An Integrated Clinical and Genetic Prediction Model for Tacrolimus Levels in Pediatric Solid Organ Transplant Recipients.

BACKGROUND: There are challenges in achieving and maintaining therapeutic tacrolimus levels after solid organ transplantation (SOT). The purpose of this genome-wide association study was to generate an integrated clinical and genetic prediction model for tacrolimus levels in pediatric SOT. METHODS: In a multicenter prospective observational cohort study (2015-2018), children <18 years old at their first SOT receiving tacrolimus as maintenance immunosuppression were included (455 as discovery cohort; 322 as validation cohort). Genotyping was performed using a genome-wide single nucleotide polymorphism (SNP) array and analyzed for association with tacrolimus trough levels during 1-y follow-up. RESULTS: Genome-wide association study adjusted for clinical factors identified 25 SNPs associated with tacrolimus levels; 8 were significant at a genome-wide level (P < 1.025 × 10-7). Nineteen SNPs were replicated in the validation cohort. After removing SNPs in strong linkage disequilibrium, 14 SNPs remained independently associated with tacrolimus levels. Both traditional and machine learning approaches selected organ type, age at transplant, rs776746, rs12333983, and rs12957142 SNPs as the top predictor variables for dose-adjusted 36- to 48-h posttacrolimus initiation (T1) levels. There was a significant interaction between age and organ type with rs776476*1 SNP (P < 0.05). The combined clinical and genetic model had lower prediction error and explained 30% of the variation in dose-adjusted T1 levels compared with 18% by the clinical and 12% by the genetic only model. CONCLUSIONS: Our study highlights the importance of incorporating age, organ type, and genotype in predicting tacrolimus levels and lays the groundwork for developing an individualized age and organ-specific genotype-guided tacrolimus dosing algorithm.

Next-generation HLA typing of 382 International Histocompatibility Working Group reference B-lymphoblastoid cell lines: Report from the 17th International HLA and Immunogenetics Workshop.

Extended molecular characterization of HLA genes in the IHWG reference B-lymphoblastoid cell lines (B-LCLs) was one of the major goals for the 17th International HLA and Immunogenetics Workshop (IHIW). Although reference B-LCLs have been examined extensively in previous workshops complete high-resolution typing was not completed for all the classical class I and class II HLA genes. To address this, we conducted a single-blind study where select panels of B-LCL genomic DNA samples were distributed to multiple laboratories for HLA genotyping by next-generation sequencing methods. Identical cell panels comprised of 24 and 346 samples were distributed and typed by at least four laboratories in order to derive accurate consensus HLA genotypes. Overall concordance rates calculated at both 2- and 4-field allele-level resolutions ranged from 90.4% to 100%. Concordance for the class I genes ranged from 91.7 to 100%, whereas concordance for class II genes was variable; the lowest observed at HLA-DRB3 (84.2%). At the maximum allele-resolution 78 B-LCLs were defined as homozygous for all 11 loci. We identified 11 novel exon polymorphisms in the entire cell panel. A comparison of the B-LCLs NGS HLA genotypes with the HLA genotypes catalogued in the IPD-IMGT/HLA Database Cell Repository, revealed an overall allele match at 68.4%. Typing discrepancies between the two datasets were mostly due to the lower-resolution historical typing methods resulting in incomplete HLA genotypes for some samples listed in the IPD-IMGT/HLA Database Cell Repository. Our approach of multiple-laboratory NGS HLA typing of the B-LCLs has provided accurate genotyping data. The data generated by the tremendous collaborative efforts of the 17th IHIW participants is useful for updating the current cell and sequence databases and will be a valuable resource for future studies.

Quality control project of NGS HLA genotyping for the 17th International HLA and Immunogenetics Workshop.

The 17th International HLA and Immunogenetics Workshop (IHIW) organizers conducted a Pilot Study (PS) in which 13 laboratories (15 groups) participated to assess the performance of the various sequencing library preparation protocols, NGS platforms and software in use prior to the workshop. The organizers sent 50 cell lines to each of the 15 groups, scored the 15 independently generated sets of NGS HLA genotyping data, and generated "consensus" HLA genotypes for each of the 50 cell lines. Proficiency Testing (PT) was subsequently organized using four sets of 24 cell lines, selected from 48 of 50 PS cell lines, to validate the quality of NGS HLA typing data from the 34 participating IHIW laboratories. Completion of the PT program with a minimum score of 95% concordance at the HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1 loci satisfied the requirements to submit NGS HLA typing data for the 17th IHIW projects. Together, these PS and PT efforts constituted the 17th IHIW Quality Control project. Overall PT concordance rates for HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRB1, HLA-DRB3, HLA-DRB4 and HLA-DRB5 were 98.1%, 97.0% and 98.1%, 99.0%, 98.6%, 98.8%, 97.6%, 96.0%, 99.1%, 90.0% and 91.7%, respectively. Across all loci, the majority of the discordance was due to allele dropout. The high cost of NGS HLA genotyping per experiment likely prevented the retyping of initially failed HLA loci. Despite the high HLA genotype concordance rates of the software, there remains room for improvement in the assembly of more accurate consensus DNA sequences by NGS HLA genotyping software.

Performance of an allele-level multi-locus HLA genotype imputation tool in hematopoietic stem cell donors from Quebec.

INTRODUCTION: Donor-recipient HLA compatibility is an important determinant of transplant outcomes. Allele-group to allele-level imputations help assign HLA genotypes when allele-level genotypes are not available during donor selection. METHODS: We evaluated the performance of HaploStats, an allele-level multi-locus HLA genotype imputation tool from the National Marrow Donor Program, in a cross-sectional study including hematopoietic stem cell donors (HSCD) from Quebec, Canada. A total of 144 self-identified Caucasian HSCD genotyped at the allele-group and allele-level for HLA-A, -B, -C, -DRB1, and -DQB1 loci were studied. We compared allele-level genotypes imputed by HaploStats to those obtained by the reference standard, sequenced-based typing (SBT). RESULTS: Imputation performance, determined by allele-level genotype recall (fraction of matching imputed and sequenced genotypes) was 97%, 96%, 95%, 84%, and 81% for HLA-A, -B, -C, -DRB1, and -DQB1 loci, respectively. Our sample deviated from Hardy-Weinberg equilibrium only at the HLA-DRB1 locus. Residual ambiguity, determined by typing resolution scores (TRS), was greatest for HLA class II loci (average TRS 0.65 and 0.80 for DRB1 and DQB1, respectively). In contrast, average TRS of 0.88, 0.84, and 0.92 was observed for HLA-A, -B, and -C, respectively. CONCLUSIONS: HLA allele imputation from ambiguous genotypes demonstrate satisfactory prediction accuracy for HLA class I but modest prediction accuracy for HLA class II loci in self-identified Caucasian HSCD from Quebec. While consideration of high-resolution allele and haplotype frequencies in the Quebec population may improve the performance of available allele-level multi-locus genotype imputation tools in Quebec, this study suggests that genotyping at the first two field level should be conducted whenever possible.

Guidance on platelet transfusion for patients with hypoproliferative thrombocytopenia.

Patients with hypoproliferative thrombocytopenia are at an increased risk for hemorrhage and alloimmunization to platelets. Updated guidance for optimizing platelet transfusion therapy is needed as data from recent pivotal trials have the potential to change practice. This guideline, developed by a large international panel using a systematic search strategy and standardized methods to develop recommendations, incorporates recent trials not available when previous guidelines were developed. We found that prophylactic platelet transfusion for platelet counts less than or equal to 10 × 10(9)/L is the optimal approach to decrease the risk of hemorrhage for patients requiring chemotherapy or undergoing allogeneic or autologous transplantation. A low dose of platelets (1.41 × 10(11)/m2) is hemostatically as effective as higher dose of platelets but requires more frequent platelet transfusions suggesting that low-dose platelets may be used in hospitalized patients. For outpatients, a median dose (2.4 × 10(11)/m2) may be more cost-effective to prevent clinic visits only to receive a transfusion. In terms of platelet products, whole blood-derived platelet concentrates can be used interchangeably with apheresis platelets, and ABO-compatible platelet should be given to improve platelet increments and decrease the rate of refractoriness to platelet transfusion. For RhD-negative female children or women of child-bearing potential who have received RhD-positive platelets, Rh immunoglobulin should probably be given to prevent immunization to the RhD antigen. Providing platelet support for the alloimmunized refractory patients with ABO-matched and HLA-selected or crossmatched products is of some benefit, yet the degree of benefit needs to be assessed in the era of leukoreduction.

Utility of cross-matched platelet transfusions in patients with hypoproliferative thrombocytopenia: a systematic review.

BACKGROUND: Multiply transfused hypoproliferative thrombocytopenic (HT) patients with alloimmune transfusion refractoriness require specially selected platelets (PLTs). Cross-matching apheresis PLTs is a popular support option, avoiding requirements for large panels of typed donors for HLA-based selection. We undertook a systematic review of the utility of various cross-matching techniques on mortality reduction, prevention of hemorrhage, alloimmunization and refractoriness, and improvement in PLT utilization or count increments. STUDY DESIGN AND METHODS: A systematic review to December 2012 was conducted of MEDLINE, EMBASE, and Cochrane databases along with a bibliographic search of pertinent references. RESULTS: Of 146 retrieved citations, 20 met inclusion criteria. Eleven more were chosen from bibliographies, describing 29 unique cohorts. All but five enrolled transfusion-refractory, predominantly alloimmunized patients. Cross-match impact on mortality and hemorrhage could not be assessed from these studies. Two studies demonstrated durable corrected count increments and/or breadth of alloimmunization throughout cross-match support; none addressed development or persistence of refractoriness. In alloimmunized refractory patients and nonrefractory cohorts with greater than 25% alloimmunization, higher increments were seen with cross-match-compatible PLTs than incompatible or un-cross-matched units. In two nonrefractory, nonalloimmunized cohorts, the lack of utility of cross-match was reflected by test sensitivity of less than 20%. Comparison of cross-matched PLT success with that of HLA-identical units revealed inferior success rates for the former in one study and equivalent rates in another. No trend was observed regarding relative utility of the various commonly employed techniques. CONCLUSION: Cross-matched PLTs are useful in increasing PLT counts in alloimmunized, transfusion-refractory HT patients, but data about their impact on hemorrhage and mortality are lacking.

Efficacy of HLA-matched platelet transfusions for patients with hypoproliferative thrombocytopenia: a systematic review.

BACKGROUND: HLA-matched platelets (PLTs) are widely used to transfuse patients but the effectiveness of HLA matching has not been well defined and the cost is approximately five times the cost of preparing the random-donor PLTs. The objective of this systematic review was to determine whether HLA-matched PLTs lead to a reduction in mortality; reduction in frequency or severity of hemorrhage; reduction in HLA alloimmunization, refractoriness, or PLT utilization; or improvement in PLT count increment in patients with hypoproliferative thrombocytopenia. STUDY DESIGN AND METHODS: We conducted a literature search of MEDLINE, Cochrane Controlled Register of Clinical Trials, EMBASE, and PubMed databases to April 2012. RESULTS: A total of 788 citations were reviewed and 30 reports were included in the analysis. Most studies did not include technologies currently in use for HLA typing or detection of HLA antibodies as 75% were conducted before the year 2000. None of the studies were adequately powered to detect an effect on mortality or hemorrhage. HLA-matched PLTs did not reduce alloimmunization and refractoriness rates beyond that offered by leukoreduction, and utilization was not consistently improved. HLA-matched PLTs led to better 1-hour posttransfusion count increments and percentage of PLT recovery in refractory patients; however, the effect at 24 hours was inconsistent. CONCLUSION: The correlation of the PLT increment with other clinical outcomes and the effect of leukoreduction on HLA-matched PLT transfusion could not be determined. Prospective studies utilizing current technology and examining clinical outcomes are necessary to demonstrate the effectiveness of HLA-matched PLT transfusion.

Blood donors implicated in transfusion-related acute lung injury with patient-specific HLA antibodies are more broadly sensitized to HLA antigens compared to other blood donors.

BACKGROUND: The prevalence of HLA antibodies in randomly surveyed blood donors was compared to the prevalence of antibody in donors who were associated with transfusion-related acute lung injury (TRALI) cases reported to Canadian Blood Services (CBS). STUDY DESIGN AND METHODS: Current operating procedure mandates that the CBS TRALI Medical Review Group (TMRG) refer possible TRALI cases to the (CBS) Platelet Immunology Laboratory for investigation. Donor samples from these TRALI cases were screened for HLA antibodies. In parallel, a survey was conducted to screen serum samples from blood donors who were not associated in TRALI cases. A comparison analysis of HLA antibody profiles in the two groups of donors was performed. RESULTS: We studied 121 TRALI-associated donors (TDs) who were recalled in a total of 44 cases reported to CBS and classified by TMRG. We also studied 149 survey donors (SDs) who were deferred for donation for varied reasons and consented to participate in a survey for HLA antibody screening. Twenty-two percent of SDs and 50.4% of TDs tested positive for HLA antibodies. In addition, TDs who were implicated in TRALI demonstrated broader sensitization and higher level of quantitative HLA antibody compared to nonimplicated TDs and SDs. CONCLUSION: Patient-specific Class I and II HLA antibodies are directly related to the risk of TRALI. Moreover, it supports the concept that HLA antibody strength is directly related to the risk of TRALI when the HLA antibody is patient specific; however, no clear cutoff as defined by mean fluorescence intensity is evident.

Transfusion-related acute lung injury prevention measures and their impact at Canadian Blood Services.

BACKGROUND: Blood operators have taken measures to reduce transfusion-related acute lung injury (TRALI). We classified suspected TRALI cases reported to Canadian Blood Services from 2001 to 2009 and assessed the impact of TRALI reduction measures. STUDY DESIGN AND METHODS: Using Canadian Consensus Conference definitions, cases were reviewed by two experts or, from 2006 to 2009, a TRALI Medical Review Group (TMRG). Detection of HLA antibodies was performed using the Luminex system starting in 2008. Measures implemented from 2007 to 2009 included use of predominantly male plasma, suspension of buffy coat platelets in male plasma, and deferral of females with a pregnancy history from plateletpheresis. The buffy coat production method was implemented from 2005 to 2008. RESULTS: Reporting of all suspected TRALI cases, as well as cases classified as definite or possible, increased from 2001 to 2004, was stable from 2004 to 2007, and declined in 2008 to 2009. The decline was most marked for plasma-associated cases, but occurred for all components. TMRG consensus on classification was achieved in 56% of cases. Cases identified as definitive or possible TRALI were significantly more likely to have donor antibody against a corresponding recipient antigen, compared to other cases. CONCLUSION: Hemovigilance data demonstrated an initial increase in TRALI cases, likely due to increased adverse event reporting and awareness of TRALI, followed by a decrease in cases related to all components. TRALI prevention measures and possibly the switch to the buffy coat production method may have contributed to the decline. Classification of cases remains challenging.

Two cases of platelet transfusion refractoriness associated with anti-CD36.

BACKGROUND: Antibodies to platelet (PLT) glycoprotein (GP) IV (CD36) have been implicated in rare cases of PLT refractoriness, particularly in non-Caucasians. We report two cases of PLT transfusion refractoriness linked to anti-CD36. STUDY DESIGN AND METHODS: A 5-year-old female of Lebanese descent and a 70-year-old male of Chinese descent both failed to respond to HLA-matched PLT transfusions during acute myelogenous leukemia induction therapy. Antibody screening was performed using a PLT antibody solid-phase kit (PAKPLUS, GTI Diagnostics), followed by the monoclonal antibody-specific immobilization of PLT antigen (MAIPA) test and, for the second case, the modified antigen capture enzyme-linked immunosorbent assay (MACE). RESULTS: Both patients demonstrated antibody to GP IV (CD36) on the PAKPLUS assay. On MAIPA testing, both phenotyped as CD36 negative. Anti-CD36 was demonstrated by MAIPA in the first case. In the second case, antibodies were not detected by MAIPA and variably detectable by MACE, depending on the mouse monoclonal antibody (MoAb) used. Because no Canadian CD36-negative donors were available, antigen-negative plateletpheresis units from the BloodCenter of Wisconsin were successfully transfused. CONCLUSION: Two cases of clinically significant CD36 antibodies are reported. Investigation of one case was complicated by steric inhibition of binding in the MAIPA and MACE assays with certain MoAbs. The cases demonstrate the importance of maintaining an ethnically diverse pool of rare donors and the value of international cooperation in the management of these patients.