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. 2021 Apr 22;16(4):e0249934. doi: 10.1371/journal.pone.0249934

Intensity of de novo DSA detected by Immucor Lifecodes assay and C3d fixing antibodies are not predictive of subclinical ABMR after Kidney Transplantation

Dominique Bertrand 1,*, Rangolie Kaveri 2, Charlotte Laurent 1, Philippe Gatault 3, Maïté Jauréguy 4, Cyril Garrouste 5, Johnny Sayegh 6, Nicolas Bouvier 7, Sophie Caillard 8, Luca Lanfranco 9, Antoine Thierry 10, Arnaud François 11, Françoise Hau 2, Isabelle Etienne 1, Dominique Guerrot 1, Fabienne Farce 2
Editor: Stanislaw Stepkowski12
PMCID: PMC8062066  PMID: 33886604

Abstract

De novo donor-specific antibodies (dnDSA) are associated with antibody-mediated rejection (ABMR) and allograft loss. We tested Immucor* (IM) Luminex Single-antigen beads (LSAB) assay and C3d-fixing antibodies in the setting of dnDSA and subclinical (s) ABMR. This retrospective multicentric study included 123 patients biopsied because of the presence of subclinical de novo DSA detected by One Lamda* Labscreen (MFI > 1000). In 112 patients, sera of the day of the biopsy were available and tested in a central lab with IM Lifecodes LSAB and C3d fixing antibodies assays. In 16 patients (14.3%), no DSA was detected using Immucor test. In 96 patients, at least one DSA was determined with IM. Systematic biopsies showed active sABMR in 30 patients (31.2%), chronic active sABMR in 17 patients (17.7%) and no lesions of sABMR in 49 KT recipients (51%). Intensitity criteria (BCM, BCR and AD-BCR) of DSA were not statistically different between these 3 histological groups. The proportion of patients with C3d-fixing DSA was not statistically different between the 3 groups and did not offer any prognostic value regarding graft survival. Performing biopsy for dnDSA could not be guided by the intensity criteria of IM LSAB assay. C3d-fixing DSA do not offer added value.

Introduction

Antibody mediated rejection (AMR) is a major cause of graft loss after kidney transplantation (KT) [1] and is mainly associated with preformed anti-HLA donor specific antibodies (DSA) (developing in the presensitized patient early posttransplantation: phenotype 1) or de novo DSA (dnDSA) (developing late posttransplantation, mostly in relation to noncompliance: phenotype 2) [2]. Even if the histological lesions are similar in the 2 phenotypes, graft survival seems to be lower in case of dn DSA [3]. AMR is a process with an early stage called “subclinical AMR” (sAMR), in which histological lesions are present in the kidney graft without clinical graft dysfunction [4]. The monitoring of dnDSA post-KT paired with a systematic biopsy in case of appearance, even in the absence of graft dysfunction, is not part of a routine clinical practice in all KT centers. In a relatively large multicentric cohort [5] (n = 123 KT recipients with subclinical dnDSA), we recently reported that a systematic biopsy performed for dnDSA in the absence of graft dysfunction leads to a diagnosis of sAMR in over 40% of cases. This screening strategy could be guided by the MFI of dnDSA. Our study shows that the MFI of dnDSA on the day of the biopsy holds a prognostic value: the higher the MFI at biopsy (iDSA, >4000; sDSA, >6300), the higher the incidence of active sAMR. In the latter study, identification of dnDSA was exclusively performed with One Lambda (OL) Labscreen Luminex Single-antigen assay from OL (LSAB)). These data confirm the results of other team with comparable strategy [6–8].

To date, studies comparing the performance and accuracy to detect DSA in the posttransplantation period of the 2 currently available SAB tests (OL Labscreen SAB and Immucor (IM) Lifescodes SAB, class I and II), are very scarce. We compared recently, we reported [9] the performance of the 2 tests for the detection of DSA and the diagnosis of AMR in the posttransplant setting. This study showed a good correlation and agreement between OL MFI and IM BCM. However, the results of IM Lifecodes LSAB cannot be interpreted with OL usual criteria and OL Labscreen LSAB seems to be better for detecting low intensity antibodies to confirm AMR diagnosis, but clearly has a lower specificity in stable situations. Nevertheless, the study population was very heterogeneous mixing patients with early and late ABMR, and active or chronic active ABMR.

Thus, to add new elements to the ongoing discussion, we performed the present study to evaluate Immucor LSAB assay in a more homogenous cohort of KT recipients presenting with subclinical dnDSA, all detected by OL LSAB. We tried to verify that IM intensity criteria hold the same prognostic value found with One Lambda MFI and evaluate the value of C3d fixing DSA.

Material and methods

Patients

Patients from 9 French KT centers from the Spiesser group included in a previous study [5] were retrospectively enrolled based on the following inclusion criteria:

  • Kidney transplant recipients over 18 years old

  • dnDSA at any time post transplantation, defined as any DSA detected with OL Labscreen LSAB after transplant that reached MFI >1000 and absent on the day of KT [5].

  • Kidney graft biopsy performed between 2008 and 2016 within 6 months of DSA detection, without graft dysfunction (serum creatinine variation <20% above baseline between DSA occurrence and DSA detection and proteinuria/creatininuria ratio <0.5 g/g).

  • No specific treatment for dnDSA detection begun before kidney biopsy.

Basic demographic information, date of KT, and immunosuppressive therapy were recorded from medical charts and from the ASTRE database from the Spiesser group (final agreement from the French commission of the CNIL, decision DR-2012-518, October 29, 2012). We recorded the histology of kidney biopsies, DSA information, as well as patient and graft survival. We calculated eGFR using the Modification of Diet in Renal Disease equation [10] at 1, 3, and 5 years after biopsy, when patients reached this point. We defined graft failure as eGFR <5 mL/min/1.73 m2 and/or return to chronic dialysis.

Assessment of dnDSA with IM Lifecodes LSAB

We tested in our central lab all patients with available sera of the day of the biopsy with IM Lifecodes LSAB class 1 and class 2.

Briefly, 10 μL of each serum sample and 40 μL of HLA class I or class II LSA were mixed and incubated in the dark for 30 minutes at room temperature. After washing with the wash buffer, 50 μL of phycoerythrinconjugated goat antihuman IgG was added to the beads and incubated for 30 minutes in the dark at room temperature. The fluorescence intensities of the samples were measured by using a Luminex 200 system. Data analysis was performed by using Match IT software provided by the manufacturer (IM). The positive control beads were coated with human IgG and were designed to yield mean fluorescence intensity (MFI) values greater than 10000 when incubated with the positive or negative control sera. Conversely, the negative control beads typically yield low MFI values (lot specific) when treated with the positive or negative control sera. To identify positive bead reactions, the background MFI value was subtracted from the raw MFI value to generate the adjusted value 1 (background corrected MFI: BCM) for each individual bead. Adjusted value 1 was then divided by the MFI value from the calculated control (CalcCON) of its respective locus to generate adjusted value 2 (BCR: BCM divided by the raw MFI of the lowest ranked bead for a locus). The CalcCON for each locus was considered the raw MFI value of the lowest ranked antigen bead for that locus. Adjusted value 3 (AD-BCR: antigen density corrected BCR values) was generated by normalization of adjusted value 2 to the amount of antigen on each bead, as indicated in the lot-specific recording sheet. Positive results were assigned when 2 of 3 criteria (BCM > 1500, BCR > 5.0, or AD-BCR > 5.0) were fulfilled. We used the the most recent kits available, composed of 96 class I and 96 class II specific beads and incorporated 1 positive control and 1 negative control beads for each set. Identification of antibody specificity was carried out using a LABScan 200 Flow analyzer (Luminex Corporation, Austin, TX). The reagents used were LABScreen Single Antigen HLA Class I and Class II (OL, Canoga Park, CA) and LIFECODES LSA Class I and Class II (IM, Stamford, CT). The tests were carried out according to the manufacturers’instructions, and the analysis was performed with IM software. The same lot number for the Luminex products was used for all sera to avoid any lot to lot variability:

  • LSAB Class I 3005909–3005872

  • LSAB Class II 3006250–3006249

Immunodominant DSA (iDSA) was defined as the dnDSA with the highest intensity. The sum of the DSA (sDSA) was defined as the sum of the MFI of all dnDSA in the patient.

DSA C3d binding assay

In the same serum previously tested with IM SAB IgG, the presence of DSA was also assayed in our central lab, using a commercially available kit which detects C3d-binding DSA to LSAB on a Luminex platform according to the manufacturer protocol (Lifecodes C3d assay; Immucor, Stamford USA). The C3d protocol is based on the same protocol as identification and the same lot (same antigen-coated beads) was used. The first step consists in the incubation of 1 μL of C3d-positive control beads with 40 μL of HLA class I and class II LSAB and 10μL of patient sera. After 30 minutes of incubation in the dark, 30μL of negative serum (from male donors) is added (source of complement). After another 30 min of incubation in the dark and 3 washes with the wash buffer, 50 μL of phycoerythrinconjugated anti-human C3d was added to the beads and incubated for 30 minutes in the dark at room temperature. The fluorescence intensities of the samples were measured by using a Luminex 200 system. Positive results were assigned when 2 of 3 criteria (Background Adjusted MFI (Bg Adj) > 1500, R-Strength > 5.0, or BCR-Neg > 5.0) were fulfilled.

Biopsy assessment

Kidney biopsy tissue was processed for light microscopy analysis, performed after fixation in Dubosq Brazil solution, 2-μm paraffin section, and coloration by Masson trichrome, hematoxylin & eosin, periodic acid-Schiff and Marinozzi silver stainings. C4d staining was performed on paraffin sections by immunohistochemical (IHC) analysis using a rabbit anti-human C4d polyclonal IgG, or by immunofluorescent (IF) staining performed on 4-μm frozen sections with monoclonal mouse anti-human C4d antibody. Peritubular C4d was considered positive if C4d staining was identified in at least 10% of peritubular capillaries (C4d2 or C4d3) by IF or in any peritubular capillaries by IHC (C4d score, >0). All transplant biopsies for dnDSA were blinded and reviewed for the study and scored according to the 2017 Banff classification [11] by a single experienced pathologist (A.F.).

Ethical requirements

This study was in accordance with the Helsinki declaration, and was approved by the local ethics board for non-invasive health research (Comité d’Ethique pour la Recherche Non Interventionnelle CERNI N°E2021-06, for the Centre de Protection des Personnes Nord-Ouest-I, Rouen University Hospital, Rouen, France), which waived the need for informed consent in this retrospective analysis.

Statistical analysis

Statistics were performed using Statview version 5.0 (SAS Institute Inc., Brie Comte Robert, France). Quantitative variables were expressed as mean ± SD, whereas qualitative variables were expressed in numbers and percentages. Categorical variables were compared using the chi-square test, and the Student t test was used for continuous variables. A 2-sided P value of <0.05 was considered to be statistically significant. Patient and graft survival data were assessed by Kaplan–Meier analysis. Logrank test was used to compare survival between groups. To demonstrate a correlation between MFI and BCM for the DSA detected, we performed a Pearson correlation test, with a correlation coefficient (r): r < 0.25 indicating low correlation, 0.25 < r < 0.5 moderate correlation, 0.5 < r < 0.75 strong correlation, and r > 0.75 excellent correlation.

Results

Detection of dn DSA with IM Lifescodes SAB

Among the 123 patients of our previous study [5], 112 had sera tested with IM Lifescodes SAB, class I and II. Within these 112 sera, 16 (14.3%) were negative for DSA and 96 (85.7%) were positive for at least one DSA.

Patients tested negative for DSA with IM Lifescodes SAB

In the group of patients tested negative for DSA (n = 16) with IM test, mean MFI of iDSA with OL test was 3420 ± 2914 (1017–10957) and MFI of sDSA was 3700 ± 3335 (1017–11858). Table 1 shows the specificity of the DSA positive with OL and negative with IM; MFI, BCM, BCR and AD-BCR were also reported. Missed iDSA were class I DSA in 4 cases and class II DSA in 12 cases. MFI of sDSA were between 1000 and 3000 in 10 cases, between 3 000 and 10 000 in 4 cases and above 10 000 in 2 cases, with OL test. In this group systematic biopsies showed active sABMR in 1 patient (6.25%) and chronic active sABMR in 2 patients (12.5%).

Table 1. Antigen specificity of the immunodominant DSA in patients with DSA positive with Labscreen SAB and negative with Lifecodes SAB.
Antigen specificity of the dn DSA Nb of KTR MFI (OL) BCM (IM) BCR (IM) AD-BCR (IM)
A2 1 1600 99 1.2 1.19
A3 1 1300 35 0.32 0.33
B37 1 5802 0 0 0
B8 1 2400 206 0.83 0.75
DQ2 3 1017–3750–8882 312–0–0 1.13–0–0 1.3–0–0
DQ5 4 1556–2000–2443–10957 35-292-13-82 0.86–3.21–2.33–1.64 0.45–3.02–2.07–1.79
DQ6 1 5500 0 0 0
DQ7 3 1608–1980–2000 0-167-202 0–1.96–1.92 0–1.85–1.80
DR13 1 1924 0 0 0
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KTR: kidney transplantation recipents; dn DSA: de novo donor specific antibody; iDSA: immunodominant DSA; MFI: mean fluorescence intensity; OL: One Lambda. SAB: single antigen beads; IM: Immucor; BCM: background corrected MFI; BCR: BCM divided by the raw MFI of the lowest ranked bead for a locus; AD-BCR: antigen density corrected BCR values.

Patients tested positive for DSA with IM Lifescodes SAB

In the group tested positive for DSA (n = 96), iDSA were the same with the 2 vendors in 80 patients out of 96 (83.3%). Fig 1 (panel A) shows the statistically significant but moderate correlation between BCM (IM) and MFI (OL) of iDSA, in this subgroup of 80 patients (r = 0.48). Correlation for the 2 most represented DSA, anti DQ7 (n = 27) and anti DQ2 (n = 12), was also reported in Fig 1, in panel B and C, respectively.

Fig 1.

Fig 1

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A. Correlation between BCM (Immucor Lifecodes SAB) and MFI (One Lambda Labscreen SAB) of iDSA, in the subgroup of 80 patients tested positive for the same iDSA. B. Correlation between BCM (Immucor Lifecodes SAB) and MFI (One Lambda Labscreen SAB) for anti DQ2 specificity. C. Correlation between BCM (Immucor Lifecodes SAB) and MFI (One Lambda Labscreen SAB) for anti DQ7 specificity. iDSA: immunodominant; MFI: mean fluorescence intensity; SAB: single antigen beads.

General characteristics of the 96 patients are presented in Table 2. Systematic biopsies showed active sABMR (group A) in 30 patients (31.2%), chronic active sABMR (group C) in 17 patients (17.7%) and no lesions of sABMR (group N) in 49 KT recipients (51%). BCM, BCR and AD-BCR of iDSA or sDSA were not statistically different between the 3 groups (Fig 2):

Table 2. Baseline characteristics.
n = 96
Sex M/F n (%) 64/32 (66.7/33.3)
Age (years). mean ± SD 50.6 ± 13.1
Cause of ESRD. n (%)
Diabetes 3 (3.1)
Glomerulonephritis 23 (23.9)
Hypertension 4 (4.2)
Cystic renal disease and genetic nephropathy 31 (32.4)
CTIN and uropathy 15 (15.6)
Other 5 (5.2)
Unknown 15 (15.6)
Prior solid organ transplant. n (%) 2 (2.1)
Donor type. n (%)
Deceased donor 90 (93.8)
Living donor 6 (6.2)
Class I antibodies before KT n (%) 7 (7.3)
Class II antibodies before KT n (%) 8 (8.3)
eGFR on the day of the biopsy ml/min/1.73 m2 53.3 ± 18.9
Proteinuria/creatininuria ratio g/g 0.18 ± 0.15
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ESRD: end stage renal disease; CTIN: chronic tubule-interstitial nephropathy; KT: kidney transplantation; eGFR: estimated glomerular filtration rate; M: male; F: female.

Fig 2. BCM, BCR and AD-BCR (Lifecodes SAB) of iDSA and sDSA according to histological diagnosis on the biopsy: active sABMR (A), chronic active sABMR (C) and no sABMR (N).

Fig 2

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sABMR: subclinical ABMR; DSA: donor specific antibodies; iDSA: immunodominant DSA; sDSA: sum of the DSA; SAB: single antigen beads. In all the box plots, the top of the box represents the 75th percentile, the bottom of the box represents the 25th percentile, and the middle line represents the 50th percentile. The whiskers represent the highest and the lowest values that are not outliers or extreme values. Black dots represent outliers.

  • for iDSA, mean BCM was 5455 ± 4685 in group A, 6428 ± 6957 in group C and 6514 ± 6261in group N, and were not statistically different.

  • for sDSA, mean BCM was 7014 ± 6080 in group A, 7255 ± 7900 in group C and 7153 ± 6654in group N, and were not statistically different.

Fig 3 shows the proportion of the 3 histological diagnoses according to different ranges of intensity of sDSA with IM LSAB assay. The percentage of active sABMR was similar with BCM below 3000 or above 10000 (27% vs 25.7%).

Fig 3. Proportion of the 3 histological diagnoses according to different range of intensity (BCM) of sDSA according to Immucor Lifecodes SAB assay.

Fig 3

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The numbers on the top of each bar refer to the percentage of each histological pattern. sDSA: sum of the DSA; SAB: single antigen beads.

Detection of C3d-fixing DSA

In the subset of 96 patients tested positive for DSA by the 2 assays, 53 (55.2%) were positive and 43 (44.8%) were negative for C3d-fixing DSA. The proportions of patients with positive C3d-fixing DSA were not statistically different in patients with active sABMR, chronic active sABMR or without sABMR (56.7% vs 47.1% vs 57.1%, p = 0.74). In the group of 47 patients with sABMR, Banff lesions were not different between positive and negative C3d-fixing DSA and peritubular capillary C4d staining was also similar (Fig 4). In the 49 patients without sABMR, Banff lesions were similar but C4d staining was higher in the C3d-fxing DSA group without reaching significance (20.7% vs 5%, p = 0.08).

Fig 4.

Fig 4

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Banff lesions and peritubular capillary C4d staining according to C3d-fixing DSA status in (A) the 47 patients witch sABMR and in (B) 49 patients without sABMR. sABMR: subclinical ABMR; DSA: donor specific antibodies. For the Banff score: g: glomerulitis, i: interstitial inflammation, t: tubulitis, ptc: peritubular capillaritis, cg: allograft glomerulopathy, ci: interstitial fibrosis, ct: tubular atrophy, cv:fibrous intimal thickening. Each item is rated 0, 1, 2 or 3. For the C4d staining: each number on the top of the bar refers to the percentage of peritubular capillary C4d positive.

Death censored graft survival was not different between the 2 groups (C3d positive or negative DSA) as presented in Fig 5. eGFR was not different at 1, 3- and 5-years post biopsy according to the C3d-fixing status in the subset of patients with sABMR or without sABMR (Fig 6):

Fig 5. Death censored graft survival according to C3d-fixing DSA status.

Fig 5

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DSA: donor specific antibodies.

Fig 6. eGFR at 1, 3- and 5-years post biopsy according to the C3d-fixing DSA status in the subset of patients with sABMR or without sABMR.

Fig 6

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eGFR, estimated glomerular filtration rate; MDRD, Modification of Diet in Renal Disease; sABMR: subclinical antibody mediated rejection; DSA: donor specific antibodies.

  • In patients with sABMR, delta of eGFR between D0 and 5 years was -15.8 mL/min/1.73 m2 in patients with C3d-binding DSA and -23.9 mL/min/1.73 m2 in patients without C3d-binding DSA (p = 0.25).

  • In patients without sABMR, delta of eGFR between D0 and 5 years was -3.7 mL/min/1.73 m2 in patients with C3d-binding DSA and +0.6 mL/min/1.73 m2 in patients without C3d-binding DSA (p = 0.37).

The intensity (BCM) of iDSA and sDSA were statistically higher in the C3d-fixing DSA group (Fig 7):

Fig 7. Intensity (BCM) of iDSA and sDSA according to the C3d-fixing DSA status.

Fig 7

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dnDSA: de novo DSA; iDSA: immunodominant DSA; sDSA: sum of the DSA. In all the box plots, the top of the box represents the 75th percentile, the bottom of the box represents the 25th percentile, and the middle line represents the 50th percentile. The whiskers represent the highest and the lowest values that are not outliers or extreme values. Black dots represent outliers.

  • for iDSA, mean BCM was 9020 ± 6284 in patients with C3d-fixing DSA, 2652 ± 2665 in patients without C3d-fixing DSA (p< 0.0001).

  • for iDSA, mean BCM was 10087 ± 6909 in patients with C3d-fixing DSA, 3480 ± 4016 in patients without C3d-fixing DSA (p< 0.0001).

Discussion

To the best of our knowledge we report here the first study evaluating the LSAB from IM in a context of dnDSA detected by OL LSAB paired with systematic kidney biopsy to detect early stage of humoral rejection. Recently [5], we demonstrated, as other teams did [7, 8] that a protocol biopsy performed for a subclinical dn DSA (MFI > 1000 with OL SAB) led to the diagnosis of sABMR in up to 40% of the biospies: we showed that the diagnosis of acute sABMR was statistically associated with the MFI of iDSA and sDSA with LSAB from OL [5]. In the present study, we failed to find such association with IM test. Neither BCM, BCR nor AD-BCR were significantly different between patients with or without sABMR.

A major part of the literature on HLA antibodies, DSA detection and intensity cutoff are based on the OL test [12]. There are scarce data in the literature comparing the detection of DSA between IM and OL [13, 14]. Clerkin et al. [15] compared detection of HLA antibodies by the 2 providers in heart and lung transplant recipients. Most antibodies with moderate to high MFI titer (> 4000) were detected by both assays. Although correlation between the assays was present, significant variance was shown. Reed et al. [16] reported MFI variation to be greater with OL than IM. As We also reported that the results of Lifecodes LSAB test cannot be interpreted with OL usual criteria [9]. In the present study correlation between MFI and BCM was low, even regarding the most common specificity of DSA (r<0.5). Furthermore, in 14.3% SAB from IM did not detect any DSA. One could also argue that some class 1 DSA detected with Labscreen SAB but not with Lifecodes SAB are against denatured antigen and so, are not pathogen [17, 18]. Nevertheless, in the present study, in patients positive with Labscreen SAB but negative with Lifecodes SAB, 3 patients experienced sABMR, all in stable situation. In a study on DSA negative ABMR, Senev et al. reported a large part of DSA negative ABMR 123/208 (58.2%), possibly positive in an uncertain proportion with OL test [19]. We have to be very cautious not to interpret studies dealing with IM tests them as if they were performed with OL SAB.

Despite the fact that SAB is not a quantitative test, Schinstock et al. [8] depicted a particular risk of active sAMR associated with a sum MFI of dnDSA over 3000. In the context of preformed DSA, Lefaucheur et al. [20] reported that the relative risk for graft loss in patients who underwent transplantation with peak HLA–DSAs >3000 was 3.8. Wiebe et al. [21] found that dnDSA MFI sum at the time of dnDSA detection predict the risk of post-dnDSA graft loss. This suggests that performing protocol biopsy for dnDSA could be guided by the MFI of the DSA. Unfortunately, we did not observe this effect in our study with intensity criteria of Lifecodes SAB: with IM, the proportion of patients with active sABMR is comparable below 3000 and above 10 000 of BCM (Fig 3). To our knowledge there is very scarce literature reporting on the predictive quantitative effect of BCM, BCR or AD-BCR on the risk of ABMR and graft survival. Recently, Senev et al. [22], reported a large cohort of 1000 KT recipients, screened for DSA with IM test. This study provided insights on the risk of ABMR and the predictive role of intensity criteria such as BCM. They validated a MFI cut‐off value of 1400 units for HLA‐DSA positivity proposed by the STAR working group and standardization study, and confirmed that patients with preformed DSA of background‐reduced MFI <1400 had excellent allograft survival [23]. However, this should be interpreted cautiously because the thresholds depend on the vendor kit and on the instrument used, and perhaps on clinical characteristics. Therefore, it is not possible to simply generalize the thresholds found in their cohort. Furthermore, there was no cut off value regarding the risk of ABMR reported in this study.

In the last few years, there has been a large interest for complement-fixing DSA. In 69 KT recipients who fullfilled the diagnostic criteria for AMR Sicard et al. [24] reported that C3d-binding capacity of DSA at the time of AMR diagnosis allows for identification of patients at risk for allograft loss. Loupy et al. [25] reported in a larger cohort that the presence of complement-binding donor-specific anti-HLA antibodies (C1q fixing complement) after transplantation was associated with a risk of graft loss (hazard ratio, 4.78; 95% confidence interval [CI], 2.69 to 8.49) when adjusted for clinical, functional, histological, and immunologic factors. These antibodies were also associated with an increased rate of antibody-mediated rejection, a more severe graft injury phenotype with more extensive microvascular inflammation, and increased deposition of complement fraction C4d within graft capillaries. In 2018, a meta-analysis published by Bouquegneau et al. [26], concluded that circulating complement-activating anti-HLA DSA had a significant deleterious impact on solid organ transplant survival and risk of rejection. Unfortunately we did not observe in the present any association between C3d-fixing DSA proportion and the diagnosis of sABMR. Furthemore, the intensity of the lesions of Banff score and C4d deposition were similar between C3d positive and C3d negative sABMR. C3d-fixing DSA were not predictive of eGFR at 5 years post biopsy nor of graft survival. In this context of subclinical dnDSA, Yamamoto et al. [6] found in a small cohort that C1q-binding DSA was a significant subclinical AMR-related factor, whereas Wiebe et a. [27] did not find any association between C1q status and AMR occurrence. Lastly, previous studies have shown a relationship between higher IgG intensity and either C3d [24] or C1q [28, 29]. We found the same correlation between BCM and C3d fixing complement. The pathogenic role of complement fixation in indolent sABMR remains to be defined.

Although our study is multicentric, with a relatively important sample, this is a retrospective study with potential bias. First of all, the fact that the Immucor tests were performed years after serum storation while the OL results were done at the time of biopsy could in a certain level of variance that might explain differences between the two systems. Secondly, no agent were added to the sera to rule out any interference of prozone / complement interference with IM LSAB. Nevertheless there is a distinct profile between the 2 tests and OL test shown clearly a more important prozone effect than IM LSAB [30]. Regarding the analysis of the sera of KT recipients with IM, a very important point is that all sera were tested by the same laboratory, experienced with the test, and overall with the same and most recent kits. It would also be interesting to test a cohort of patients with Immucor DSA + with OL test to evaluate the rate of patients with negative DSA with OL. Finally, regarding complement-fixing DSA, we did not test the sera with C1q binding DSA, which could have been interesting, to compare with C3d-biding DSA results.

In conclusion, in this relatively large multicentric study, we reported that IM Lifecodes SAB intensity criteria (BCM, BCR and AD-BCR) of DSA are not predictive of sABMR when a systematic graft biopsy is performed for dnDSA without graft dysfunction. Furthermore, a significant proportion of patients with OL positive DSA were negative with Lifecodes SAB test with clinical and histological impact. Morever, this study did not identify C3d-fixing DSA as a potential predictive factor for sABMR diagnosis or graft survival. These results suggest that identification and quantification of dnDSA with the 2 tests, OL and IM, are not similar in the setting of dnDSA monitoring post-transplant.

Abbreviations

eGFR

estimated glomerular filtration rate

MDRD

Modification of Diet in Renal Disease

KT

kidney transplantation

ABMR

antibody mediated rejection

sABMR

subclinical ABMR

DSA

donor specific antibodies

dnDSA

de novo DSA

iDSA

immunodominant DSA

sDSA

sum of the DSA

CNI

calcineurin inhibitors

MMF

mycophenolate mofetil

S

steroids

Mtor-I

mammalian target of rapamycin inhibitors

LSAB

Luminex single antigen beads

OL

One Lambda

IM

Immucor

IHC

immunohistochemical

IF

immunofluorescent

Data Availability

All relevant data are within the paper.

Funding Statement

The authors received no specific funding for this work.

References

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Decision Letter 0

Stanislaw Stepkowski

1 Mar 2021

PONE-D-21-00625

Intensity of de novo DSA detected by Immucor Lifecodes Assay and C3d fixing antibodies are not predictive of subclinical ABMR after Kidney Transplantation.

PLOS ONE

Dear Dr. Bertrand,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The study examined the role of de novo subclinical DSA and their potential C3-fxing antibody. Based on over 100 patients, the authors conclude that no correlation was observed between subclinical antibody mediated rejection (sAMR) and the detection of C3-fixing antibody. The reviewer # 1 requested that the authors make mor conclusive statement based on their analysis. Indeed, the statement that more study should be performed is not satisfactory as almost every study needs a continuation. The Reviewer # 2 indicated at several editorial changes which need to be addressed. 

The authors need to address points raised by the reviewers 1 and 2:

Reviewer # 1:

Bertrand and colleagues conducted a retrospective, multicenter, study to test potential correlation between the C3D-fixing antibody and IgG SAB by Imucor in a cohort of patients previously tested by the IgG SAB assay by One Lambda (MFI > 1000).

The authors confirmed previous report showing some differences between data obtained by the two vendors (Immucor and One Lambda), looking either at immunedominant DSA or sum of DSAs. They further attempted comparison between histological diagnosis on the biopsy and antibody characteristics and show no correlation

Biopsies were obtained with significant time difference from sera test (within 6 months for some samples) and the purpose for obtaining them is not clear – does not look like the same time point from transplant for all patients – the only thing in common is that they did not correlate with graft dysfunction.

Overall, this is a relatively small study cohort, utilizing patients from 9 different centers, with no systematic approach to obtaining either biopsies or serum samples. With that – it is not clear what the clinical value of their finding is. 

Reviewer # 2:

Review Comments to the Authors Manuscript title: Intensity of de novo DSA detected by Immucor Lifecodes Assay and C3d fixing antibodies are not predictive of subclinical ABMR after Kidney Transplantation. 

The rationale and objective of this study is clear. Moreover, authors successfully describe the contribution of this work to the field, while recognizing its limitations and noting outstanding questions. However, the manuscript requires much improvement to the writing quality and data presentation and interpretation. The manuscript lacks organization and focus due to run-on and awkwardly structured sentences. In addition, figures are missing crucial details required to interpret the data. 

Several results are stated without a clear connection to the supporting data. Authors should address the following points: 

Readers could benefit from a more detailed description of HLA donor specific antibodies and de novo DSA in the introduction, and how this is relevant to transplant pathology. Please explain rationale for accepting positive results for the IM Lifecodes LSAB after 2 of 3 criteria were met, instead of all requiring all criteria to be met. Please indicate negative control beads for the IM Lifecodes LSAB. 

Authors should specify concentration of beads, phycoerythrin conjugated goat antihuman IgG, anti-human C3d, instead of, or in addition to the volume used, as volume is arbitrary without knowing this information. 

Please describe methods for light microscopy and immunofluorescence staining of kidney tissue. In statistical analysis, authors should indicate a Student t-test was used. Please describe the advantages/disadvantages of the iDSA and DSA parameters. For example, why were both used, and is one superior to the other? 

Please indicate all abbreviations used in Table 1. Authors should consider reformatting the methods and results sections from bullet points to paragraphs. For reader’s ease, authors should consider labeling Figure 1 panels A-C to indicate the correlation described (as indicated in the Figure legend). For Figure 2, please define the descriptive statistics reflected in the box and whisker plot. Please label y-axes in Figures 2, 3, 4, 5, and 6. Please specific what the numbers refer to on the top of each bar in Figure 3. In addition, please explain in more detail the significance of Figure 3. Figure 4 is not clearly described and thus, difficult to interpret. To address this, please indicate the abbreviations used (g+cpt, i+t, ci+ct, cv, and cg) in the figure legend. Please define the numbers indicated on top of the bar graphs in the figure legend. 

Authors should indicate the Figure showing the data supporting the following results: “The proportions of patients with positive C3d-fixing DSA were not statistically different in patients with active sABMR, chronic active sABMR or without sABMR (56.7% vs 47.1 % vs 57.1%, p=NS).” 

Please indicate the figure showing the data supporting the following results that begin with the sentence: “The intensity (BCM) of iDSA and sDSA were statistically higher in the C3dfixing DSA group…” 

Please be consistent with reporting non-significant results as either “NS” (without a p-value) or reporting a p-value greater than 0.05. Preference is to report p-values. Authors should also consider reporting actual p-values for significant data (instead of indicating p 

The discussion should be condensed to be more concise and direct because it lacks clear focus. The discussion should also better delineate between the findings from the group’s previous work and the findings form the current study. Finally, authors should be cautious about describing data as “perfectly comparable” to previous studies. 

“Finally” is spelled incorrectly in the discussion.

Please submit your revised manuscript by Apr 10 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Stanislaw Stepkowski

Academic Editor

PLOS ONE

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6.Thank you for submitting the above manuscript to PLOS ONE. During our internal evaluation of the manuscript, we found some minor occurrences of overlapping text with the following previous publication(s), some of which you are an author, which needs to be addressed:

- https://journals.lww.com/transplantjournal/Abstract/2021/03000/Complex_Linkage_Disequilibrium_Effects_in_HLA_DPB1.27.aspx?context=FeaturedArticles&collectionId=23

- https://onlinelibrary.wiley.com/doi/full/10.1111/ajt.15414

- https://www.nejm.org/doi/10.1056/NEJMoa1302506

- https://journals.lww.com/transplantjournal/Fulltext/2019/03000/Comparison_of_Two_Luminex_Single_antigen_Bead_Flow.28.aspx

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Please revise the manuscript to quote or rephrase the duplicated text and cite your sources for text outside the methods section. Please note that further consideration is dependent on the submission of a manuscript that addresses these concerns about the overlap in text with published work.

Additional Editor Comments:

The study examined the role of de novo subclinical DSA and their potential C3-fxing antibody. Based on over 100 patients, the authors conclude that no correlation was observed between subclinical antibody mediated rejection (sAMR) and the detection of C3-fixing antibody. The reviewer # 1 requested that the authors make mor conclusive statement based on their analysis. Indeed, the statement that more study should be performed is not satisfactory as almost every study needs a continuation. The Reviewer # 2 indicated at several editorial changes which need to be addressed.

The authors need to address points raised by the reviewers 1 and 2:

Reviewer # 1:

Bertrand and colleagues conducted a retrospective, multicenter, study to test potential correlation between the C3D-fixing antibody and IgG SAB by Imucor in a cohort of patients previously tested by the IgG SAB assay by One Lambda (MFI > 1000).

The authors confirmed previous report showing some differences between data obtained by the two vendors (Immucor and One Lambda), looking either at immunedominant DSA or sum of DSAs. They further attempted comparison between histological diagnosis on the biopsy and antibody characteristics and show no correlation

Biopsies were obtained with significant time difference from sera test (within 6 months for some samples) and the purpose for obtaining them is not clear – does not look like the same time point from transplant for all patients – the only thing in common is that they did not correlate with graft dysfunction.

Overall, this is a relatively small study cohort, utilizing patients from 9 different centers, with no systematic approach to obtaining either biopsies or serum samples. With that – it is not clear what the clinical value of their finding is.

Reviewer # 2:

Review Comments to the Authors Manuscript title: Intensity of de novo DSA detected by Immucor Lifecodes Assay and C3d fixing antibodies are not predictive of subclinical ABMR after Kidney Transplantation.

The rationale and objective of this study is clear. Moreover, authors successfully describe the contribution of this work to the field, while recognizing its limitations and noting outstanding questions. However, the manuscript requires much improvement to the writing quality and data presentation and interpretation. The manuscript lacks organization and focus due to run-on and awkwardly structured sentences. In addition, figures are missing crucial details required to interpret the data.

Several results are stated without a clear connection to the supporting data. Authors should address the following points:

Readers could benefit from a more detailed description of HLA donor specific antibodies and de novo DSA in the introduction, and how this is relevant to transplant pathology. Please explain rationale for accepting positive results for the IM Lifecodes LSAB after 2 of 3 criteria were met, instead of all requiring all criteria to be met. Please indicate negative control beads for the IM Lifecodes LSAB.

Authors should specify concentration of beads, phycoerythrin conjugated goat antihuman IgG, anti-human C3d, instead of, or in addition to the volume used, as volume is arbitrary without knowing this information.

Please describe methods for light microscopy and immunofluorescence staining of kidney tissue. In statistical analysis, authors should indicate a Student t-test was used. Please describe the advantages/disadvantages of the iDSA and DSA parameters. For example, why were both used, and is one superior to the other?

Please indicate all abbreviations used in Table 1. Authors should consider reformatting the methods and results sections from bullet points to paragraphs. For reader’s ease, authors should consider labeling Figure 1 panels A-C to indicate the correlation described (as indicated in the Figure legend). For Figure 2, please define the descriptive statistics reflected in the box and whisker plot. Please label y-axes in Figures 2, 3, 4, 5, and 6. Please specific what the numbers refer to on the top of each bar in Figure 3. In addition, please explain in more detail the significance of Figure 3. Figure 4 is not clearly described and thus, difficult to interpret. To address this, please indicate the abbreviations used (g+cpt, i+t, ci+ct, cv, and cg) in the figure legend. Please define the numbers indicated on top of the bar graphs in the figure legend.

Authors should indicate the Figure showing the data supporting the following results: “The proportions of patients with positive C3d-fixing DSA were not statistically different in patients with active sABMR, chronic active sABMR or without sABMR (56.7% vs 47.1 % vs 57.1%, p=NS).”

Please indicate the figure showing the data supporting the following results that begin with the sentence: “The intensity (BCM) of iDSA and sDSA were statistically higher in the C3dfixing DSA group…”

Please be consistent with reporting non-significant results as either “NS” (without a p-value) or reporting a p-value greater than 0.05. Preference is to report p-values. Authors should also consider reporting actual p-values for significant data (instead of indicating p

The discussion should be condensed to be more concise and direct because it lacks clear focus. The discussion should also better delineate between the findings from the group’s previous work and the findings form the current study. Finally, authors should be cautious about describing data as “perfectly comparable” to previous studies.

“Finally” is spelled incorrectly in the discussion.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The rationale and objective of this study is clear. Moreover, authors successfully describe the contribution of this work to the field, while recognizing its limitations and noting outstanding questions. However, the manuscript requires much improvement to the writing quality and data presentation and interpretation. The manuscript lacks organization and focus due to run-on and awkwardly structured sentences. In addition, figures are missing crucial details required to interpret the data. Several results are stated without a clear connection to the supporting data.

Please see attached document for remaining comments.

Reviewer #2: Bertrand and colleagues conducted a retrospective, multicenter, study to test potential correlation between the C3D-fixing antibody and IgG SAB by Imucor in a cohort of patients previously tested by the IgG SAB assay by One Lambda (MFI > 1000).

The authors confirmed previous report showing some differences between data obtained by the two vendors (Immucor and One Lambda), looking either at immunedominant DSA or sum of DSAs. They further attempted comparison between histological diagnosis on the biopsy and antibody characteristics and show no correlation

Biopsies were obtained with significant time difference from sera test (within 6 months for some samples) and the purpose for obtaining them is not clear – does not look like the same time point from transplant for all patients – the only thing in common is that they did not correlate with graft dysfunction.

Overall, this is a relatively small study cohort, utilizing patients from 9 different centers, with no systematic approach to obtaining either biopsies or serum samples. With that – it is not clear what the clinical value of their finding is.

**********

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Reviewer #1: No

Reviewer #2: No

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Attachment

Submitted filename: Review Comments to the Author_Intensity of de novo DSA detected by Immucor Lifecodes Assay and C3d fixing antibodies are not predictive of subclinical ABMR after Kidney Transplantation.pdf

Click here for additional data file. (27.6KB, pdf)
PLoS One. 2021 Apr 22;16(4):e0249934. doi: 10.1371/journal.pone.0249934.r002

Author response to Decision Letter 0

16 Mar 2021

The reviewer # 1 requested that the authors make more conclusive statement based on their analysis. Indeed, the statement that more study should be performed is not satisfactory as almost every study needs a continuation.

Thank you for your analysis. We tried to make more conclusive statement in our revised manuscript.

The Reviewer # 2 indicated at several editorial changes which need to be addressed.

• Readers could benefit from a more detailed description of HLA donor specific antibodies

We added in the introduction a more detailed description on HLA DSA and dn DSA

• and de novo DSA in the introduction, and how this is relevant to transplant pathology.

We added in the introduction a more detailed description on HLA DSA and dn DSA.

• Please explain rationale for accepting positive results for the IM Lifecodes LSAB after 2 of 3 criteria were met, instead of all requiring all criteria to be met.

Thank you for your comment. Accepting positive results for the IM Lifecodes LSAB after 2 of 3 criteria were met is used according to the manufacturers' instructions. Nevertheless, when BCM is low (below 1500) but the amount of antigen in the bead is also low, result could be positive because BCR and AD-BCR are positive.

• Please indicate negative control beads for the IM Lifecodes LSAB.

As indicated in the methods section, the negative control beads typically yield low MFI values (lot specific) when treated with the positive or negative control sera.

• Authors should specify concentration of beads, phycoerythrin conjugated goat antihuman IgG, anti-human C3d, instead of, or in addition to the volume used, as volume is arbitrary without knowing this information.

We completely understand your comment. Unfortunately, all these components are provided with LIFECODES LSAB and LIFECODES C3d kits by the manufacturer and the concentration of beads, phycoerythrin conjugated goat antihuman IgG, anti-human C3d are not available.

• Please describe methods for light microscopy and immunofluorescence staining of kidney tissue.

Thank you for your comment. Light microscopy analysis was performed after fixation in Dubosq Brazil solution, 2-μm paraffin section, and coloration by Masson trichrome, hematoxylin & eosin, periodic acid-Schiff and Marinozzi silver stainings. C4d staining was performed on paraffin sections by immunohistochemical (IHC) analysis using a rabbit anti-human C4d polyclonal IgG, or by immunofluorescent staining performed on 4-μm frozen sections with monoclonal mouse anti-human C4d antibody. We added this point in the revised manuscript.

• In statistical analysis, authors should indicate a Student t-test was used.

This point was added in the statistical analysis section.

• Please describe the advantages/disadvantages of the iDSA and DSA parameters. For example, why were both used, and is one superior to the other?

Thank you for your comment. Both iDSA and sDSA were used in this study because some kidney transplant recipients had more than 1 DSA. Even if the intensity of the iDSA is not sufficient to predict subclinical ABMR, the combination of all DSA would be.

Réf: Bertrand D, Farce F, Laurent C, et al. Comparison of Two Luminex Single-antigen Bead Flow Cytometry Assays for Detection of Donor-specific Antibodies after Renal Transplantation. Transplantation. 2019;103(3):597–603. doi:10.1097/TP.0000000000002351

• Please indicate all abbreviations used in Table 1.

We indicate all abbreviations used in Table 1 in the revised manuscript.

• Authors should consider reformatting the methods and results sections from bullet points to paragraphs.

Thank you for your help to improve the methods and results sections, presented in the revised manuscript.

• For reader’s ease, authors should consider labeling Figure 1 panels A-C to indicate the correlation described (as indicated in the Figure legend).

We corrected this point in the manuscript

• For Figure 2, please define the descriptive statistics reflected in the box and whisker plot.

We defined the descriptive statistics reflected in the box and whisker plot.

• Please label y-axes in Figures 2, 3, 4, 5, and 6.

We labeled y-axes in Figures 2, 3, 4, 5, and 6.

• Please specificy what the numbers refer to on the top of each bar in Figure 3. In addition, please explain in more detail the significance of Figure 3.

Thank you for your comment. We made these corrections in the revised manuscript.

• Figure 4 is not clearly described and thus, difficult to interpret. To address this, please indicate the abbreviations used (g+cpt, i+t, ci+ct, cv, and cg) in the figure legend. Please define the numbers indicated on top of the bar graphs in the figure legend.

Thank you for your comment. We made these corrections in the revised manuscript.

• Authors should indicate the Figure showing the data supporting the following results: “The proportions of patients with positive C3d-fixing DSA were not statistically different in patients with active sABMR, chronic active sABMR or without sABMR (56.7% vs 47.1 % vs 57.1%, p=NS).”

Thank you for your comment. We did not add an extra figure for this point meaning that it was not useful for the readers.

• Please indicate the figure showing the data supporting the following results that begin with the sentence: “The intensity (BCM) of iDSA and sDSA were statistically higher in the C3dfixing DSA group…”

Thank you for your comment. Figure 7 about the intensity (BCM) of iDSA and sDSA according C3d fixing DSA positivity was added in the revised manuscript.

• Please be consistent with reporting non-significant results as either “NS” (without a p-value) or reporting a p-value greater than 0.05. Preference is to report p-values. Authors should also consider reporting actual p-values for significant data (instead of indicating p<0.0001).

We modified this point in the new manuscript.

• This sentence is awkwardly written: “In our previous published study 4, we demonstrated that this strategy led to the diagnosis of sABMR in up to 40 %.”

We completely agree with your comment. We modified this sentence in the text.

• The discussion should be condensed to be more concise and direct because it lacks clear focus. The discussion should also better delineate between the findings from the group’s previous work and the findings form the current study. Finally, authors should be cautious about describing data as “perfectly comparable” to previous studies.

Thank you for this comment. We modified the discussion in order to be more concise and direct. We also tried to delineate between the findings from our group’s previous work and the findings form the current study.

• “Finally” is spelled incorrectly in the discussion.

We modified this point in the new manuscript.

Attachment

Submitted filename: Response to Reviewers.docx

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Decision Letter 1

Stanislaw Stepkowski

29 Mar 2021

Intensity of de novo DSA detected by Immucor Lifecodes Assay and C3d fixing antibodies are not predictive of subclinical ABMR after Kidney Transplantation.

PONE-D-21-00625R1

Dear Dr. Bertrand,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Stanislaw Stepkowski

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Acceptance letter

Stanislaw Stepkowski

12 Apr 2021

PONE-D-21-00625R1

Intensity of de novo DSA detected by Immucor Lifecodes Assay and C3d fixing antibodies are not predictive of subclinical ABMR after Kidney Transplantation.

Dear Dr. Bertrand:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Stanislaw Stepkowski

Academic Editor

PLOS ONE

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