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Saudi Journal of Kidney Diseases and Transplantation
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Year : 2018  |  Volume : 29  |  Issue : 3  |  Page : 698-704
A case report of successful renal transplantation in an ABO incompatible patient with a preformed donor-specific antibody and negative CDC human leukocyte antigens crossmatch

1 Department of Transfusion Medicine, Indraprastha Apollo Hospital, New Delhi, India
2 Department of Nephrology, Indraprastha Apollo Hospital, New Delhi, India

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Date of Submission31-Dec-2016
Date of Acceptance31-Mar-2017
Date of Web Publication28-Jun-2018


ABO incompatibility and preformed antibodies against the human leukocyte antigen (HLA) are two impermissible barriers to a successful renal transplantation, especially in highly sensitized patient population. With the availability of effective desensitization regimens, good patient and graft outcomes have been reported. As transfusion medicine specialists we report our experience, where patient presented with dual histocompatibility barriers i.e. ABO incompatibility along with preformed donor-specific antibodies (DSA) and negative complement dependent lymphocytotoxicity (CDC) HLA crossmatch. The desensitization strategy followed for our patient included rituximab (375 mg/m2), bortezomib (1.3 mg/m2) and eleven pre-transplant therapeutic plasma exchange (TPE) followed by intravenous immunoglobulin (100 mg/kg per TPE session). Anti-B titer of 1:1 and negative Luminex crossmatch (LumXm) class II DSA (less than 1000 mean fluorescence intensity; MFI), was achieved prior to renal transplantation. Fifteen months post-transplant, patient is doing well with serum creatinine level of 0.8 mg/dL with repeat LumXm class II DSA negative (891 MFI). The desensitization regimen followed proved to be effective in our case.

How to cite this article:
Chowdhry M, Makroo RN, Kakkar B, Jasuja S, Sagar G, Thakur Y. A case report of successful renal transplantation in an ABO incompatible patient with a preformed donor-specific antibody and negative CDC human leukocyte antigens crossmatch. Saudi J Kidney Dis Transpl 2018;29:698-704

How to cite this URL:
Chowdhry M, Makroo RN, Kakkar B, Jasuja S, Sagar G, Thakur Y. A case report of successful renal transplantation in an ABO incompatible patient with a preformed donor-specific antibody and negative CDC human leukocyte antigens crossmatch. Saudi J Kidney Dis Transpl [serial online] 2018 [cited 2022 Jul 1];29:698-704. Available from: https://www.sjkdt.org/text.asp?2018/29/3/698/235194

   Introduction Top

The complement dependent lymphocytetoxicity (CDC) assay is a classical method for detection of antibodies against human leukocyte antigens (HLA) in patients awaiting solid organ transplantation. With the emergence of Luminex-based assay, the detection and characterization of HLA antibodies are easier due to greater sensitivity, in comparison to CDC and enzyme-linked immunoassay.[1] Both HLA alloimmunization and ABO incompatibility are considered two major histocompatibility barriers to successful organ transplantation and graft survival.[2],[3],[4] Whether the coexistence of anti-A/B antibody and preformed donor-specific antibody (DSA) have an impact on the development of antibody-mediated rejection (AMR) is still unclear. Although several studies quote that the presence of either ABO or HLA incompatibility, increases the risk of AMR,[4] however, advances in immunosuppression and desensitization strategies have led to successful outcomes in solid organ transplantation such as renal transplantation.[5] Prior history of blood transfusion, multiple pregnancies, and previous transplantation can result in the development of HLA antibodies.[6] Even if pretransplant CDC crossmatch is negative, the presence of preformed DSA can affect the outcome of the transplant as well as lead to inferior graft outcomes. Therefore, desensitization strategies comprising of therapeutic plasma exchange (TPE), intravenous immunoglobulin (IVIg), rituximab and newer therapeutic agents such as bortezomib and eculizumab, have revolutionized the management strategies for patients awaiting renal transplantation across immunologic barriers.[7]

   Materials and Methods Top


The patient presenting with ABO incompatibility and positive Luminex crossmatch (LumXm) was reviewed, desensitized, and later transplanted. The patient was offered the option of “swap program” before going for incompatible transplant.

Antibody testing

Complement-dependent cytotoxicity, antihuman globulin augmented (CDC-AHG) cross match was performed using patient's sera and donor lymphocytes (B and T cells were separated). Standard (½ h before and 1 h after addition of complement) and extended (double of standard) incubation timings at room temperature were used. Appropriate controls (positive and negative) were used. The CDC-AHG crossmatch was interpreted as positive when more than 20% of donor lymphocytes were lysed in excess of the baseline rate.

LumXm is a bead-based immunoassay used for the detection of IgG antibodies in the patient's sera against donor's HLA class I and class II antigen. It is based on the method of Luminex xMAP® Technology using the Life codes Class I ID and Life code Class II ID v2 (Immucor). In this method, donor lymphocytes isolated from peripheral blood were used as a source material for HLA. The isolated cells were solubilized with a nonionic detergent. Following a centrifugation step to remove cell debris and fragments, the lysate was used. LumXm includes a single blend of Luminex beads which when mixed with lysate will capture the solubilized HLA, making a donor-specific HLA target for antibodies in the serum sample. After the capture of the donor HLA, the beads were transferred to a filter plate and washed. Serum diluted in the specimen diluent was then added and incubated with the beads for 30 min. Following another wash, the diluted anti-human IgG-phycoerythrin (PE) conjugate was added to the beads. After a final 30 min incubation, wash buffer was added to the wells, the plate is loaded in the Luminex instrument, and the data are collected for analysis. As per the departmental standard operating procedure, LumXm was considered negative when mean fluorescence intensity (MFI) was <1000, borderline or weak positive when MFI was 1000-1500 and MFI above 1500 was considered as positive.

HLA antibody screening and panel reactive antibody (PRA) was based on the method of Luminex xMAP® Technology using the Life codes LSATM MIC, Life codes Life Screen Delux and Life codes class I ID and Life code class II ID v2 (Immucor), respectively. For HLA antibody screening and PRA, a raw MFI <1000 was considered as negative. In case, the value of the control beads was high, suggesting background error, average adjusted MFI values were taken as the final value and interpreted accordingly.

HLA typing included isolation of DNA from peripheral leukocytes using Invitrogen DNA isolation kit (Invitrogen USA PureLink™ Genomic DNA Mini Kit Catalog No. K1820-01) according to the manufacturer's instruction, and DNA was diluted with 150 μL elution buffer and stored at -20°C until analysis. Molecular typing was carried out by polyme-rase chain reaction-sequence-specific primer (PCR-SSP) method (AllSet+™ Gold SSP HLA-ABDR, Life Technologies, USA) utilizing allele-specific primers along with the control primers to identify the respective allele.

ABO titration was performed by the serial doubling dilution of patient's sera using conventional tube technique in AHG phase. The target pretransplant titer was anti-A/B <1:4. The ABO titration was performed before and after every session of TPE/IVIg.

Desensitization protocol

The patient received triple sequential immu-nosuppression - mycophenolate mofetil (MMF; dose - 500 mg/day) and tacrolimus (dose - 3.5 mg; adjusted to a 12 h trough levels of 8-12 ng/mL) and solumedrol. The patient also received rituximab (RTX; dose - 375/mg/m2), bortezomib (dose - 1.3 mg/m2), TPE (11 preoperative sessions) followed by IVIg (dose - 100 mg/kg per TPE session).

TPE was performed using haemonetics MCS plus (Braintree, USA) cell separator using central venous access. A single-use, sterile disposable set, REF 981E (Haemonetics, USA) was used. ACD-A was used as anticoagulant with ACD-A: blood ratio of 1:14-1:16. The whole blood inlet flow rate and collection flow rate were adjusted by the system according to size of the patient (sex, height, and weight). All procedures were done after due consent from the patient. A standard 1.5 plasma volume TPE with 5% human albumin (20% human albumin reconstituted in saline) and/or fresh frozen plasma (FFP) was performed. A minimum of 12 h gap was given after IVIg dose and before the start of the next TPE.

   Case Report Top

A 42-year-old female, hypertensive since 25 years (controlled on medication) complained of progressive dyspnea on exertion, loss of appetite, generalized weakness with on and off pedal edema since one month. The patient was evaluated at a private hospital and was found to have deranged renal parameters with high serum creatinine levels (8.5 mg/dL). The patient was referred to our hospital with the above-mentioned complaints for further evaluation and treatment. On examination, blood pressure was 160/100 mm Hg, heart rate was 78/min, pallor present, there was no pedal edema, and obstetric history revealed two live births (7 and 8 years back) and one spontaneous abortion. History revealed recurrent urinary tract infection during childhood. There was no history of blood transfusion. Baseline investigations showed low hemoglobin (7.5 g/dL) and deranged renal profile (blood urea - 222 mg/dL, serum creatinine - 10.5 mg/dL, serum sodium - 142 mEq/L, and serum potassium - 3.9 mEq/L). Renal Doppler revealed bilateral small kidneys, bilateral loss of cortico- medullary differentiation, and small calibration with relatively high velocity of the right renal artery. The patient was started on maintenance hemodialysis, and transplant workup was started.

Patient's blood group was O positive and her prospective renal donor's blood group was B positive (mother). The baseline ABO titer for anti-A was more than 1:1024 and for anti-B was 1:32.

CDC-AHG crossmatch was negative. PRA class I - 0%, PRA class II - 58%. LumXm was performed to detect the preformed DSA and was found to be negative (293 MFI) for class I, whereas class II was positive (9883 MFI), indicating that patient had HLA class II DSA.

HLA typing of the patient and donor (HLA - A, HLA - B, and HLA - DR) showed a 3/6 match [Table 1].
Table 1: HLA typing of patient and the donor.

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Since the patient was ABO incompatible along with positive LumXm, a rigorous desen- sitization protocol was initiated, in view of living donor-related renal transplantation. Injection RTX was given at day 15 pre- transplant followed by administration of injection bortezomib at day 12 pretransplant. TPE /IVIg therapy was started on day 15 pre- transplant. Following four consecutive day sessions of TPE/IVIg, a fall in LumXm HLA class II MFI was noted from 9883 to 2436, following which alternate day sessions of TPE/IVIg were performed. Despite being on TPE/IVIg therapy, a gradual rise in LumXm class II MFI levels was noted from 2436 to 7134 MFI till day 4 pretransplant. On day 4 pretransplant, the patient was started on tacro- limus along with MMF (dose doubled on day 3 pretransplant) and the continuation of TPE/IVIg therapy led to a decrease in the HLA Class II MFI levels and a repeat LumXm HLA Class II level on day 1 pretransplant was less than 1000 MFI. All this while, the patients' anti-B titer remained well below the target value [Table 2]. During the pretransplant phase, a total of 11 TPE were performed, followed by IVIg administration. [Table 2] and [Figure 1] show the details of desensitization regimen followed for our patient.
Figure 1: Desensitization regimen (pretransplant till time of discharge).
TPE: Therapeutic plasma exchange, IVIg: Intravenous immunoglobulin, TAC: Tacrolimus, MMF: Mycophenolate Mofetil, ATG: Anti-thymocyte globulin, DSA: Donor-specific antibody, MFI: Mean fluorescence intensity, Bote: Bortezomib, ABO titers: 1:32 to 1:1.

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Table 2: Details of desensitization regimen.

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In view of dual histocompatibility barriers, prophylactically two sessions of TPE/IVIg were performed postoperatively on day 1 and day 2. A repeat LumXm HLA class II was found to be negative (767 MFI) on postoperative day 3. No adverse effects related to TPE procedure were noted during the pre- and post-transplant phase.

The patient had slow graft function (low urine output and slow decline in serum creatinine levels), therefore, graft biopsy was done on postoperative day 6 which revealed border-line changes of acute cellular rejection (ACR). The patient was given injection antithymocyte globulin (ATG) in a dose of 100 mg over two consecutive days. From postoperative day 7, serum creatinine showed a declining trend along with increase in urine output from 40 to 100-150 mL/h. The patient was discharged on postoperative day 14 in a hemodynamically stable condition, on triple immunosuppression (mycophenolate mofetil, tacrolimus, and steroids) with serum creatinine 1 mg/dL, 150200 mL hourly urine output and ABO titer (anti-B- 1:1).

The Patient has been on follow up since postoperative day 1, with regular monitoring of renal profile, posttransplant DSA levels and clinical status. Fifteen months posttransplant, the serum creatinine was 0.8 mg/dL and repeat LumXm class II DSA was also found to be negative (891 MFI) and ABO titer well below the target value.

   Discussion Top

Renal transplantation has emerged as a leading treatment modality for end-stage renal disease patients.[8] We present a case where the patient was ABO incompatible and had preformed DSA class II antibodies. These two re-present formidable barriers and in the present scenario, would represent a contraindication to renal transplantation.

However, pretransplant assessments along with effective desensitization strategies are the key factors for a successful renal transplantation in such patients. Desensitization protocol varies from center to center[1],[6],[8] and from clinicians experience within a center. The desensitization protocol employed for our patient included rituximab, bortezomib administration along with TPE/IVIg therapy and triple immunosuppression.

TPE is one of the simplest and most common methods employed for desensitization, as it helps in effective immunoglobulin removal. When combined with low-dose IVIg, it permits the rapid depletion of circulating antibodies and producing durable elimination of DSA. One of the major disadvantages of performing TPE is that it leads to depletion of clotting factors and requires replacement with FFP. With the discontinuation of TPE, a rebound in alloantibodies levels can be seen, but simultaneous administration of IVIg benefits in a long run by suppressing the rebound of such antibodies.[9] Several studies have reported rebound of DSA before transplantation and were found to be associated with poor graft outcome.[1],[5],[7],[8] Similar finding of rebound in DSA levels was also seen in our patient, for which rigorous TPE/IVIg therapy was followed, along with administration of potent immunosuppressant. Graft failures due to insufficient immunosuppression during the post- transplant phase have been reported due to development of de novo DSA and autoantibodies against the new antigenic epitopes.[1] However, this was not observed in our patient, as patient was on triple immunosuppression and the posttransplant LumXm class II DSA was negative (767 MFI) as well as the ABO titers (anti-B 1:1) well below the target levels. A state of accommodation can be seen in renal transplant recipients, where desensitized patients continue to have circulating DSA and stable kidney function along with normal kidney transplant biopsy. Whether these patients are still at a risk of developing chronic rejection in future is unclear. The current scenario depicts the need to closely follow-up such patients, and in case of, worsening graft function or increasing MFI values, a repeat graft biopsy can help in determining the further course of treatment.[8]

We observed that our patient had features of biopsy-proven ACR, which was effectively managed with ATG infusion and methylpred- nisolone pulse therapy. Huh et al, also reported that renal transplant recipients with positive LumXm and a negative CDC-AHG crossmatch have a higher immunological risk for the development of ACR. They suggested that ATG induction in the pretransplant phase might reduce the risk of ACR in LumXm positive patients and also reported that the rituximab administration in the pretransplant phase, might be the reason for the absence of biopsy-proven AMR in these patients.[10] However, in our case, no ATG was given in the pretransplant phase, while rituximab was administrated.

Rituximab, anti-CD20 monoclonal antibody, has an added advantage over splenectomy that it effectively inhibits the B-cell proliferation and induces cellular apoptosis by antibody- dependent cell mediated cytotoxicity mediated by complement activation.[9] The study done by Fuchinoue et al, reported that patients receiving rituximab pretransplant, had a lower incidence of AMR along with better five-year graft out- come.[11] Bortezomib, a proteosome inhibitor, inhibits antibody production by plasma cell apoptosis via nuclear factor-kappa B pathway inhibition. In comparison with rituximab, it is the only drug that leads to plasma cell apop- tosis.[8] Case series of three patients using bortezomib along with rituximab/TPE/IVIg as desensitization regimen showed significant fall in the target antibody titers in highly sensitized and ABO-incompatible renal transplant patients in whom the conventional therapy failed.[12] Similar protocol was followed in our case.

Our study was not without limitations. First, it represents the data from only one patient, and a larger longitudinal study would be more beneficial in terms of explaining the effectiveness of desensitization strategy to be followed for such patients. Second, graft biopsies were not done to document the reversal in the histo- logical features and C4d positivity to document the effectiveness of the treatment given or rejection of the graft. Third, Luminex-based single antigen bead testing was not available, which could have ascertained the specificity of the HLA antibody.

This study, however, reinforces that with effective desensitization strategy and post- transplant DSA monitoring, renal transplants are still feasible in high-risk renal transplant recipients presenting with impermissible barriers, i.e., ABO incompatibility and preformed DSA, which under the current scenario are considered a contraindication for successful renal transplantation.

Conflict of interest: None declared.

   References Top

Süsal C, Opelz G, Morath C. Role and value of luminex(®)-detected HLA antibodies before and after kidney transplantation. Transfus Med Hemother 2013;40:190-5.  Back to cited text no. 1
Lefaucheur C, Loupy A, Hill GS, et al. Preexisting donor-specific HLA antibodies predict outcome in kidney transplantation. J Am Soc Nephrol 2010;21:1398-406.  Back to cited text no. 2
Sicard A, Amrouche L, Suberbielle C, et al. Outcome of kidney transplantations performed with preformed donor-specific antibodies of unknown etiology. Am J Transplant 2014;14:193-201.  Back to cited text no. 3
Chung BH, Joo YY, Lee J, et al. Impact of ABO incompatibility on the development of acute antibody-mediated rejection in kidney transplant recipients presensitized to HLA. PLoS One 2015;10:e0123638.  Back to cited text no. 4
Garonzik Wang JM, Montgomery RA, Kucirka LM, et al. Incompatible live-donor kidney transplantation in the United States: Results of a national survey. Clin J Am Soc Nephrol 2011;6:2041-6.  Back to cited text no. 5
Al Meshari K, Pall A, Chaballout A, et al. Outcome of desensitization in human leukocyte antigen- and ABO-incompatible living donor kidney transplantation: A single- center experience in more than 100 patients. Transplant Proc 2013;45:1423-6.  Back to cited text no. 6
Morath C, Opelz G, Zeier M, Süsal C. Prevention of antibody-mediated kidney transplant rejection. Transpl Int 2012;25:633- 45.  Back to cited text no. 7
Marfo K, Lu A, Ling M, Akalin E. Desensitization protocols and their outcome. Clin J Am Soc Nephrol 2011;6:922-36.  Back to cited text no. 8
Shin M, Kim SJ. ABO incompatible kidney transplantation-current status and uncertainties. J Transplant 2011;2011:970421.  Back to cited text no. 9
Huh KH, Kim MS, Kim HJ, et al. Renal transplantation in sensitized recipients with positive luminex and negative CDC (complement-dependent cytoto-xicity) crossmatches. Transpl Int 2012;25: 1131-7.  Back to cited text no. 10
Fuchinoue S, Ishii Y, Sawada T, et al. The 5- year outcome of ABO-incompatible kidney transplantation with rituximab induction. Transplantation 2011;91:853-7.  Back to cited text no. 11
Yang KS, Jeon H, Park Yet al. Use of bortezomib as anti-humoral therapy in kidney transplantation. J Korean Med Sci 2014;29:648-51.  Back to cited text no. 12

Correspondence Address:
Dr. Mohit Chowdhry
Department of Transfusion Medicine, Indraprastha Apollo Hospital, New Delhi 110 076
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/1319-2442.235194

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  [Table 1], [Table 2]


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