A retrospective observational study to estimate the risk of HLA alloimmunization with blood transfusion: Can the risk be reduced by leucodepletion?

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.

Detection of donor-specific HLA antibodies: A retrospective observation in 350 renal transplant cases.

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, respectively. 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.

Effects of different sensitization events on HLA alloimmunization in renal transplant cases; a retrospective observation in 1066 cases.

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.

Significance of Luminex-crossmatch assay and its mean fluorescence intensity - a retrospective observation in 380 renal transplant cases.

<b>Introduction:</b> 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. </br></br> <b> Aim:</b> 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. </br></br> <b> Methods:</b> 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. </br></br> <b>Results:</b> 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. </br></br> <b>Conclusion:</b> 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.

Outcome of desensitization in human leukocyte antigen and ABO incompatible living donor kidney transplantation: Single center experience of first 200 incompatible transplants.

BACKGROUND AND AIMS: Although desensitization is well established, concerns about graft outcome, patient survival and rejection still exist. The present study aims at comparing outcomes of renal transplant recipients across simultaneous ABO and human leukocyte antigen (HLA) incompatibility barriers to those with ABO or HLA incompatibility alone. MATERIALS AND METHODS: This was a retrospective study conducted from October 2015 to December 2018. All patients with a clinical diagnosis of chronic kidney disease, who were prospective HLA incompatible (HLAi) and/or ABO incompatible (ABOi) renal transplant recipients were included. A total of 400 cases including 36 ABOi transplants, 154 HLAi transplants, 10 simultaneously ABO and HLA incompatible transplants, and 200 ABO (ABOc) and HLA (HLAc) compatible kidney transplants from living donors were included. RESULTS: There were significantly more number of blood transfusions, previous transplants and pregnancies in HLAi transplant recipients relative to the ABOi or the control group. Mean number of therapeutic plasma exchange procedures per patient and mean plasma volume processed per procedure were slightly higher in the ABOi + HLAi category. The incidence of graft dysfunction due to suspected antibody-mediated rejection during first year was highest in the ABOi + HLAi group, followed by ABOc + HLAi and ABOi + HLAc, lowest in the ABOc + HLAc category. Mean time to first episode of graft dysfunction was significantly shorter with incompatible transplants. There were no kidney transplant recipient deaths in the study. CONCLUSION: Patient outcome and graft outcomes observed with incompatible transplants were not worse than those observed with compatible transplants.

Comparative analysis of complement-dependent lymphocytotoxicity crossmatch and flow cytometry crossmatch results versus Luminex single-antigen bead-based donor-specific IgG class I antibody MFI values in live related renal transplant cases; a retrospective observation in 102 cases.

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.

Therapeutic apheresis in ABO-incompatible kidney and liver transplantation: A single-center experience of 50 patients.

ABO antigens play an important role in solid organ transplantation. Desensitization for ABO incompatibility offers patients awaiting transplant a larger donor pool. The aim of this study was to assess outcome of desensitization using the institutional preconditioning protocol in ABO-incompatible solid organ transplants. A retrospective analysis of ABO-incompatible solid organ transplants between October 2015 and June 2018, at a tertiary healthcare center was performed. The preconditioning regimen consisted of immunosuppression and therapeutic apheresis (TA). Pre- and post-TA titers were performed, until a target titer of 8 or below was achieved, at which transplant was performed. Follow-up data till 1 year was analyzed. A total of 50 ABO-incompatible solid organ transplantations, including 14 liver transplants and 36 renal transplants were analyzed. The median baseline anti-A and anti-B titers were 192 and 256, respectively. A total of 150 therapeutic plasma exchange (TPE) procedures were performed for renal transplant recipients; 19 TPE and eight immunoadsorption procedures (five preoperative and three intraoperative) were performed for liver transplant recipients. Five (10%) patients experienced minor adverse events. Biopsy revealed antibody-mediated rejection was observed in three cases in the immediate posttransplant phase and in three (6.67%) cases over 1 year. There was one death due to transplant-associated thrombotic microangiopathy. Graft survival for renal transplant was 100% and death-censored graft survival for liver transplant was 100%. Despite difficulties, ABO-incompatible transplants can be performed without antibody-mediated rejection with the use of an appropriate protocol.

Clinical outcome of living donor kidney transplantation across simultaneous ABO and HLA incompatibility: Single center experience of first ten cases.

BACKGROUND AND AIMS: Preconditioning using different protocols has been tested to prevent antibody mediated rejection (ABMR) individually for ABO and HLA incompatibility. However, simultaneous presence of both barriers is still less explored. The aim of this study was to report outcomes of institutional desensitization protocol in renal transplant recipients with simultaneous ABO and HLA incompatibility. MATERIALS AND METHODS: This was a retrospective study conducted from October 2015 to December 2018. All patients with a clinical diagnosis of dialysis dependent chronic kidney disease (CKD), who were prospective coexistent HLA and ABO incompatible renal transplant recipients were included in the study. Patients were followed up and graft function and patient survival was assessed at 1 y from the date of transplant. RESULTS: Median and mode baseline anti-A titers were 64, while median and mode baseline anti-B titers were 256. All recipients were discharged by tenth postoperative day. None of the patients had any bleeding complications. Post transplant infection rate was found to be 20 %. A total of 54 therapeutic plasma exchange (TPE) procedures were performed before transplant and 8 were performed after transplant. Graft survival and patient survival was 100 % at 3, 6, 9, and 12 months. Range and mean follow-up period was 15-42 months and 23 months respectively. Mean glomerular filtration rate (GFR) at 1 y using the CKD-EPI equation was 85.25 ± 13.76 mL/min. Biopsy proven ABMR was observed in one case only which was managed with TPE and immunosuppression. CONCLUSION: Simultaneous ABO and HLA incompatibility in renal transplant recipients can be managed successfully with adequate preconditioning and careful monitoring.

Significance of Luminex-based single antigen class II bead assay and its mean fluorescence intensity in renal transplant cases; a retrospective observation in 97 cases.

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.

Role of plasma exchange in management of patients clinically diagnosed of postpartum thrombotic microangiopathies: A retrospective observation from a tertiary health-care center.

INTRODUCTION: Diagnosis of postpartum thrombotic microangiopathies in pregnancy is a challenge, but plasma exchange (PE) is life-saving in such cases. This study was conducted with the aim to find the result of the early start of PE in such patients. MATERIALS AND METHODS: There were a total of seven clinically diagnosed cases of post partum thrombotic microangiopathies (PP-TMA) where PE was done. The diagnosis of PP-HUS and decision to start PE in such cases were based on the classical triad of microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. All the PE procedures were done using fully automatic COM.TEC (Fresenius Kabi, Germany). RESULTS: Immediately before the start of PE, the mean platelet count and serum lactate dehydrogenase (LDH) and hemoglobin (Hb) were 53.1 × 109/L, 10,943 IU/L, and 6.4 gm%, respectively. After seven sessions of PE, platelet count improved to 158 × 109/L and LDH dropped to 609 IU/L, and Hb improved to 10.3 gm% (P < 0.05). We got a positive renal response in four patients in whom serum creatinine value reached within normal range while in the remaining three patients, no positive renal response was obtained and serum creatinine remained above normal range. Thus, the response of PE was shown to be inadequate in three patients. Compliance to PE was good. Patients were discharged after 20 days (mean) of hospital admission. CONCLUSION: PE is life-saving in PP-HUS. High degree of clinical suspicion to it and early start of PE were crucial for successful outcome in our patient population.

Renal transplantation in HLA sensitized patients: Traversing the immunological barrier.

Successful renal transplantation across HLA barrier in sensitized individuals has been on the rise during the past decade, primarily due to improved desensitization regimes. The aim of this study was to share outcome of desensitization in renal transplant recipients with donor-specific anti-HLA antibodies (DSA). This was a retrospective analysis of all HLA immunized individuals who were prospective renal transplant recipients. All such patients underwent preconditioning as per the institutional desensitization protocol. Complement-dependent cytoxicity-based crossmatch (CDC-XM), luminex-based crossmatch (LM-XM) and flowcytometry-based crossmatch (FC-XM) were done in all cases. If any of these tests turned out positive, single antigen bead assay (SAB) was performed. Desensitization for DSA was performed in 55 patients and all patients were followed-up for 1 year to assess graft function and patient outcome. CDC-XM being a less sensitive assay, could not detect incompatibility in 29 (52.73%) cases. After desensitization, even though SAB and LM-XM results revealed an MFI within acceptable range, FC-XM being an extremely sensitive assay, continued to give a positive result in eight (14.55%) cases. The mean ± SD number of pretransplant TPE were 3.44 ± 0.98 (2-11). Out of 55, there were 10 patients who were lost to follow up. Patient and graft survival of 45 patients at 1 year was found to be 100%. Preconditioning for renal transplants in the form of immunosuppression with TPE is an extremely useful auxiliary for transplantation in HLA sensitized renal transplant recipients.

Short-course metronidazole-induced reversible acute neurotoxicity in a renal transplant recipient.

Neurotoxic manifestations due to chronic metronidazole intake are well known, but neurotoxicity due to short-term use of metronidazole is very rare. We present a case of acute neurotoxicity due to short course of injectable metronidazole given in usual doses to a renal allograft recipient for persistent diarrhea. It responded to withdrawal of the offending drug. Tacrolimus trough concentration did not increase during neurotoxicity, thereby ruling out any metronidazole-tacrolimus interaction. Magnetic resonance imaging of the brain showed widespread osmotic demyelination and its recovery after drug withdrawal. This is the first reported case of a renal transplant recipient developing acute neurotoxicity due to short-term use of metronidazole, without any increase in tacrolimus trough concentrations.

Florid diabetic complications in impaired glucose tolerance.

We report a 56-year-old gentleman who had a history of impaired fasting glucose 4 years earlier but spontaneously reverted to normoglycemia. He subsequently presented with impaired glucose tolerance and proteinuria. Detailed evaluation revealed florid complications of diabetes, including nodular glomerulosclerosis of the kidney. Such complications in pre-diabetes have rarely been reported. We need to search for them early to prevent further morbidity.