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ORIGINAL ARTICLE
Year : 2022  |  Volume : 16  |  Issue : 4  |  Page : 390-396

Impact of donor-specific anti-human leukocyte antigen antibodies in haploidentical hematopoietic stem-cell transplantation: A single-center retrospective study


1 Department of Transfusion Medicine and Histocompatibility and Immunogenetics, Kokilaben Dhirubhai Ambani Hospital and Medical Research Institute, Mumbai, Maharashtra, India
2 Department of Paediatric Haemat-Oncology, Kokilaben Dhirubhai Ambani Hospital and Medical Research Institute, Mumbai, Maharashtra, India
3 Department of Haemat-Oncology, Kokilaben Dhirubhai Ambani Hospital and Medical Research Institute, Mumbai, Maharashtra, India
4 Department of Histocomptibility and Immunogenetics, Kokilaben Dhirubhai Ambani Hospital and Medical Research Institute, Mumbai, Maharashtra, India

Date of Submission03-Jan-2022
Date of Acceptance18-Sep-2022
Date of Web Publication30-Dec-2022

Correspondence Address:
Santanu Sen
No. 7th Floor, Department of Transfusion Medicine and Histocompatibility and Immunogenetics, Kokilaben Dhirubhai Ambani Hospital and Medical Research Institute, Andheri West, Mumbai - 400 053, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijot.ijot_2_22

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  Abstract 


Introduction: While donor-specific anti-human leukocyte antigen (HLA) antibodies (DSA) have been implicated in graft rejection in solid organ transplantation, their role in hematopoietic stem-cell transplantation (HSCT) remains unclear. Aim: The aim of this study is to study the role of DSA for proper donor selection and its effect in the setting of allogeneic HSCT. Methodology: HLA A, B, C, DRB1, and DQB1 high-resolution typing, and DSA cross match (XM) of patients (n = 73) and their prospective donors (n = 74) were assessed. A case–control study was designed retrospectively to evaluate the effect of pre- existing DSAs on engraftment. Thirty-five cases with 5/10 HLA alleles mismatches and 38 cases with full HLA matched, these two controls were selected for comparison. These were matched for disease, graft type, conditioning regimen, age, gender, blood group, and sensitizing events. DSAs were tested with solid-phase assay (Luminex 100/200 platform). Results: DSAs were detected in six of 35 patients (17%); however, donors selected for transplantation were all negative for DSA crossmatch. These six patients who underwent haploidentical (HI) transplants had antibodies against Class I and II. One patient carried antibodies against both classes. A patient who experienced primary graft failure had a second HI transplant. No other known factors that could negatively influence engraftment were associated with the development of graft failure in this patient. Conclusions: DSAs are not associated with graft rejection in patients undergoing HI stem-cell transplantation. Anti-HLA sensitization should be evaluated routinely in HSCT with HLA mismatched donors for a better outcome.

Keywords: Anti-human leukocyte antigen, donor specific antibodies, graft kinetics, haploidentical, hematopoietic stem cell transplantation, human leukocyte antigen antibodies, lysate crossmatch


How to cite this article:
Sawant RB, Sen S, Tulpule SA, Naker DY. Impact of donor-specific anti-human leukocyte antigen antibodies in haploidentical hematopoietic stem-cell transplantation: A single-center retrospective study. Indian J Transplant 2022;16:390-6

How to cite this URL:
Sawant RB, Sen S, Tulpule SA, Naker DY. Impact of donor-specific anti-human leukocyte antigen antibodies in haploidentical hematopoietic stem-cell transplantation: A single-center retrospective study. Indian J Transplant [serial online] 2022 [cited 2023 Feb 3];16:390-6. Available from: https://www.ijtonline.in/text.asp?2022/16/4/390/364615




  Introduction Top


Allogeneic hematopoietic stem-cell transplantation (HSCT) is the only curative treatment for hematologic malignancies. In past, the main limitations of this treatment modality were high rate of graft failure and graft-versus-host disease (GVHD), which occurs due to alloreactive reactions due to the human leukocyte antigen (HLA) mismatch between the recipient and the donor. The presence of donor-specific anti-HLA antibodies (DSA) is associated with increased risk of graft failure in haploidentical (HI) stem-cell transplantation. Assessment of the presence or absence of circulating HLA antibodies and of their specificity and strength is an important step in donor selection procedure. While the role of donor-specific antibodies in solid organ transplantation is well established, their importance in HSCT is becoming clear now. Recently developed technology using solid phase immunoassays has greatly improved the ability to detect and classify DSAs.

The sensitivity and specificity of current solid-phase assays for HLA antibody detection are high, correlation with graft failure remains uncertain. Consensus guidelines from the European Society for Blood and Marrow transplantation has set a median fluorescence intensity (MFI) >1000 as a cutoff for DSA positivity. HLA-DSAs are preformed antibodies in the recipient directed against the donor's Class I and/or Class II HLA antigens. The Class I antigens, HLA-A, -B, and -C, are expressed on most cells, and the Class II antigens, HLA-DR, -DQ, and -DP, are restricted primarily to antigen-presenting cells. Patients can form antibodies to foreign HLA antigens after exposure to foreign cells or tissue. Common exposures include pregnancy, blood product transfusion, and previous transplantation. Therefore, HLA antibody evaluation requires monitoring in the pre- and post-transplantation period.

The aim of this study was to analyze the impact of DSA on the risk of graft failure and poor graft function, as well as on major outcomes in a consecutive cohort of patients who were systematically screened for DSA before HI-HSCT in comparison to full-match HSCT.


  Methodology Top


We retrospectively evaluated the correlation of DSA's with graft failure in 73 consecutive patients with hematologic malignancies treated at tertiary care multispeciality hospitals between January 2016 and May 2019. The diagnosis of patients is mentioned in [Table 1] and [Table 2]. All patients provided informed consent. According to the institutional policy, patients who were candidates for transplantation underwent simultaneous sibling and parent donor searches. HI donor selection was done based on the absence of DSA. Other factors taken into account were patient and donor cytomegalovirus status, blood group, sex, age, and weight. The presence of DSA was considered an exclusion criterion for potential donors, except in patients with no other donor options.
Table 1: Distribution of patients as per the diagnosis

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Table 2: Distribution of patients as per disease type

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High-resolution HLA typing for recipient and donor and lysate crossmatch were performed pretransplant. HLA high-resolution typing for HLA Class I A, B, and C and Class II DRB1 and DQB1 was done using Illumina MiSeq. The presence of DSA was tested using the Luminex technology. The recipiemt's serum samples were screened for Class I and Class II immunoglobulin G HLA antibodies using the commercially available LIFECODES® Donor-Specific Antibodies kit (Immucor). In this assay, the patient's serum is incubated with soluble HLA antigens bound to a solid matrix. These tests are used to assess the presence and relative strength of HLA-specific antibodies. Results were interpreted using MFI values. The MFI values of the reporter signal are proportional to the amount of antibody bound to each bead and are used as a measure for estimation of antibody levels. All samples with an MFI value >1200 were considered positive. The main advantages of these methods are the high specificity, sensitivity, semiquantitation, and multiplexing capability. Results were interpretated as MFI < 800 as negative, MFI 800-1200 as indeterminate, MFI > 1200 as positive.

Transplant Conditioning Regimen

All patients received a HI graft from mismatched related donors and doses of CD34+ stem cells. Apheresis technique was used for stem cell collection using the COMTEC® (Fresenius Kabi). A minimum acceptable CD34+ cell dose was 8 × 106 CD34+ cells/kg for HI transplant. If the patients had DSA, they were treated with a combination of bortezomib and therapeutic plasma exchange in an attempt to decrease DSA level and prevent graft rejection.

Statistical analysis

Probabilities of survival, i.e., overall survival (OS) and disease-free survival (DFS) were estimated using Kaplan–Meier curves and Log rank test by comparing the differences between groups in a univariate analysis.

Hazards ratios with 95% confidence intervals were estimated using univariate Cox regression and the impact of antibody status was assessed in multivariate Cox proportional hazards regression analysis.

Two-sided P values were presented and a probability <0.05 was considered statistically significant.

Analyses were performed using R-Statistical software - version 3.5.2 (2018) GraphPad prism version 8.0 (2018) both are free software for statistical computing.

Declaration of patient consent

The authors certify that patient consent has been taken for participation in the study and publication of clinical details and images. Patients understand that their names and initials would not be published, and all standard protocols will be followed to conceal their identity.

Ethics statement

Since anonymized retrospective patient data were analyzed without any active intervention, the Ethical Committee approval was not needed. However, informed consent was obtained from all patients and donors included in the study.


  Results Top


Seventy-three patients who underwent HSCT were treated with almost similar conditioning regimens as mentioned above. The median of various factors such as age, gender, source of stem cells, and engraftment kinetics was considered [Table 3], [Table 4], [Table 5] and [Chart 1], [Chart 2], [Chart 3]. Out of 73 patients, 38 were full-matched HSCT (FM-HSCT) and 35 were HI-HSCT. The white blood cell (WBC) engraftment occurred on 12–15 days post peripheral blood stem-cell infusion in FM-HSCT and 14–16 days in HI-HSCT. Platelet engraftment occurred on 16–18 days for FM-HSCT and 18–21 days for the HI-HSCT group. Graft engraftment was observed to be 89% (34/38) for FM-HSCT and 80% (28/35) for HI-HSCT. Graft failure was found to be 11% (4/38) for FM-HSCT and 20% (7/35) for HI-HSCT. The OS was 87% for FM-HSCT and 69% for HI-HSCT. The posttransplant survival analysis for time to event was observed to be >250 days for HI-HSCT and FM-HSCT falling with the same interval for a longer duration suggesting constant probability of survival [[Figure 1] – Kaplan–Meier curve]. The hazard ratio graph suggests that in time frame between 0 and 50 days both FM-HSCT and HI-HSCT have nearly equal risks involved and beyond 100 days the risk factor influence seems to be increasing in HI-HSCT compared to FM-HSCT [Figure 2]. The odds ratio for FM-HSCT and HI-HSCT for DSA Cross match (XM) status pretransplantation was calculated and found to be similar for both groups [Table 6].
Figure 1: Overall survival analysis for time to events and outcome. Considering 95% CI; appropriate time (days) frame with defined status (events) and probabilities of survival (Kaplan–Meier curve). Each drop in the survival function represents a failure. It's been observed that >250 days HI and FM HSCT falling with the same interval for longer duration suggesting constant probability of survival. HI: Haploidentical, FM-HSCT: Full matched-hematopoietic stem-cell transplantation, CI: Confidence interval

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Figure 2: Hazard proportion: Compared between FM versus HI-HSCT. Log–log plot (5% level of significance): To asses proportional hazard assumption. The graph suggests that in time frame between 0 and 50 days both FM and HI-HSCT have nearly equal risks involved and beyond 100 days the risk factor influence was seems to be increasing in HI-HSCT compared to FM-HSCT. FM: Full matched, HI-HSCT: Haploidentical-hematopoietic stem-cell transplantation

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Table 3: Median values of different parameters

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Table 4: Median age of patients in two groups

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Table 5: Median age of donors in two groups

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Table 6: Risk estimate in haploidentical versus full matched hematopoietic stem-cell transplantation as per the donor-specific antibodies XM status pretransplantation

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Six patients in the HI group were identified to have DSA's [Table 7]. We identified DSA against antigens of HLA Class I, Class II, or both. In this group, 3/6 patients experienced primary GVHD with DFS. One out of six patients experienced primary graft failure. The same patient underwent a second HI-HSCT with mother as a donor and was engrafted successfully [[Table 8] - Patient 4]. 5/6 patients were successfully engrafted, and one death occurred due to infection/sepsis.
Table 7: Donor-specific antibodies-XM status of D-R pair in transplants

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Table 8: Characteristics of six patients who received hematopoietic stem cell transplantation and had human leukocyte antigen antibodies

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To exclude other potential causes for primary graft failure, we analyzed the degree of HLA matching, median of age, gender effect, and disease status at transplant between the study and the control group. No significant differences were identified for any of these parameters [Table 9]. The P value for FM-HSCT and HI-HSCT is displayed in [Table 9].
Table 9: Haploidentical transplant for transplant outcome with respect to different factors

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  Discussion Top


There are several factors that are considered for donor selection such as ABO compatibility, young male donor, natural killer cell alloreactive donors, non - inherited Maternal antigen mismatched donor, and DSA screening, which is one of the most important factor [Figure 3]. Solid-phase assays have revolutionized the approach to pretransplant screening by providing unparalleled sensitivity and specificity in the detection of anti-HLA antibodies. We have analyzed prospectively the relationship between the development of primary graft failure, engraftment kinetics, and OS with the presence of DSA in patients undergoing HI transplants at our institution. The criteria that constitute a prohibitive DSA are unknown; however, desensitization techniques can successfully lower DSA to range deemed safe for transplantation. The removal of preformed antibodies through plasmapheresis and intravenous gamma globulin has been used to prevent rejection in solid organ transplantation,[1] whereas rituximab, in addition to the elimination of B-cells, can decrease de novo antibody production and facilitate graft tolerance in newly transplanted organs.[2] We have treated our patient with a combination of bortezomib/rituximab and plasma exchange when high DSA level were identified. Overall, this intervention decreased the DSA level in all patients, who achieved engraftment successfully.
Figure 3: Factor considered for donor selection. HLA: Human leukocyte antigen

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While comparing the engraftment rate, it was observed that engraftment was not affected by the presence of DSA as compared to the control group which is also reported by Brunstein et al.[3] There are results and other reports that show desensitization can reduce DSA's to levels permissible for donor engraftment[4] which was also confirmed in our study. Although it is consistently stated in the literature that high level of DSA is associated with poor outcomes for HSCT, it is yet to be defined if there is a “permissible” DSA level for HI-HSCT. Although the results from various studies have confirmed that the presence of DSAs influences graft outcomes and survival in HI-HSCT, we need to bear in mind that different cutoff levels of DSAs as well as different methods of DSAs detection were used in these studies. In our study, we considered MFI ≥1200 to be positive, whereas Yoshihara et al.[5] and Ciurea et al.[6] considered MFI >1500 or 5000 to be positive in their study, respectively. Recently, Chang et al. also showed that positive DSA at MFI of 10,000 or more correlated with primary graft rejection, whereas MFI of 2000 or more was strongly associated with primary poor graft function.[7] The data analyzed by Gladstone and Bettinotti et al. show that weak- and low-level DSA, as defined by a single antigen bead (SAB) MFI <5000 to HLA-A, -B, and -DR, do not affect engraftment.[4]

In our study, the posttransplant OS period for both groups was clinically similar, in fact, the presence of DSA in the HI-HSCT group was not associated with lower survival which is in contradiction to data reported by Rugger et al.[8] The rate of graft failure was clinically similar for the FM-HSCT and HI-HSCT groups. The presence of DSA was not associated with the increased risk of graft failure in our study which is in contraindication with the findings of Cutler et al.[9]

At this point of time, it is most important to establish the permissible or acceptable DSA levels for allogeneic HI-HSCT, since recipients mostly have preformed DSA against HLA antigen mismatches with their donor resulting in deferral of donors. Luminex lysate crossmatch in combination with Class I and II screening (pooled bead assay) is useful for the detection of anti-HLA antibodies in HI-HSCT. Generally, SAB is a test of choice in developed countries; however, in resource-limited countries like India due to cost constraints, SAB is not feasible. Therefore, lysate crossmatch along with Class I and II antibody screening is found to be a cost-effective and useful strategy for the detection of clinically significant DSAs.[10]

Limitations of the study

Some of the limiting factors we faced were high background value which was sometimes directed to particular bead or to a specific lot of reagents. This was eventually reduced by additional washing steps. The test needs to be evaluated in multiple centers on larger sample size and if resources permit a parallel testing with SAB assay to conclusively establish its role. Screening for anti-HLA antibodies is must while considering donors with mismatches and attempts to reduce the antibodies levels before transplantation deserve further investigation in HSCT. Moreover, since mismatches are most frequent in the HLA DPB1 locus even for 10/10 matched unrelated donors, the impact of anti-DP antibodies deserves to be deeply investigated. We wish to highlight a few limitations of our study which are small sample size, KIR ligand data was not available with us, and DSA-XM was performed for safe donor selection only, not for monitoring during the engraftment phase.


  Conclusions Top


The presence of DSA did not correlate with graft failure and delayed engraftment in HI-HSCT. Therefore, the presence of DSA may not always be a contraindication for HI-HSCT. The identification of DSA enables clinician to make informed decisions regarding acceptance of the organ and choice of immunosuppression protocol. In cases with preformed donor-specific antibodies, an attempt to decrease the antibody levels before transplant may be required for successful engraftment. Therapeutic strategies (intravenous immunoglobulin/therapeutic plasma exchange/rituximab) for such cases need to be assessed in prospective clinical trials. A consensus agreement/standardization for cutoff values for MFI of DSAs should be practiced for a particular population.

Acknowledgment

The authors would like to convey their sincere thanks to the team of Transfusion Medicine, team of Laboratory Medicine, and hospital management for their support.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Montgomery RA, Zachary AA, Racusen LC, Leffell MS, King KE, Burdick J, et al. Plasmapheresis and intravenous immune globulin provides effective rescue therapy for refractory humoral rejection and allows kidneys to be successfully transplanted into cross-match-positive recipients. Transplantation 2000;70:887-95.  Back to cited text no. 1
    
2.
Bearden CM, Agarwal A, Book BK, Vieira CA, Sidner RA, Ochs HD, et al. Rituximab inhibits the in vivo primary and secondary antibody response to a neoantigen, bacteriophage phiX174. Am J Transplant 2005;5:50-7.  Back to cited text no. 2
    
3.
Brunstein CG, Noreen H, DeFor TE, Maurer D, Miller JS, Wagner JE. Anti-HLA antibodies in double umbilical cord blood transplantation. Biol Blood Marrow Transplant 2011;17:1704-8.  Back to cited text no. 3
    
4.
Gladstone DE, Bettinotti MP. HLA donor-specific antibodies in allogeneic hematopoietic stem cell transplantation: Challenges and opportunities. Hematology Am Soc Hematol Educ Program 2017;2017:645-50.  Back to cited text no. 4
    
5.
Yoshihara S, Maruya E, Taniguchi K, Kaida K, Kato R, Inoue T, et al. Risk and prevention of graft failure in patients with preexisting donor-specific HLA antibodies undergoing unmanipulated haploidentical SCT. Bone Marrow Transplant 2012;47:508-15.  Back to cited text no. 5
    
6.
Ciurea SO, Thall PF, Wang X, Wang SA, Hu Y, Cano P, et al. Donor-specific anti-HLA Abs and graft failure in matched unrelated donor hematopoietic stem cell transplantation. Blood 2011;118:5957-64.  Back to cited text no. 6
    
7.
Chang YJ, Zhao XY, Xu LP, Zhang XH, Wang Y, Han W, et al. Donor-specific anti-human leukocyte antigen antibodies were associated with primary graft failure after unmanipulated haploidentical blood and marrow transplantation: A prospective study with randomly assigned training and validation sets. J Hematol Oncol 2015;8:84.  Back to cited text no. 7
    
8.
Ruggeri A, Rocha V, Masson E, Labopin M, Cunha R, Absi L, et al. Impact of donor-specific anti-HLA antibodies on graft failure and survival after reduced intensity conditioning-unrelated cord blood transplantation: A Eurocord, Société Francophone d'Histocompatibilité et d'Immunogénétique (SFHI) and Société Française de Greffe de Moelle et de Thérapie Cellulaire (SFGM-TC) analysis. Haematologica 2013;98:1154-60.  Back to cited text no. 8
    
9.
Cutler C, Kim HT, Sun L, Sese D, Glotzbecker B, Armand P, et al. Donor-specific anti-HLA antibodies predict outcome in double umbilical cord blood transplantation. Blood 2011;118:6691-7.  Back to cited text no. 9
    
10.
Mishra MN, Lal V, A novel strategy for donor specific antibody detection for haploidentical hematopoietic stem cell transplantation. Austin Transplant Sci 2018;3:1009.  Back to cited text no. 10
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9]



 

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