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Background Sensitized patients awaiting heart transplantation spend a longer time on the waitlist and have higher mortality. We are now able to further characterize sensitization by discriminating antibodies against class I and II, but the differential impact of these has not been assessed systematically. Methods and Results Using United Network for Organ Sharing data (2004–2015), we analyzed 17,361 adult heart transplant patients whose class I and II panel reactive antibodies were reported. Patients were divided into 4 groups: class I and II ≤25% (group 1); class I ≤25% and class II ˃25% (group 2); class II ≤25% and class I >25% (group 3); and both class I and II >25% (group 4). Outcomes assessed were treated rejection at 1-year mortality, all-cause mortality, and rejection-related mortality. Compared with group 1, only group 4 was associated with a higher risk of treated rejection at 1 year (odds ratio 1.31, 95% confidence interval [CI] 1.05–1.64), all-cause mortality (hazard ratio 1.24, 95% CI 1.06–1.46), and mortality owing to rejection (subhazard ratio 1.84, 95% CI 1.18–2.85), whereas groups 2 and 3 were not (P > .05). Conclusions Combined elevation in class I and II panel reactive antibodies seem to increase the risk of treated rejection and all-cause mortality, whereas risk with isolated elevation is unclear.

Objectives To identify the risk of developing acute rejection, allograft fibrosis, and antibody-mediated rejection, a retrospective review of pediatric patients who underwent liver transplant between July 31, 1998 and February 29, 2016 and had donor-specific antibodies measured at time of liver biopsy was undertaken. Materials and methods HLAMatchmaker Software (http://www.hlamatchmaker.net) was used to define epitope mismatches between donors and recipients and to predict de novo donor-specific antibody risk. Epitope mismatches were evaluated for their immunogenicity. Results In our group of 42 recipients, 20 (48%) had donor-specific antibodies. Having an antibody against HLA-DQB1*02 was associated with acute rejection (66.6% vs 36%; P = .024). We found that DQ epitope mismatch load was greater in recipients with class II donor-specific antibodies (9.7 vs 3.6; P = .001). HLA-DQ (7.4 vs 3.6; P = .04) and HLA-DR (8.8 vs 3.8; P = .04) epitope mismatch loads were higher in recipients with DQ + DR donor-specific antibodies. A high portal fibrosis score was associated with higher mismatch load at the DQ locus (P = .005) and DQ + DR loci (P = .03). Having > 5 or > 6 epitope mismatch loads at the DQ locus identified a threshold above which development of DQ donor-specific antibodies would occur (area under the curve = 0.878). Mismatches for eplet 4Q, 45GE, 52PQ, and 52PL, thought to be immunodominant epitopes, were observed for several recipients. Conclusions Knowledge of epitope mismatches between recipients and donors may aid transplant physicians in devising immunosuppression strategies.

To determine the significance of low-level DSA (donor specific antibody) in patients transplanted with negative cytotoxicity AHG (antihuman immunoglobulin) crossmatch, data from 279 patients who received a kidney transplant between July 1999 and March 2006 were collected. All kidney recipients received ABO-compatible donors. A poor outcome was defined as any one of the following: death, Cr2.0 mmol/L, occurrence of a rejection episode. Luminex Screening and Single Antigen assays from Tepnel Life Codes were used to detect human leukocyte antigen antibodies on pre-transplant sera retrospectively. Twenty-four out of 279 recipients demonstrated the presence of solid-phase DSA (MFI1000) present pre-transplant. In DSA+ group, the accumulated good versus poor outcome rate was 0.30 versus 0.70, respectively. These rates were 0.49 and 0.51, respectively, in the DSA- group. The difference in composite poor outcome between DSA+ versus DSA- group was significant (p=0.030). The DSA- group had no difference in patient survival as compared to the DSA+ group (p=0.061). There is no statistically significant difference for either mortality or outcome results between high MFI (2000) and low MFI (≤2000) groups. Our data suggest that solid-phase antibodies which are not strong enough to elicit a positive T-AHG crossmatch may influence long-term graft outcome.

We hypothesized that de novo donor-specific antibody (DSA) causes complement-dependent endothelial cell injury in kidney transplants, as assessed by expression of endothelial cell-associated transcripts (ENDATs), that may be attenuated through complement inhibition. In total, 15 participants (five control, 10 treatment) with DSA and deteriorating renal function were enrolled. The treatment group received 6 mo of eculizumab followed by 6 mo of observation, whereas controls were observed. The primary end point was percentage change in estimated GFR (eGFR) trajectory over the treatment period. The treatment group had an improved eGFR trajectory versus control, based on our predetermined two-sided 0.10 significance level (p = 0.09). Within-subject analysis of treated participants at 6-mo intervals did not show significant change (p = 0.60). Modeling C1q status showed that C1q-positive patients had significantly higher mean eGFR than patients with negative C1q (p = 0.04). Biopsies revealed elevated renal ENDATs in most participants, but ENDATs were not reduced with complement inhibition. Our data suggest that eculizumab treatment may stabilize kidney function in patients with chronic persistent DSA based on our pilot a priori significance threshold. ENDAT expression predicative of acute humoral injury is not reduced with complement inhibition in this chronic setting. Further studies will be necessary to determine which patients may benefit from eculizumab.

Introduction Pretreatment of serum with ethylenediaminetetraacetic acid (EDTA) eliminates interfering components and provides a clearer, more accurate antibody (Ab) profile in luminex single antigen (SA) antibody detection assays. Some patient sera do not show obvious signs of interference but have high background (i.e. binding to NC bead devoid of HLA antigen). We report the case of a highly sensitized patient with hidden reactivity to class I antigens revealed by EDTA, but confounded by treatment to reduce high background. Methods DC is a 64 yo Asian male who received a deceased donor (DD) kidney transplant (Tx) in 1984, but since 2005 has had MPGN and is currently listed for a second kidney Tx. His antibody profile has remained stable the past few years (cPRA 49–57%). His serum has been historically treated with Adsorb Out™ (AO) to remove high background. The strongest Ab reactivity in AO treated sera has been against class I: A3, A11, A25, A26, A29, A43. However, recent flow crossmatches (XMs) against two DD cells were strong B and T cell positive in the absence of any strong or discernible DSA, with delta channel shifts (DCS) of 350–400. One DD typed for A1, the other A11 so we suspected an epitope/CREG reactivity since the actual DSA levels to A1 and A11 were low to moderate (MFI = 616–3,155), which did not correlate with the strong positive flow XMs. We decided to treat the patient’s sera with EDTA, instead of the routine AO treatment, for the SA assay and discovered a significant increase in A1 Ab (MFI = 23,274) and A11 (MFI = 20,622). In fact, all Abs previously detected by AO treatment, displayed significantly higher MFI levels after EDTA treatment. Although the background reactivity to the NC bead was higher in EDTA treated compared to AO treated sera, it was within acceptable limits of the assay. We then EDTA and AO treated this patient’s serum and found no significant difference compared to the EDTA alone treated sample (A1 MFI = 23,226, A11 MFI = 21,672). Conclusion Despite what may appear as clear, consistent patient Ab data over the course of months to years, it is imperative to continually monitor Ab profiles carefully, to reveal possible epitopes and changing trends. Ab testing by SA assays must also be interpreted in concert with XM data to ensure proper unacceptable antigens are in UNET and to ultimately provide an optimal Tx outcome.

To increase the donor options for our active living kidney donor program, candidates who are blood group B may receive a living donor transplant from a non-A1 or non-A1B donor. ABO incompatible donors (ABOi) are no longer an absolute contraindication for kidney transplantation as long as the recipient titer against ABO A is low. It is well known that there are several variations of non-A1 blood groups, and therefore measurement of anti-A titer against commercial A1 or A2 cells may not provide an accurate measurement of donor reactive IgG antibody to blood group A antigens. Therefore, we have initiated a strategy of assessing anti-A titers using red cells from the prospective non-A1 donor being evaluated. The candidate will have initial non-A1 titers performed with commercially produced A2 red cells as well as red cells from the intended living donor. This will help identify the rare instance of a more or less immunogenic ABO A subgroup. Recipient sera is serially diluted with saline from 1:1 to 1:32 in a standard tube assay. One drop of RBC suspension (50 ul) is added to 2 drops (100 ul) of each sera dilution. Tubes are incubated at RT for 30 min, centrifuged and agglutination macroscopically observed and recorded. This provides measurement of the IgM non-A1 titer present in the recipient. Tubes are then incubated at 37C for 30 min, washed 4 times with saline, IgG added, then centrifuged and agglutination recorded again to provide IgG non-A1 titer results. To be considered compatible for non-A1 or non-A1B donors, the IgG titer obtained with A2 reagent cells should be no higher than 1:16, and no greater than 1:8 with the intended living donor. Titers must be repeated within 48 h prior to transplant. Consistent with initial testing, IgG non-A1 titer results at the time of transplant are considered acceptable if the titer is no greater than 1:16 against commercial A2 red cells, and 1:8 against the red cells of the intended deceased donor. As of the date of this submission we have performed two such ABOi transplants using this testing methodology, with no rejection episodes occurring thus far.

Our lab has reported discrete results into Epic EMR since the initial Epic Go Live allowing for access to electronic data for clinical and research use. YNHH initiated the LIS update from SoftLab to Epic Beaker in 2016. This update dramatically changed our workflow. Initially, the Epic Beaker team set up our lab with a workaround to report results back into Epic without completing the standard Beaker sample workflow. Generic sample labels and incomplete specimen collection information often led to confusion and samples were redrawn or orders were automatically canceled in Epic before results were posted to the EMR. In 2017, a project was initiated to incorporate the HLA lab into the Epic Beaker workflow. This project faced three primary challenges. First, Beaker lacked support for Epic order panels with multiple tests and sample types, which were initially set up to improve efficiency when ordering multiple concurrent tests. As a solution, these panels were split into orders based on the various tube types necessary to perform the test and order sets were created in Beaker. The second challenge was the Beaker required update of the HL7 orders and results interfaces to support the data transfer between Epic and HistoTrac. The final hurdle was that every result message needed to be updated with the Beaker sample ID. This prompted the HLA Lab to consolidate our Epic orders removing organ specificity and reducing orders from 28 to 10 unique order codes. Furthermore, Beaker enforced a one to one relationship between orders and results. To accommodate this rule, SystemLink added status logic to the results message. All results are sent as preliminary until the last outstanding test result is signed off in HistoTrac and sent as final. Prior to the HLA lab Go Live the Epic team trained the sample collection staff to process our orders with the standard Beaker workflow, updated the clinicians with an Epic splash screen message, and trained the HLA staff to receive samples in Beaker before processing in HistoTrac. All Epic outstanding orders, standing orders and preference lists were updated by the Epic consolidation team. Post-Go Live optimization has included filing results for all pre-Go Live outstanding tests in HistoTrac, and identifying sample collection sites needing updated order transmittal rules which add the Beaker sample ID at collection. As a result of the Epic Beaker implementation order specific labels are printed for all samples, sample status is updated in real time, and samples are processed with the standard Beaker workflow.

Introduction Donor Specific Antibodies (DSA) can be a contraindication for kidney transplants. The clinical relevance of DSA to DP antigens and correlation with crossmatch (XM) remain unclear. We report the conversion of a negative XM to positive due to development of DSA to DP6, most likely caused by an unreported sensitizing event. Methods A 38 yo female received a deceased donor (DD) kidney transplant (Tx) after negative T/B cell flow XMs using sera drawn 35 and 44 days pre Tx (day −35 and −44). Delta channel shifts (DCS) in these XMs were: T = 0 for both sera; B = 37 and 31 for days −35 and −44. These sera were negative for DSA. We received a serum drawn at admission for Tx (day −1). The patient received blood transfusions within the previous week (day −6). The day-1 serum had high level DSA to DP6 (MFI = 13,723), compared to day −44 (1,321) and day-35 (1,653). By flow XM, the day −1 serum was T negative, B positive (DCS = 228), likely due to the increase in DSA to DP6. In addition to standard immunosuppression, this recipient also received thymoglobulin and basiliximab once the program was notified of high level DSA and positive flow XM. Post Tx monitoring showed an immediate decline in DSA to DP6: post Tx day 6 (MFI = 7,380), day 12 (5,669), day 21 (4,042), day 63 (485). The DSA to DP6 remained low (MFI 400) for five years. However, the recipient had BK viremia and a kidney biopsy five years post Tx showed “arteriolar hyalinosis and arteriosclerosis related to possible CNI toxicity”. The recipient is re-listed in UNET for another kidney Tx, the primary reason for graft failure deemed transplant glomerulopathy. Discussion This case emphasizes the importance of timely antibody testing in relation to XM testing, awareness of sensitizing events, and updated HLA typing. Our lab now employs stricter guidelines to ensure more frequent receipt of patient sera, especially following potential sensitizing events. At the time of this patient’s Tx, DP typing was not performed on DD. After discovering the increase in antibodies at pre Tx day −1, but absence of DSA, we performed DP typing. Finding DP6 as a DD antigen helped explain the change to positive XM, as the level of DSA to DP6 significantly increased following the sensitizing event pre-Tx. This early peri and post Tx antibody response may have contributed to reduced graft survival and transplant glomerulopathy.

Aim The significance of HLA antibody in liver transplantation has been unclear, although recent studies have described an increase in the diagnosis of antibody mediated damage in this population. Our specific aim is to correlate the development of de novo HLA antibody including C1q testing, and AT1R antibody levels with graft dysfunction to determine if there is a significant association of these antibodies with graft function. Methods 63 patients at our center from 7/31/1998 to 2/29/16 were included for analysis, of which 42 were screened for Anti-HLA DSA, C1q and AT1R. DSA >1000 mfi was considered positive and patients were evaluated retrospectively for ACR. Specificity of antibody locus as well as alleles were analyzed and correlated with liver dysfunction. Available biopsies were evaluated for rejection grade, mean fibrosis score as well as portal, central, and sinusoidal fibrosis. Patient characteristics were summarized using mean, standard deviation, median and range for continuous variable, frequency and percentage for categorical variable. Wilcoxon Rank Sum or Kruskal Wallace test was conducted to check differences of continuous variables. Two sided p value less than 0.05 was considered as statistically significant. Results Portal fibrosis scores were significantly lower in patients without Class II DQ DSA post transplant ( p p p p Conclusions There is significant development of Class II DSA in pediatric liver transplantation. These antibodies were associated with increases in fibrosis scoring and ACR. DQ2 mismatches in particular were shown to be more immunogenic, the significance of which is currently under study.

Detection of donor specific HLA antibodies (DSA) is currently an important component of the Banff criteria for diagnosis of antibody mediated rejection. There is a perception of significant variations in anti-HLA antibody (HLA-Ab) testing practices and interpretation algorithms among different laboratories. In this study, we aimed to survey large transplant centers worldwide to evaluate similarities and differences in HLA-Ab testing practices . METHOD An online survey was sent electronically to 120 histocompatibility laboratories and we received data from 72 laboratories (60% response rate). The survey focused on defining sensitization status, method of reporting HLA-Abs, and post-transplant DSA monitoring practices. Responses from laboratories that support more than 25 transplants per year were analyzed ( n = 66). RESULTS: More than 80% of surveyed laboratories use Luminex based single antigen bead assay to test for HLA-Abs. In non-sensitized patients, absence of HLA-Abs was defined by screening beads (11% of labs), phenotype beads (11%) by single antigen beads (30%) and 2 different assays (48%). For sensitized patients 54% of the laboratories used a combination of assays. Definition of highly sensitized patients varied widely among labs with thresholds ranging from 30% to 80% CPRA. Fifty-five percent of the labs reported MFI values associated with HLA-Abs while 30% reported relative strength (weak, moderate, strong) of the antibody. The definition of relative strength varied among labs with MFI ranges of 500–5000 and 3000–10,000 defining weak and strong antibodies, respectively. Only 39% of the laboratories routinely evaluated sera for the presence of interfering substances/prozone effect. Only 44% of the labs routinely screened for DSA post-transplant; 24% screened only at the time of for-cause and surveillance biopsies and 9% do not monitor for DSA at all. CONCLUSION There is great variability in testing, interpretation and reporting practices among laboratories. Similarly, the definition of a highly sensitize patient varies from center to center. Lack of consistency in practices and definitions hinder our ability to compare patient outcomes from one center to another and highlights the need for standardization and evaluation of clinical relevance of different practice patterns.

HLA antibody (Ab) detection and characterization is a critical step in transplantation. With high sensitivity and allele level Ab identification, the Luminex-based single beads assay is an essential tool in all HLA labs. One of the challenges with the current testing is the potential detection of cryptic or not-physiologically expressed epitopes. Here we report an interesting case of cryptic epitopes and describe the decision making process in preparation for virtual crossmatch requests. A 2 y/o female patient being evaluated for small bowel transplant was tested for presence of HLA Ab. Her class II profile was positive for all DR except DR10, with strongest reactions to alleles with epitopes 98E (DR4,7,9) and 48YQ6 (DR53). The patient typed as DR7,10,53 and the sample was immediately flagged as a possible false pattern of reactivity. Single antigen beads from 2 different vendors showed similar profiles; the same reactivity pattern was also detected by multi-antigen beads. Upon dilution the Ab profile was weaker but similar to the undiluted sample (figure). The pattern “all DR but DR10” could be explained by the patient carrying a rare DR7 allele or a non-expressed DR7 variant. However, no patient cells were available for serological DR7 detection. Molecular typing by SSO and/or SSP may detect a rare DR7 but could not exclude the possibility of a SNP or frameshift mutations creating a premature stop codon behind exon 2. Therefore, the sample was tested by NGS. By sequencing past exon 2 we confirmed that the recipient expresses DRB1∗07:01:01 and no premature stop codons were present in exons 3 or 4. To determine the functional characteristics of the Ab, surrogate donors were tested by flow cytometry crossmatch (FCXM). One donor was DR7 homozygous and the other was DR4, DR7. No reactivity was detected against these donors’ DR antigens, despite the presence of 4 potential targets (figure). This case demonstrates the usefulness of additional testing by FCXM to confirm the presence of false pan-DR reactivity, probably due to a denatured epitopes. Download : Download high-res image (533KB) Download : Download full-size image

Aim To evaluate NGS (Next Generation Sequencing) platforms for high resolution HLA typing in a clinical setting. HLA mismatches can have a significant effect on transplantation outcome. Current low resolution HLA typing techniques are unable to provide coverage for the growing number of known alleles. Sanger sequencing, the standard for high resolution typing, cannot provide full gene coverage or phasing. Newly developed Next Generation Sequencing kits accomplish full length gene sequencing and allele phasing. NGS reduces ambiguity in HLA genotyping, providing the best possible results for transplant matching. Full gene sequencing also allows for the discovery of novel alleles. Method Two assay platforms are being evaluated. Whole genes are amplified by long range polymerase chain reaction, yielding amplicons ∼3–10 Kilobases in length. Long amplicons are randomly fragmented using proprietary enzymatic methods. Unique “barcode” sequence adapters are ligated to fragment ends. Fragments are combined to create a paired end sequencing library. Size selection is performed for best quality sequencing reads. Libraries are sequenced on the Illumina MiSeq (40 h). Analysis software assembles reads and assigns HLA typing based on IMGT (international ImMunoGeneTics project)/HLA database information. Results Our first sequencing run (12 samples – 96 loci) yielded 97% concordance. Data points were eliminated due to: no amplification or sequencing due to possible degraded DNA (4), novel variant (1), full reference sequence unknown (2), analysis errors since resolved with software development (6). Issues requiring troubleshooting include amplification failure at one locus and uneven sequence coverage for Class I loci. Additional sequencing runs will be analyzed and compared. Limitations of NGS include blocking of SNP positions due to primer placement and lack of full IMGT sequences of some alleles. Conclusion Workflow complexity, turnaround time, and cost of instrumentation and consumables must be considered in a decision to adopt NGS in a clinical HLA laboratory. Meticulous techniques which can impact outcome must be mastered. NGS is useful to sequence multiple samples using DNA barcodes which makes it viable for routine use, which may significantly improve transplantation outcome.

ABO incompatibility (ABOi) is no longer an absolute contraindication for kidney transplantation. Recipients who are either blood group O or B have successfully received kidney transplants from donors who are blood group A, as long as their titer against the donor A antigen is low. Although the predominant A antigen subtype in the population is A1, less than 20% of blood group A individuals are of other subtypes, with weaker antigen expression, A2 being the most common of these. Accurate quantification of the amount of Anti A antibody titer in the recipient against the intended donor is important to determine compatibility for ABOi transplants. A review of the literature demonstrated that there is a high degree of technique variability, especially concerning the target cells utilized to determine such titers. Accordingly, we decided to use A2 and donor RBCs as well as our standard A1 RBCs in our assays, in order to determine the differences in titer when various A subtype cells are utilized as the target. Our case is a 54 year old male; 0% CPRA, blood group O recipient, whose donor was ABO typed as a non-A1. Flow T and B cell crossmatch negative. The transplant program decided to move forward with the ABO incompatible transplant since no other live donors were available. Determination of the Anti-A titer was as follows: Recipient sera was serially diluted with saline from 1:1 to 1:512. One drop of RBC suspension is added to 2 drops (100 ul) of each sera dilution. Three different titrations were performed using commercially produced A1 and A2 RBC as well as donor RBC. Tubes were incubated at RT for 30 minutes, centrifuged and agglutination macroscopically observed and recorded. Tubes were then incubated at 37C for 30 minutes, washed 4 times with saline, IgG added then centrifuged and agglutination recorded again. This procedure allows for determination of both IgM and IgG antibodies against blood group A antigens. IgG titers were 1:16 with A1 RBC; 1:8 with A2 RBC; and 1:8 with donor RBC. Our transplant program considers an IgG titer less than or equal to 1:8 acceptable and the transplant was performed. Recipient is out 11 weeks with no rejection episodes.

Aim YNHH initiated EPIC Go Live to enable electronic medical record (EMR) data capture for all patients last year. Since an attractive feature of this system is the ability to provide electronic access to data not only for clinical but research use, the aim of our project was to develop the process for all histocompatibility data to populate EPIC. Methods The Laboratory uses Histotrac (Systemlink), as the laboratory management system. An interface (HL7 manager) was required for data migration into EPIC, and Histotrac latest version software was updated. Significant firewall issues were encountered since the Lab is university based and data required two way migration between hospital and university, as well as vendor. Significant steps were taken to ensure that the data was represented in intelligible form by EPIC, which required significant programming, test definition, and error tracking. All existing patients required new medical record numbers as a consequence of EPIC adoption, and every patient sample received without an electronic order required review and problem solving prior to testing. Order sets were created to simplify the ordering process for coordinators and reduce order entry errors. Extensive code and definitions were developed to allow for antibody testing (single antigen, ID, cytotoxicity), along with titration, pretreatment, and donor specific antibody data in a presentation format that is informative to clinicians. Results All histocompatibility data is now available in EPIC. This project has required one full time FTE, as well as training of all staff on use of EPIC. Hurdles encountered include ensuring all patient samples (including monthly sera from dialysis) have EPIC orders in place, identifying when discrete data elements can be transmitted and when imaged data is preferred. Solutions involving PDF creation, transmission and representation were necessary for specific data elements. Conclusion In conclusion, all data on active patients is now available in EPIC, and technologists use the EMR as the source for all data, reducing transcriptional errors. Next steps include paper data elimination, billing through EPIC, and creating a strategy to handle deceased donor data reporting. G. Manley: Employee; Company/Organization; SystemLink.

Aim Post-PCR amplification products can pose a problem in clinical laboratories. Great care is needed to ensure post and pre-PCR areas minimize the risk of unwanted amplicons affecting results. Linkage Biosciences designed the LinkS e ¯ q™ real-time PCR HLA test to include deoxyuridine triphosphate (dUTP) rather than the standard deoxythmidine triphosphate (dTTP) in the PCR reaction as a preventive measure so that Uracil DNA Glycosylase enzyme (UDG) can be used as an optional additive to eliminate unwanted uracil-containing amplicons during PCR set-up. The aim of this study was to evaluate the effectiveness of adding UDG to the LinkS e ¯ q™ test in a laboratory setting with False Positive and False Negative issues. Methods To validate the usefulness of UDG reagent (provided by Linkage Biosciences) we selected 10 clinical DNA samples that contained aberrant primer dimer peaks which may have contributed to False Positive and False Negative allele amplification. All samples were typed with the standard LinkS e ¯ q™ protocol and with the UDG protocol. The 2 real-time kits that were used were the LinkS e ¯ q™ 384 well tray, Cat #1575 and the 192 well tray, Cat#1554 with SYBR Green dye for melting curve analysis. All samples were amplified using the Roche LightCycler® 480. Results All 10 samples showed improved results in the reduction of False Positives and removal of dimeric peaks using the UDG reagent. The data shows a total reduction of 93.6% of the False Positives, 66.6% reduction in False Negatives, and 86.7% reduction of software forced calls. The samples demonstrated concordance with PCR-SSP (Invitrogen), or UNET reported results. Conclusions In summary, the LinkS e ¯ q™ real-time PCR assay is a fast and reliable HLA typing method. The UDG reagent works well when necessary to remove suspect peaks which may interfere with SureTyper Software data analysis.