry crossmatch, and (iii) HLA-DSA detection using solid-phase immunoassay based on a Luminex platform. The study was approved by the Institutional Ethics Committee, and written informed consent was obtained from all recipients and donors.
Complement-dependent cytotoxicity (CDC) crossmatch: T-cell CDC crossmatch assay was performed using the standard two-stage National Institute of Health (NIH) technique 12 , and a score of ≥4 was considered positive. Human leucocyte antigen (HLA)-donor-specific antibody (DSA) detection using solid-phase immunoassay (SPI): Serum samples from the recipients were analyzed for Class I and Class II IgG HLA antibodies using the commercially available LABScreen SAB assay kit (One Lambda, Inc., Canoga Park, CA, USA) on a Luminex platform (Bio-Plex 200, Bio-Rad Laboratories, USA) 14 . The procedure was performed according to the manufacturer's instructions, and samples were analyzed using Luminex 100 IS v 2.3 software (Luminex Corporation, USA) for data acquisition. Data analysis was done with HLA Fusion software (One Lambda, Inc.). Results were interpreted using raw MFI values.
HLA typing: Low-resolution HLA typing of all recipients and donors included in this study was performed for A, B and DR locus alleles using the polymerase chain reaction-sequence-specific primer (PCR-SSP) method 15 .
Statistical analysis: For categorical data, Chi-square test was applied and odds square with 95 per cent confidence interval (CI) and P value were calculated. Receiver operating characteristic (ROC) analysis was performed, ROC curve was plotted between the true-positive fraction (sensitivity) against the falsepositive fraction (1-specificity) at various MFI values for Class I DSA and area under the curve (AUC) was calculated. For CDC and FCXMs, classifiers were simply a positive or negative crossmatch result. All calculations were performed using SPSS for windows, version 20.0 (SPSS Inc. Chicago, USA).

Results
The outcome of CDC crossmatch, FCXM and HLA-DSA results by SAB performed on all 116 recipients included in this study are presented in     remaining seven (63.64%) were negative. However, in the group with DSA MFI >7000, nine (90%) of the 10 patients were CDC positive and only one was negative. This negative patient had MFI value of 9986. When SAB-C1q assay was performed on the serum sample of this patient, it was found to be negative indicating the presence of non-complement-fixing antibodies.
It was also found that MFI values were not homogeneous with respect to CDC crossmatch results (Chi square=21.07, df=3, P<0.001, Yates' P<0.001 and Fisher's exact P≤0.001) ( Table III). Since the values in some of the cells were extremely low (<1), the data were compressed in two categories with a MFI cut-off value of 7000 (  (Fig. 3B). MFI cut-off of 4644 exhibited a diagnostic sensitivity of 76.9 per cent and specificity of 91.3 per cent for predicting positive CDC crossmatch. Sensitivity decreased to 69.2 per cent each for MFI cut-off value of 4886 and 7507 while the specificity remained same for MFI cut-off of 4886 and increased to 95.7 per cent at MFI of 7507. None of the patients had DSA MFI between 4886 and 7507.

Discussion
This study was carried out with the main aim of correlating Luminex-based SAB-MFI values with that of cell-based assays so as to determine the actuarial MFI cut-off value above which the CDC and FCXM would predictably be positive. An attempt was made to explore the correlation of HLA Class I DSA detected by Luminex-based SAB with the IgG T-cell FCXM and CDC crossmatch results. We did not include B-cell FCXM as its results were influenced by both Class I and II HLA-DSA. Moreover, B-cell crossmatch is prone to false positivity due to non-specific binding of immunoglobulins to Fc receptors. Data on IgM crossmatch were not considered since Luminex-based assay detects only IgG antibodies.
Our results indicated that Luminex-based single antigen MFI cut-off of 3000 significantly correlated with FCXM positivity while a MFI value of 7000 and Table III   above was predictive of a positive CDC crossmatch. Although the numbers were small, an almost 100 per cent concordance reached between the two techniques i.e., CDC and Luminex-based SAB for the MFI values <3000 and >7000. Our results were consistent with those of others 10,16 . In fact, the latter group of investigators reported that in their experience, all CDC-positive crossmatches had a MFI value of >6000, but not all serum samples with high MFI were positive for CDC. They attributed this finding to the possible presence of non-complement-fixing antibodies. In our study also a similar case was seen in whom the CDC crossmatch was negative despite having DSA with MFI value of 9986. We found this to be due to the presence of non-complement-fixing antibodies by performing Luminex-based C1q assay for the detection of complement-fixing anti-HLA antibodies (C1qScreen, One Lambda). In the study by Ellis et al 16 , no such test was performed to confirm the presence of non-complement-fixing nature of antibodies.
The MFI cut-off value for FCXM in our study was much lower than those reported in earlier studies 9,11 . For example, Moreno et al 11 demonstrated a cut-off MFI value of 6500 for predicting a positive FCXM result with a sensitivity of 85 per cent and specificity of 82 per cent. This could be due to the differences in the MCS defined for positive T-cell FCXM which was taken as 25 or more in our laboratory as compared to 40 in the earlier two studies. Moreover, the cumulative effect of antibodies against more than one antigen was possible, and this could have influenced the MFI values to be lower as observed in our study. Lobashevsky 17 reported unpublished data suggesting a positive predictive value of 97 per cent for a MFI cut-off of 2600 and T-cell-positive FCXM results. This was in line with our cut-off value of 3000 with a positive predictive value of 95.2 per cent. However, in this study 17 , the details on cut-off values for MCS for positive T-cell FCXM have not been given.
In our study there were two cases in whom the DSA was not identified by Luminex-SAB assay while the flow-based crossmatch for IgG T-and B-cell was found to be positive. This could be due to the presence of non-HLA antibodies. In one of the cases, the presence of DSA against major histocompatibility complex Class I-related chain A (MICA) antigen have been found (data not shown). It is presumed that the observed FCXM positivity could well be because of the presence of MICA antibodies. It is known that the expression of MICA molecule can be induced on activated lymphocytes 18 .
Our study had some limitations, the main being that we included participants having DSA against more than one Class I antigen and this cumulative effect of different antigen-antibody interaction could have affected the MFI cut-off value and thus the correlation with the strength of cell-based crossmatch. Another limitation was non-inclusion of HLA-C antigens in the study for the determination of Class I DSA. This is because currently, typing for HLA-C locus is not a part of the donor selection criteria under the renal transplantation protocol 19 and hence our cut-off applies to anti-HLA Class I antibodies limited to HLA-A and HLA-B loci only.
To conclude, our study correlated MFI values detected by Luminex-based single antigen with the strength of CDC and flow-based crossmatch results. Only when data from other centres and larger cohorts become available, it will be possible to explore the nearaccurate MFI cut-off values predicting the strength of cell-based crossmatch results. The MFI cut-off values of determining donor-specific anti-HLA antibodies on Luminex platform could serve as a surrogate marker for CDC and FCXM test results and help in resolving the limitations of different cell-based techniques.