FCXM Results Research Articles

P.180: Results of CDC-XM and FCXM cross-match in highly sensitized patients with CKD.

P.180: Results of CDC-XM and FCXM cross-match in highly sensitized patients with CKD.

Intravenous Immunoglobulins Alone for the Desensitization of Lung Transplant Recipients with Preformed Donor Specific Antibodies and Negative Flow Cytometry Crossmatch.

The lack of evidence regarding optimal desensitization strategies for lung transplant candidates with preformed donor specific anti-human leukocyte antigen antibodies (DSAs) has led to varying approaches among centers towards this patient group. Our institution's desensitization protocol for recipients with preformed DSAs and negative flow cytometry crossmatch (FCXM) consists of intravenous immunoglobulin (IVIG) as the sole therapy. The study aimed to determine outcomes using this approach. This retrospective study included adults who underwent lung-only transplantation for the first time between January 2015 and March 2022 at a single center. We excluded patients with positive or missing FCXM results. Transplant recipients with any DSA ≥ 1000 MFI on latest testing within three months of transplant were considered DSA-positive, while recipients with DSAs <1000 MFI and those without DSAs were assigned to the low-level/negative group. Graft survival (time to death/retransplantation) and chronic lung allograft dysfunction (CLAD)-free times were compared between groups using Cox proportional hazards models. Thirty-six out of 167 eligible patients (22%) were DSA-positive. At least 50% of preformed DSAs had documented clearance (decrease to <1000 MFI) within the first 6 months of transplant. Multivariable Cox regression analyses did not detect a significantly increased risk of graft failure (aHR 1.04 95%CI 0.55-1.97) or chronic lung allograft dysfunction (aHR 0.71 95%CI 0.34-1.52) in DSA-positive patients compared to patients with low-level/negative DSAs. Incidences of antibody-mediated rejection (p=1.00) and serious thromboembolic events (p=0.63) did not differ between study groups. We describe a single-center experience of administering IVIG alone to lung transplant recipients with preformed DSAs and negative FCXM. Further studies are required to confirm the efficacy of this strategy against other protocols.

Impact of COVID-19 pandemic on transplant laboratories: How to mitigate?

A positive flow cytometry crossmatch (FCXM) due to donor specific antibodies (DSA) constitutes a risk for kidney transplantation; such a finding may indicates an unacceptable donor for this patient. However, positive FCXM in the absence of DSA is considered discordant and need further investigations. During COVID-19 pandemic, we observed 22% discordant results out of 445 FCXM performed during eight months period in our laboratory and another 7% were invalid due to high background negative control (NC). No study has addressed the impact of COVID-19 pandemic on FCXM and the overall pre-kidney transplant workups or described a solution to deal with these non-specific reactivities. Herein, we analyzed all FCXM results in SARS-CoV-2 seropositive patients and addressed how this pandemic affected significantly the pre-kidney transplant workups, highlighting both technical and financial implications. We also shared our modified FCXM procedures using dithiotheritol (DTT) sera treatment or blocking donor cells with negative control human serum (NCS) which we found to be successful to abrogate 98% of all discordant FCXM results and to validate all invalid results due to high background NC.In conclusion, COVID-19 pandemic has affected our HLA laboratory significantly by creating many false positive or invalid crossmatch results. Transplant laboratories must consider this before test interpretations and immune risk assessments. We recommend the use of DTT serum treatment to remove nonspecific bindings in the sera of kidney transplant candidates and the use of NCS-blocked donor cells to correct high background when performing FCXM in transplant candidates or donors with recent history of SARS-CoV-2 immunization respectively.

The Halifax flow Crossmatch Protocol Results According to the HLA Class and MFI of the DSA

Introduction: The main strategy to reduce the immunogenicity of transplanted grafts is to seek maximum compatibility between alloantigens in the donor-recipient pair. It is understood that the virtual crossmatch (VXM) can be a good tool in the evaluation of the donor / recipient pair and that a broader application of these protocols will improve the pre-transplant immunological risk assessment. The aim of this study was to correlate the physical flow crossmatches results, looking at different mean fluorescence intensity (MFI) values of the donor specific antibodies (DSA), and the probability of a positive crossmatch. We also aimed to validate this tool using a well standardized flow crossmatch protocol. Methods: We performed a total of 15,217 FCXM between 2015 and 2019. All were tested by the Halifax Flow Cytometer Crossmatch (FCXM) protocol, with cells from deceased donors and serum from renal recipients. For this analysis we selected only samples that had one or two DSA per locus (N = 1,081), when the MFI was above 1,000, and they were divided according to the allelic group recognized by the antibody (anti-HLA-A, B, C, DR or DQ) looking at the probability of a positive crossmatch with different MFI values of the DSAs. Combinations among them were also analyzed in the same way (N=175). Results: In the presence of an exclusive DSA against the allelic groups A, B and DR, with an index MFI above 5,000, all the FCXM were positives. With exclusively antibodies against groups C or DQ, all cases with DSAs above 15,000 MFIs were positives. With two or more DSAs anti A and/or B, and/or DR, when their MFIs sums exceeded 5,000, all FCXM results against B cells were positive. The presence of anti-Class I DSAs (A, B and A+B), regardless of the MFI value, was responsible for 71% (N=424 of 601) of the T cell positivity and 77% (N=460 of 661) in the B cell crossmatches. The presence of only anti-Class II DSAs (DR and DQ) accounted for 55% (N=168 of 303) of FCXM positivity in B cells. The overall mean of the MFI of the DSAs was higher in the FCXM positive group when compared to negative crossmatch. The group with sum of DSAs A (N=217) showed that when the sum was 4,000 MFI or higher, there was a 24 times higher probability for a positive B cell crossmatch when compared with lowers MFIs. Conclusions: Our results show a strong association between the DSA MFI and the FCXM result. The data here presented confirm the results of our previous studies, justifying the VXM standard used by our center, proving to be a good tool to streamline the selection of transplant recipients and facilitate the sharing of organs from national donors.

Correlation of Luminex-Based Single Antigen Based Results With Complement-Dependent Cytotoxicity Crossmatch and Flow Cytometry Crossmatch Results: A Single-Center Experience From Istanbul

This study aimed to retrospectively investigate the correlation of mean Class I donor-specific antibody (DSA) intensity values detected in Luminex-based techniques with the results of complement-dependent cytotoxicity crossmatch (CDC-XM) and flow cytometry crossmatch (FC-XM) results. A total of 335 patients with kidney failure and their living donors whose CDC-XM, FC-XM, and single antigen based (SAB) tests were studied between 2018 and 2020 for transplant preparation from living donor candidates were included in the study. Patients were divided into 4 groups according to their mean fluorescence intensity (MFI) values of SAB assay. Anti-HLA antibodies (class I and/or class II) were detected using SAB in 91.6% patients included in the study (MFI >1000). Class I DSA was positive in 34.8% of patients with anti-HLA antibodies. When CDC-XM and FC-XM results were evaluated in the 4 groups separated according to MFI values, 3 patients with DSA MFI <1000 had negative CDC-XM and T-B-FC-XM results. Of 32 patients with DSA-MFI between 1000 and 3000, 93.75% (n=30) had T-B-FC-XM or CDC-XM-negative results, and 6.25% (n=2) had B-FC-XM-positive results. The CDC-XM, T, and B-FC-XM were negative in all 17 patients with DSA-MFI between 3000 and 5000. Our results showed that MFI >5834 DSA values were significantly correlated with positive T-FC-XM (P < .001), and MFI >6016 values were significantly correlated with positive CDC-XM (P=.002). In addition, MFI values >5000 were associated with both CDC-XM and FC-XM in our study. The MFI values >5000 correlated with both CDC-XM and FC-XM.

The utility of cytolytic flow cytometry crossmatch before kidney transplantation

The donor/recipient matching in kidney transplantation is based on approved laboratory tests, which are complement-dependent cytotoxic crossmatch (CDC-XM) and flow cytometry crossmatch (FCXM). Both have some disadvantages: CDC-XM has low sensitivity, whereas FCXM does not differentiate between lytic vs. non-lytic alloantibodies. To find an improved method, we have developed a new crossmatch technique of cytolytic flow cytometry crossmatch (cFCXM), which allows for sensitive detection of clinically relevant complement-binding antibodies. The cFCXM assay detects dead cells with viability dye that ensue from the binding of allospecific lytic antibodies. In our study, 135 unsensitized kidney transplant recipients were recruited based on the CDC-XM and FCXM results and the clinical utility of cFCXM was evaluated. The 5-year follow-up for acute rejection incidents revealed that cFCXM could verify the clinical relevance of positive FCXM results as recipients with positive FCXM but negative CDC-XM had the same risk of rejection as patients with both negative CDC-XM/FCXM results. These findings suggest that cFCXM assay may provide more precise immunological risk assessment in kidney transplant recipients.

Examination of the discrepancy of the results of FCXM and anti-HLA antibodies in ABO-incompatible kidney transplantation

Examination of the discrepancy of the results of FCXM and anti-HLA antibodies in ABO-incompatible kidney transplantation

P143 Development and validation of LSAB MFI cutpoints for use in the virtual crossmatch test to predict flow cytometry crossmatch (FCXM) and cdc crossmatch (CDC XM) results

Aim Luminex Single Antigen bead assay (LSAB) derived mean fluorescence intensity (MFI) values of anti-HLA antibodies directed at the potential donor’s HLA are the primary parameter used in the virtual crossmatch to predict physical crossmatch (XM) outcome. We aimed to develop statistically validated LSAB MFI cutpoints for predicting CDC XM and FCXM results. Methods We leveraged the ASHI proficiency testing (PT) 80% consensus results of 7156 T FCXM, 6758 B FCXM, 2917 T CDC XM and 2233 B CDC XM as the reference results to investigate whether LSAB MFI of IgG anti-HLA antibodies predict validated physical XM results. LSAB MFI was determined in our laboratory using One Lambda Single Antigen HLA Class I and Class II beads. The 80% consensus results are from 8 consecutive challenges during 2013 to 2016, and 107 laboratories across USA tested sera and HLA typed cells distributed by ASHI, and reported the results to ASHI for assessment of the laboratory’s proficiency. Data analysis included: summing of MFI of IgG antibodies directed at HLA-A, B and C for investigating their association with T FCXM and T CDC XM results; summing of MFI of antibodies directed at HLA-A, B, C, DR, DQ and DP for investigating their association with B FCXM and B CDC XM results; assigning alternate ASHI challenges to the Discovery set and the Validation set; investigating the association between LSAB MFI and physical XM results by logistic regression analysis corrected for overdispersion; and identification of LSAB MFI cutpoint for maximizing the sum of sensitivity and specificity. LSAB MFI cutpoints derived from the Discovery set to predict FCXM and CDC XM results were investigated in an independent validation set. Results Table 1 demonstrates that LSAB MFI cutpoints from the Discovery set predicts XM outcomes in the Validation set (ROC AUC range from 0.974 to 0.999). Conclusions We have developed and validated LSAB MFI cutpoints for use in the virtual crossmatch to accurately predict physical T FCXM, B FCXM, T CDC XM and B CDC XM results.

P140Proper setting of cut-off value for the lymphocyte crossmatch by flow cytometry in kidney transplantation

Aim The flow cytometric lymphocyte crossmatch (FCXM) has become a routine test performed prior to kidney transplantation. Many studies have reported that a proportion of transplant recipients experienced early graft loss despite a negative complement-dependent cytotoxicity (CDC) crossmatch test. The FCXM is able to detect Human Leukocyte Antigen (HLA) antibodies of low titer or of a non-complement binding nature that may be responsible for graft loss. It is important to report positive and negative results using appropriate cut-off values. We reviewed the results of FCXM with CDC and panel reactive antibody (PRA) and attempted to set clinically useful cut-off values for FCXM. Methods From January 2015 to October 2017, 518 kidney recipients’ results of FCXM, CDC, and PRA (Screening test, Panel kit, and single antigen bead assay, LABScreen Luminex kits, One Lambda, Canoga Park, CA, USA) were analyzed. The PRA test was performed simultaneously from 403 patients, and 159 patients who were negative for HLA antibodies in the all PRA tests. Fifty-seven patients were positive for CDC test results and 37 patients were both positive in T cells and B cells. In 20 patients, only B cells CDC results were positive, and 13 patients were negative for FCXM test results. Results The final interpretation of FCXM result was decided through CDC, PRA result and existence of donor specific antibody (DSA). T cell FCXM result was interpreted as negative in 451 patients and positive in 67 patients according to DSA. The appropriate cut-off value of the Mean fluorescent intensity (MFI) ratio using the ROC curve for 518 patients was 1.86 (sensitivity 100%, specificity 98.2%, Youden’s index 0.982). However, patients who underwent desensitization treatment due to DSA or ABO blood type incompatibility were included. The cut-off value obtained using the ROC curve for 446 patients who have not experienced desensitization treatment was 2.45 (sensitivity 100%, specificity 99.3%, Youden’s index 0.993). Generally used, Median + 2 SD value of MFI ratio of patients judged as negative FCXM was 1.60. Conclusions Based on the statistical method used, various cut-off values could be obtained. It is important to determine the clinically significant cut-off value based on the data accumulated in each laboratory, not the simple median value from negative patients.

P145 Further evidence of the relevance of DP epitope analysis in the virtual and actual crossmatch in solid organ transplantation. A case report

Aim Epitope-based HLA matching has been developed as a better matching method in solid organ transplantation. Here we present a highly sensitized patient with 100% cPRA but low donor specific antibody (DSA) level at the DP locus. Virtual crossmatch (vXM) with a cadaveric donor predicted a negative result but the actual crossmatch gave an unexpected positive result in B cells maybe due to the presence of a cross-reactive epitope. The clinical relevance of antibodies directed against HLA-DP antigens is discussed in the literature with conflicting reports. Methods Single antigen bead assay (SAB) was used to detect the levels of alloantibodies in the patient serum. Flow crossmatches were performed with 3 conjugated antibodies: FITC-anti-Human IgG, CD3-RD1 and CD19-PC5. Serum-incubated cells were stained with fluorochrome conjugated antibodies prior to running on Navios instruments. Test was done using current and historical sera from the patient. Results SAB analysis showed high levels of alloantibodies in both Class I and Class II but only a DSA for DP6. In our lab, DSA are considered “Positive” for HLA-DP when MFI values are ⩾ 15,000. MFI values for current and historical sera were 7536 and 10520 respectively suggesting a negative crossmatch. FCXM results were negative in both T and B cells for the last serum but unexpected positive crossmatch only in B cells was observed in the historical serum. Retrospective analysis of previous sera showed a well-defined antibody to the DP epitope, 56E, with gradual decreasing levels of MFI. The apparent discrepancies between the virtual and actual crossmatch results can be probably due to the “peanut butter” effect of this cross-reactive epitope. The patient was transplanted based on the negative crossmatch from current serum followed by immunosuppression protocol. Conclusions Our results provide evidence to the impact of epitope analysis in the vXM and its effect in the actual crossmatch. Implementation of epitope-based HLA matching in our lab will help to better select compatible donors. Characterization and matching DP epitopes using computational methods seems to be more relevant than common matching alleles. Transplant in the presence of DSA at the epitope level needs to be closely monitored to determine their true clinical relevance in solid organ transplantation.

P137 Go pro(nase)! the importance of donor lymphocyte treatment with pronase in flow cytometric crossmatch testing

Aim Flow cytometric crossmatch (FCXM) is performed to confirm donor/recipient histocompatibility. Previous multicenter study (Liwski et al. ASHI 2012) suggested that protocol differences contribute to FCXM result variability and that pronase treatment improves B cell FCXM. In this study we assess the impact of pronase on FCXM results. Methods Donor lymphocytes, isolated from spleens/lymph nodes, were treated with pronase (4.7 U/ml) or saline. FCXM were performed at Santa Casa Lab in Porto Alegre using the Halifax protocol, acquired on BD Canto II and analyzed using a median channel fluorescence (MCF; 1024 channel) scale. 3SD cutoffs were determined by testing neg ctrl (NC) sera against 63 donor cells. Pos ctrl (PC) sera were used in each FCXM. A total of 240 FCXM (122 predicted neg; 118 predicted pos) were performed in parallel using pronase treated vs untreated cells. Pos/Neg FCXMs were predicted based on LABScreen single antigen bead testing with a 1,000 MFI cutoff. Results NC serum testing shows that pronase significantly reduced B cell background reactivity (pronase MCF = 195 +/- 32 vs untreated MCF = 363 +/- 89, p Conclusions B cell FCXM sensitivity is unacceptably low (26.3%) when untreated cell are used. Pronase treatment greatly improves B cell FCXM sensitivity (74.6%) and decreases the rate of false negative B cell reactions by reducing the background and improving signal to noise ratio.

Value of C3d assay and IgG subclass in the prediction of the flow cytometry cross-match result for renal transplantation

The aim of this study is to compare the association and the predictive capacity of DSA MFI, complement fixing capacity (C3d assay) and IgG subclasses determination in the prediction of FCxM result. MethodsWe used cryopreserved (70C) sera from potential renal transplant recipients, containing DSA against their respective potential donors. All patients showed negative AHG-CDC CxM and either positive or negative FCxM. Class I and Class II HLA-DSA were determined by Luminex SAB. C3d were detected by Luminex (Lifecodes®Immucor), DSA IgG 1–4 subclasses were evaluated using monoclonal antibodies specific for IgG subclasses (Luminex). Results93 donor/recipient tests were evaluated; 32 (35.9%) patients presented at least one C3d + Ab, of which only 11 (11.8%) were donor specific. At least one IgG subclass was identified in 45 samples (48.3%). Twenty-seven FCxM tests (29%) were positive. On multivariate analysis HLA mismatches, the IgG subclass detection, DSA MFI and class II PRA remain associated to FCxM whereas C3d + Ab was not associated. For the FCxM prediction, the IgG subclass detection in combination with the DSA-MFI > 2800, had the best negative predictive value 93.9 (CI 95%, 84.2–100). ConclusionNeither the C3d assay nor the IgG subclasses detection alone had an adequate predictive capacity for the FCxM. In the absence of IgG subclass detection and DSA-MFI < 2000, the probability of a negative FCxM was near 94%.

Reanalysis of the role of pronase treatment of B cells in the flow cytometric crossmatch assay: Fc receptor is not the primary target

Pronase, a mixture of nonspecific bacterial proteases, is used to pretreat human lymphocytes to prevent false-positive B cell results in the flow cytometric crossmatch (FCXM) assay. The target of pronase has been reported to be B cell-expressed Fc receptors, which nonspecifically bind IgG. As pronase use in FCXM can induce other complications, including degradation of HLA leading to inappropriate FCXM results, and false-positive T cell results when testing serum from HIV-positive patients, we tested whether specifically blocking Fc receptor CD32 could replace pronase. Anti-CD32 mAb 6C4 was superior to pronase for blocking binding of aggregated IgG to B cells. However, 6C4 was unable to replace pronase in clinical FCXM, as it did not prevent false-positive B cell FCXM results, or enhance sensitivity of the assay. We conclude that the functional targets of pronase in the FCXM assay are poorly understood, and that B cell-expressed Fc receptor plays an insignificant role.

P233 On the performance characteristics of luminex single antigen bead (LSAB) assay mean fluorescence intensity cutpoint in predicting flow cytometry crossmatch (FCXM) results

Aim LSAB mean fluorescence intensity (MFI) is often used to predict physical FCXM results, and LSAB MFI cutpoints are used to list unacceptable antigens. We therefore determined the accuracy of LSAB assay MFI cutpoint used to score the LSAB assay as positive or negative in foretelling FCXM results. Methods One hundred and forty-six pre-transplant sera from 146 patients with end stage renal disease were tested against potential kidney donor’s T cells and B cells using a 3-color flow cytometry clinical protocol; the same 146 sera were tested using LSAB assay and the cumulative MFI directed at donor HLA class I and/or II antigens were determined. A T cell FCXM with a median channel shift (MCS) ≥ 40 with the patients serum vs. negative control serum was classified as a positive test; a B cell FCXM with MCS ≥ 50 with the patients serum vs. negative control serum was classified as a positive test. In the LSAB assay, MFI > 2000 directed at donor HLA antigens was scored as positive DSA. These cutpoints are our laboratory’s cutpoints for both ASHI proficiency testing and for clinical use. Results Statistical analysis demonstrated a significant association between LSAB results and FXCM results (Table 1). LSAB assay results predicted T Cell FCXM results with a sensitivity of 70% and a specificity of 100% (Fischers Exact Test, P 0.0001, Positive Predictive Value (PPV = 100%, Negative Predictive Value (NPV) = 91%). LSAB assay results also predicted B Cell FCXM results with a sensitivity of 86% and a specificity of 72% (Fischers Exact Test, P 0.0001, PPV = 62%, NPV = 91%) (Table 1). In a subset of 31 patients, both donor FCXM and auto FCXM results were available and 17 of 31 donor positive FCXM were also auto B cell FCXM positive (17/17) and auto T cell FCXM positive (1/17). Conclusions LSAB assay results are significantly associated with FCXM results. Auto B cell FCXM and high resolution HLA typing should further refine the relationship between these two high sensitive assays. Download high-res image (138KB) Download full-size image

P197 Cumulative weak HLA antibodies. relevant or not. A case study

Aim The relevance of cumulative, low MFI anti-HLA specificities on equivocal virtual crossmatch (VXM) results has been discussed in the Guidelines for the detection and characterization of clinically relevant antibodies in allotransplantation of the British Society for Histocompatibility and Immunogenetics. Moreover, some labs take this phenomenon into consideration in their daily practice. However, no conclusive evidence has been published and no consensus has been reached about it. This case describes a 3 year old male awaiting cardiac transplant presenting with multiple HLA Class I and II antibodies (Ab). It demonstrates the relevance of weak HLA Ab cumulative effect on B lymphocyte flow crossmatch (FC XM) outcome. Methods Ab screening was performed according to manufacturer instructions using LabScreen Single Antigen Beads (SAB) (One Lambda). FC XM were acquired on BC FC500 and analyzed using MESF shift. MESF shift scoring validated in our lab are 100000 (strong positive). Results To assess potential for reactivity to lower MFI specificities, a mock crossmatch was performed against an individual for which the patient had Ab against 4 and 7 HLA Class I and Class II antigens, respectively. MFI for these specificities ranged between 1533 to 2908 MFI which are considered as weak positive according to the cut off stabilished in our lab for SAB test; >1500 (negative), 1500–3500 MFI (weak positive), 3500–8000 (positive), >8000 (strong positive). Cumulative MFI for Class I and Class II were 7935 and 15689 respectively. FC XM for T cells was negative (1309 ΔMESF) while it was positive for B cells (23098 ΔMESF). Conclusion In our experience, samples exhibiting specificities with MFI ranging from 1500 to 9000 detected in the SAB test remain in an uncertain zone when predicting FC XM results. The cumulative effect of specificities with weak positive MFI is not completely understood yet. This case study illustrates the contribution that this effect may have on positive B cell FC XM results, highlighting the potential for deleterious effect of multiple lower MFI HLA Ab in organ transplantation as well as the relevance of this phenomenon for VXM prediction.

P033 Cryopreserved donor cell crossmatches: Can we believe the results?

Aim In order to facilitate rapid turnaround, exchange programs such as the NKR require preliminary flow cytometric crossmatching (FCXM) using frozen cells. However, we’ve found that questionable FCXM results occur more often with frozen cells compared to results obtained with fresh donor lymphocytes. In this study, we investigated the frequency of such discrepant results. We compared 17 donor/recipient pairs who were crossmatched using both frozen and fresh donor cells. Methods Fresh lymphocytes were isolated using EasySep™ (StemCell Technologies). Frozen cells were thawed following a validated protocol (ASHI Laboratory Manual). All cells were treated with pronase. FCXM were performed using a rapid protocol (Liwski, et al., H.I. 72, 2011). In all cases, a screening FCXM was performed with frozen cells and a final FCXM was performed with fresh donor cells. For final FCXM, one specimen tested in the initial screening FCXM was included. Donor specific antibodies (DSA) were analyzed using multiple solid phase platforms. Results Six out of the 17 FCXM comparisons were discrepant. Among the discrepant FCXM, 4/6 were T & B cell positive using frozen cells and negative using fresh cells; 1/6 had elevated, but within negative range, MCS using frozen cells and clearly negative MCS ( Conclusions The problem with unreliable frozen cell crossmatches is that false positive results can lead to unnecessary donor denial and false negative results can bring about last minute exchange failure. When a recipient clearly has no DSA, a positive FCXM using frozen cells is not typically problematic and most often a repeat FCXM using fresh cells is negative. On the other hand, for patients with DSA, the intent of an exploratory FCXM with frozen cells is to rapidly evaluate whether or not DSA strength is at an acceptable level. However, in order to be clinically informative, it is important that FCXM results using frozen cells correlate with fresh cells. Download : Download high-res image (279KB) Download : Download full-size image

P030 Previously believed to be nonsensicl crossmatch results, explained by ANTI-HLA-C antibodies

Aim While trying to determine the frequency of “False Positive” Flow cytometric crossmatches (FCXM) in our laboratory, we observed that almost all cases in which we saw a Positive T-cell, Negative B-cell (T+/B-) result, a HLA-C donor specific antibody (DSA) was present. Prior to the development of assays that enabled us to accurately detect HLA-C antibodies, it was believed that a T+/B- result was likely due to a technical error, B-cell viability issue, or a Non HLA antibody. Methods FCXM results with Positive T-cell, Negative B-cell results were collected and assessed for DSA. Results We found one case where two different patients showed T+/B- results against the same donor. Both patients had Strong (>10,000 MFI) DSA to HLA-C (DSA-C). We also found a number of patients that responded this way against multiple donors. In particular, we found 1 patient that has had FCXM against 30 different donors. Of the 30 FCXM, 26 of these resulted in T+/B- and DSA-C (Median MFI = 5010) was detected in all of these cases. There was 1 FCXM with DSA-C that was T-/B- (DSA MFI 4492). The remaining 3 cases without DSA-C were T-/B-. (See Table 1). Epitope analysis of this patient’s antibody results show a clear 76VRN antibody pattern, that explained all of her Class I antibodies. Conclusions It has been recently reported that HLA-C is expressed higher on T cells than B cells. This is contrary to what many of us were taught and should now be considered as a possible explanation of an “unexpected” FCXM result, in which the T-cell is positive and the B-cell is Negative. Download : Download high-res image (69KB) Download : Download full-size image

P107 HLA expression in flow crossmatch: Cell source and pronase matter

Aim Flow cytometry crossmatch (FCXM) is an important tool in pre-transplant assessment of immunologic risk. Variation in HLA antigen density (AgD) may affect FCXM interpretation. The objectives of this study are to measure HLA AgD on lymphocytes and compare the differences in AgD between cell source, donor type, and pronase treatment. Methods AgD was measured with monoclonal antibodies (clones: W6/32, WR18 (pan-Class II), and HLA-DQ1 (binds DQB1 except DQ2)). Lymphocytes were isolated by immunomagnetic depletion from living donors (LD-PBL, n = 39), deceased donors (DD-PBL, n = 7), and spleen (SPL, n = 20). Cells were pronase treated. AgD was assessed with and without pronase ( n = 3). HLA-DQ AgD analyses were limited to DQ2-negative cells. Results There were no significant differences across the three cell sources between class I AgD on either T- or B-cells (Fig. 1A and 1B). In contrast, class II AgD was lower on SPL vs LD-PBL and DD-PBL. DQ AgD was lower on both DD-PBL and SPL (Fig. 1C and 1D). Pronase treatment enabled increased detection of class I AgD on T-cells but not B-cells with only slight increase in class II AgD (Table 1). Conclusions Although T or B cell class I AgD did not differ significantly between cell sources, B cell class II was substantially decreased, particularly in DD spleen while HLA-DQ expression was lower on all deceased donor cells. Our results suggest that FCXM results may be affected when assessing deceased donor FCXM in the context of class II DSA. Pronase treatment primarily affected the detection of class I on T cells with minimal impact on B cell AgD, but this was observed in a limited number of samples. We are continuing to collect data and will assess the impact of class/loci specific HLA antibody and the relationship to AgD. Download high-res image (167KB) Download full-size image Download high-res image (81KB) Download full-size image

P230 Automation for flow cytometry crossmatch (FCXM) lymphocyte isolation using robosep

Aim The importance of cell purity to obtain a reliable FCXM result is critical and long established. The aim of this study was to improve efficiency and implement automated cell preparation for FCXM due to the increased workload in the histocompatibility lab after the new Kidney Allocation System (KAS) was implemented. Methods The use of Robosep TM Instrument along with EasySep TM Direct Human Total Lymphocyte Isolation Kit (RLI) starting from buffy coat was evaluated in parallel with our traditional ficoll hypaque-based lymphocyte isolation method (FLI) that includes CD15 Dynabeads, lysing buffer and soft spin washes to achieve target purity criteria when applicable. FCXM was performed for all cells (n = 20) isolated in parallel using FLI & RLI. Spiked Positive Controls were used for FCXM and tested in parallel to verify sensitivity of the assay. The costs associated with both methods were evaluated by comparing labor-based expenses and reagent price. Results The purity of the cell preparations obtained by the RLI method was superior (CD45-95.4%, myeloid -0.9%, plt-0.8∗10 3 /μL) when compared with the traditional FLI technique (CD45-84.7%, myeloid -4.9%, plt-7.9∗10 textsuperscript3/μL). Lymphocyte yield and viability was equivalent by both methods. There was no significant change in the Negative Control MFI values for 90% of the isolated cell preparations, however two Negative Controls obtained using RLI showed high MFI for B cell crossmatch. The sensitivity of the FCXM proved to be unmodified when lymphocytes were isolated by RLI. The use of RLI for FCXM reduced the cost of reagents/cell isolation by 75% compared to the FLI method. The total isolation protocol time was reduced by 25% when using RLI in comparison with FLI, and the total tech hands-on time was dramatically reduced by 55%. Conclusions Our study revealed that automation using EasySep TM kit on Robosep TM markedly improves the purity of the cells with identical results and sensitivity in the FCXM. The use of RLI proved to be more efficient and cost effective, reducing not only the reagent cost but also more importantly ”hands-on” tech time.

Effect of ABO incompatibility on T-cell flow cytometry cross-match results prior to living donor kidney transplantation.

Due to its high sensitivity, the flow cytometry cross-match (FCXM) has been described as valuable tool for identifying an optimal donor. We here focused on the impact of ABO incompatibility on FCXM results. We analyzed 29 ABO incompatible and 89 ABO compatible donor-recipient pairs (73 and 175 datasets, respectively) prior to living donor kidney transplantation. In all patients, lymphocytotoxic cross-matches for B and T cells were negative. Recipients with blood group O (A to O and B to O) displayed significantly (P < 0.05) higher T-FCXM results than those with blood group A and B (A to B, B to A and AB to A), respectively. Donor-specific T-FCXM responses (ΔMFI values) were significantly higher (P < 0.05) in ABO incompatible vs. compatible pairs (ABO incompatible recipients with blood group O: 32 ± 6; with blood group A: 19 ± 7; with blood group B: 7 ± 4; recipients with ABO compatibility: 3 ± 2, respectively, data represent mean ± SEM). Consistent with the T-FCXM results donor-specific isohemagglutinins (IgG titers) were significantly higher in recipients with blood group O vs. A, both prior to rituximab treatment and plasmapheresis/immune adsorption (P = 0.004) and immediately prior to transplantation, i.e., after rituximab and antibody-depleting therapies (P = 0.04). ABO incompatibility was associated with higher T-FCXM responses, especially in recipients with blood group O. This finding has major impact on the interpretation of flow cross-match results. Current cut-off values need to be reassessed in the ABO incompatible setting. © 2016 International Clinical Cytometry Society.