AIM:Detection of anti-HLA antibodies is crucial for pre-transplant histocompatibility testing, donor selection, and graft survival. The aim of this study was to evaluate the spectrum of anti-HLA antibodies among live related renal transplant recipients from one of the largest transplant centers in north India. METHODS:In this study, retrospective data of transplant workup done in past four years were analyzed using GraphPad Prism 9 Version 9.2.0 (La Jolla, CA, USA). All samples received for pre-transplant work-up if showed positive screening results underwent Luminex single antigen bead (L-SAB) assay. Antibodies identified on L-SAB were evaluated for their specificity and the strength of their mean florescence intensity (MFI). RESULTS:A total of 1250 renal transplant samples were included for analysis. Out of these, 458 (36.64 %) samples were found positive in screening tests algorithm. All 458 samples were further analyzed for both class I and class II HLA antibodies by Luminex single antigen bead (L-SAB) assay. In this study, we observed that anti-A*24:01, anti-B*15:01 and anti-C*07*01 were the three most prevalent anti-HLA antibodies identified against HLA-class I antigens. However, anti-DRB1*11:01, anti-DQA1*05:01-DQB1*03:01 and anti-DPA1*02:01-DPB1*17:01 were the most common anti-HLA antibodies identified against HLA-class II antigen. Furthermore, our study found a significant association between anti-HLA class I antibody and the history of pregnancy. However, in re-transplant cases, we observed the presence of antibodies both against HLA class I and II antigens. CONCLUSION:For a transplant center, it is of utmost importance to have comprehensive knowledge about the prevalence of HLA antibodies, their MFI, and their association with various sensitization events. This study may immensely help transplant communities in selecting appropriate prospective organ donors, planning desensitization regimes, managing recipients' care and predicting transplant outcomes in live related renal transplantation.

BACKGROUND:In this retrospective study, our aim was to find the effect of leucodepleted (LD) blood transfusions on the formation of anti-HLA-antibodies when compared to non-leucodepleted (non-LD) transfusions using Luminex-based method. METHODS:In this study, Luminex single antigen bead assay (L-SAB) and HLA typing were performed on 310 patients. Test positivity rates (as MFI - Mean florescence intensity) were analyzed according to the different sensitization events and gender. RESULTS:Of the 310 patients included in the study, 58.06% (180) patients were male and 41.93% (130) were female. The average age of the patients was 42.86 (±12.37) years. In this study, test positivity rates were significantly lower in the patients who received LD RBC units than in those who received non-LD RBC units (28.43% = 29 of 102 Vs 55.22% = 74 of 134, p < 0.05). In our study, transfusion combined with a history of pregnancy had higher number of significant HLA antibodies compared to cases where transfusion was the only sensitization event (81.81% = 18/22 Vs 39.71% = 85/214, p < 0.05). In addition, anti-HLA-antibodies-MFI were significantly (p < 0.01) higher in non-LD patients compared to LD patients. CONCLUSION:Patients who received LD RBC units had a significantly lower rate of transfusion-associated alloimmunization compared to those who received non-LD RBC units. Multiparous women had a high risk for transfusion-related alloimmunization compared to both nulliparous women and male patient. Furthermore, class I-anti-HLA-antibodies (HLA-B and HLA-A + B) were significantly associated with pregnancy sensitization and/or blood transfusion as a single sensitization.

Background: The main objective of this study was to determine the results of the cell-based assay (CDC-XM and FC-XM), and correlate with the results of solid phase assay (L-SAB).Methods: In this retrospective study, 350 prospective renal transplant recipients were tested for the presence of HLA antibodies by CDC-XM, FC-XM and L-SAB screening with their corresponding donor.Results: T-cell-FC-XM showed a sensitivity of 71.43% and a specificity of 91.50% for detecting class I L-SAB (+), while B-cell-FCXM showed a sensitivity of 94.94% and a specificity of 61.99% for detecting class II L-SAB (+). On the other hand, T-CDC-XM showed a sensitivity of 32.14% and a specificity of 98.64% for detecting class I L-SAB (+), while B-CDC-XM showed a sensitivity of 44.30% and a specificity of 94.83% for detecting class II L-SAB (+). In this study, the results indicated that DSA class I MFI value of 2845 and above significantly (p <0.001) correlated with T-cell-FC-XM positivity, while MFI value of 4585 and above (p <0.001) showed strong predictive accuracy of a positive T-cell-CDC-XM. However, DSA class II MFI cut-off of 1988 and above significantly (p <0.001) correlated with B-cell-FC-XM positivity, while MFI value of 5986 and above (p <0.001) showed strong predictive accuracy of a positive B-cell-CDC-XM.Conclusions: Our study showed that CDC-XM has poor sensitivity, while FC-XM has poor specificity to detect DSA. L-SAB has good correlation with T-cell-FC-XM (p < 0.0001) but not with B-cell-FC-XM (P = 0.31). DSA strength >2845 and > 1988 significantly correlated with T-cell-FC-XM positivity and B-cell-FC-XM positivity, respec-tively. While, a MFI value of >4585 and > 5986 significantly correlated with T-cell-CDC-XM positivity and B-cell-CDC-XM positivity, respectively. These MFI cut-off values could serve as a surrogate marker for CDC-XM and FC-XM tests and may help in resolving the limitations of cell-based techniques. In conclusion, we found that L-SAB is more sensitive and specific than CDC-XM and FC-XM and therefore may be used as a test of choice.

Introduction: Cell-based complement-dependent cytotoxicity crossmatch (CDC-XM) and solid phase assays were introduced for assessing HLA antibodies. However, the complexity of data from cell-based and solid phase assays have led to potential confusion about how to use the results for clinical decision making. Aim: Aim of this study was to compare results of cell-based assay and solid phase assay, to evaluate the usefulness of L-XM for pretransplant detection of HLA class I and II donor-specific IgG antibodies, correlate the mean fluorescence intensity (MFI) values of class I and class II L-XM assay and with CDC-XM and L-PRA (panel reactive antibodies) results. Methods: In this retrospective study, 380 prospective renal transplant recipients were tested for the presence of HLA antibodies by CDC-XM, IgG-L-XM, IgG-L-PRA & L-SAB screening with their corresponding donor. Results: Fifty-one recipients (13.42%) had a positive CDC-XM. L-XM was positive in 125 recipients (32.89%); class I-L-XM was positive in 46 (36.80%) cases, and class II-L-XM was positive in 58 (46.4%) cases and 21 (16.8%) samples were positive for class I and class II. High background was present in 22 (5.87%) samples, the results of which were confirmed by retesting or by correlation with L-PRA and L-SAB assays. Conclusion: The introduction of more sensitive approaches for the detection of anti-HLA-IgG-antibodies, such as L-XM and L-PRA assay, has allowed the identification of anti-HLA-antibodies in recipient serum which is not usually identified by CDC-XM alone. However, L-XM has some limitations; they can be overcome if we combine this assay with L-PRA.

BACKGROUND:Patients awaiting solid organ transplantation may develop anti-HLA antibodies after sensitization events such as transfusions, pregnancies, or previous transplantations. However, the effects of a particular sensitization event on HLA alloimmunization have not been well studied in parallel using cell-based assays and solid-phase assays. In this study, we evaluated and compare how different sensitization events affect the HLA antibody screening (HLA-Ab) and donor specific antibody (DSA) status in solid renal organ transplantation patients.METHODS:HLA antibody (HLA-Ab) screening tests like complement-dependent cytotoxicity crossmatch (CDC-XM), flow cytometry crossmatch (FC-XM) and Luminex panel-reactive antibody (L-PRA) were performed in all 1066 patients (635 males and 431 females). If any of these tests turned out to be positive, a Luminex single antigen bead (L-SAB) assay was performed for DSA identification. Test positive rates and antibody strengths were analyzed according to the different sensitization events and gender.RESULTS:In this study, HLA-Ab screening tests positive rates (L-PRA, FC-XM and CDC-XM) were significantly higher in patients with previous transplantation (73.91%, 100% and 56.52% p < 0.001), previous pregnancy (57.46%, 70.14% and 18.85% p < 0.001) or blood transfusion (27.33%, 35.55% and 7.33% p < 0.001) compared with patients without a sensitizing event (6.17%, 13.58% and 1.09). In this study, re-transplantation group showed significantly stronger antibody strength (DSA) than non sensitized group (class I and II MFI 11418.04, 17,837.78 vs class I and II MFI 2659, 3329; P < 0.001) and those with single sensitization events of transfusion (class I and II MFI 11418.04, 17,837.78 vs class I and II MFI 5790.26, 6004.16; P < 0.001) or pregnancy (class I & II MFI 11418, 17,837 vs class I and II MFI 8631.71, 7253.29; P < 0.001).CONCLUSIONS:Pregnancy and blood transfused had high allo-immunization rate for class I HLA antigens. While re-transplantation patients had high allo-immunization rate for both the HLA classes (HLA- class I and HLA- class II). Re-transplantation group showed significantly stronger antibody strength, followed by pregnancy and then transfusion.

Advances in immune suppression therapies and desensitization have made possible kidney transplantation regardless of HLA incompatibility. Single antigen bead assay (SAB) is a semi-quantitative estimation of the amount of human leukocyte antigen (HLA) antibodies present in the recipient plasma, and mean fluorescence intensity (MFI) generated gives this rough estimation of the antibodies present in the recipient. Here we present a case of successful kidney transplantation in a patient who expressed DSA with high MFI. A 33-yr-old male, diagnosed with chronic kidney disease (CKD) on regular maintenance hemodialysis, opted for second kidney transplant with his sibling as prospective donor and was referred to the department of Transplant Immunology for histocompatibility testing. Patient had HLA incompatibility with multiple DSA identified by SAB. Patient undergone 20 sessions of plasma exchange till discharge and finally till 6 months graft was functioning well. The authors thus conclude that the option of a high-risk HLA incompatible kidney transplant can be offered to recipients with high MFI DSA, who wish to undergo transplantation for end stage renal disease.

Objective: The objective of the study is to assess the graft and patient outcome after desensitization in human leukocyte antigen incompatible kidney transplantation (KT) with positive baseline complement-dependent cytotoxic (CDC) crossmatch and high mean fluorescein intensity (MFI) of donor-specific antibodies (DSA). Methods: This was a retrospective study conducted at Jaypee Hospital, Noida. This study included highly sensitized patients who were transplanted with positive CDC and DSA >10,000 MFI for single antigen or >5000 MFI for multiple donor antigens. The patient's renal outcomes were documented. The desensitization protocol consisted of rituximab, therapeutic plasma exchanges (TPE), and thymoglobulin. Results: A total of five patients who had positive CDC crossmatch with very high level of preformed DSA underwent KT. Three patients had end-stage renal disease due to diabetic kidney disease while other two due to autosomal dominant polycystic kidney disease and chronic glomerulonephritis. All the patients were on dialysis. The MFI by Luminex single antigen bead assay for Class I varied from 1657 to 23440 and for Class II varied from undetectable to 11120. The mean number of pretransplant TPE sessions given per patient was 7.8 ± 2.68 and posttransplant TPE sessions per patient was 0.8 ± 0.45. The mean follow-up period was 308.2 days. Mean creatinine on the day of discharge was 0.58 ± 0.17 mg/dL. None of the patients had any postoperative infections or rejections. Conclusion: The current report showed favorable short-term patient and graft outcomes post-KT without any postoperative infections or rejections with desensitization therapy comprising of rituximab, TPE, and thymoglobulin induction.

The objective of this study was to determine the mean fluorescence intensity (MFI) values of class II Luminex single antigen bead (L-SAB) assay and compare these MFI values with cell-based complement-dependent cytotoxicity crossmatch with anti-human globulin (CDC-AHG-XM) and IgG-B-cell flow cytometry crossmatch (FC-XM) results and explore the near-accurate MFI-cutoff values of positive cell-based crossmatch results. This retrospective study was an analysis in 97 renal transplant recipients, who were tested for the presence of DSA by CDC-AHG-XM and IgG-B-cell-FC-XM methods with their corresponding donor as well as for anti-human leukocyte antigen (HLA) antibody detection using a sensitive L-SAB assay. In the group having DSA MFI values <1000, none of the patients showed positivity for FC-XM and CDC-AHG-XM; in the group having MFI values between 1000 and 3000, 35.48% showed positivity for FC-XM but none by the CDC-AHG-XM method. However, in the group having MFI values >3000, 83.33% of cases were positive for FC-XM. Further, in those groups with MFI values between 3000 and 6000, 38.09% were positive for CDC-AHG-XM, while 86.66% showed positivity in the group with MFI >6000. Our results indicated that Luminex-DSA MFI value >1995 (P < .0001) significantly correlated with IgG-B-cell-FC-XM positivity while Luminex-DSA MFI value of >4247 (P < .0006) was significantly correlated with positive CDC-AHG-XM. MFI cutoff of 1995 exhibited a diagnostic sensitivity of 97.56% and specificity of 89.29% for predicting positive IgG B-cell FC-XM and MFI cutoff of 4247 exhibited a diagnostic sensitivity of 90.48% and specificity of 97.37% for predicting positive CDC-AHG-XM. However, a cutoff MFI of >5000 and >7000 for SAB assay had a sensitivity and specificity of 100% in detecting a positive IgG B-cell FC-XM and CDC-AHG-XM, respectively.

The aim of this study was to compare the results of solid phase assay and cell-based assay, and explore the near-accurate DSA-MFI-cutoff value detected on solid phase assay above which the cell-based assay would show a positive result. In this retrospective study, 102 prospective renal transplant recipients were tested for the presence of donor-specific antibodies (DSAs) by cell-based assay (T-cell-CDC-AHG-XM and T-cell-IgG-FC-XM) and solid phase assay (class-I-IgG-L-SAB) with their corresponding donor. Among the 40 patients in the group first (L-SAB-DSA-MFI<1000), one case was positive in IgG-T-cell-FC-XM while T-cell-CDC-AHG-XM was negative in all the cases. In the second group having L-SAB-DSA-MFI values between 1000 and 3000, 19 cases were positive and the remaining 11 cases were negative in IgG-T-cell-FC-XM. T-cell-CDC-AHG-XM showed a negative reaction in all 30 cases. In the third group having L-SAB-DSA-MFI values between 3000 and 5000, IgG-T-cell-FC-XM was positive in 18 cases while, two were negative. T-cell-CDC-AHG-XM demonstrated a negative result in 14 cases while reaming six cases demonstrated a positive result. In the fourth group having L-SAB-DSA-MFI values >5000, all 12 cases showed a positive result in both IgG-T-cell FC-XM and T-cell-CDC-AHG-XM. Our results indicated that the L-SAB-DSA-MFI values >2215 were significantly (P < .001) correlated with positive IgG-T-cell-FC-XM while L-SAB-DSA-MFI values >4689 were significantly (P < .001) correlated with positive CDC-XM.