Cutaneous adverse reactions associated with antiseizure medication: clinical characteristics and implications in epilepsy treatment.

OBJECTIVE: To describe the clinical characteristics of cutaneous adverse reactions and cross-sensitivity induced by antiseizure medications and compare the pattern of use of antiseizure medications in patients with epilepsy according to skin rash history. METHODS: We analysed patients with a history of skin rash presenting for up to 12 weeks after initiating antiseizure medication. The history of skin rash was verified by medical charts, interviews, and identification of skin lesions by patients based on illustrative images. The minimum follow-up period was eight months. The control group comprised epilepsy patients with regular antiseizure medication use for at least 12 weeks without skin rash. We included 109 cases and 99 controls. RESULTS: The median (interquartile range) period from the index rash was six years (2-11). Carbamazepine was the trigger medication in 48% of cases and induced skin rashes in all patients with cross-sensitivity and carbamazepine exposure. Stevens-Johnson syndrome, toxic epidermal necrolysis, or drug reactions with eosinophilia and systemic symptoms affected 36% of cases. Carbamazepine- or oxcarbazepine-induced maculopapular exanthema occurred earlier (median: one week) than that induced by other antiseizure medications (median: three weeks) (p=0.006). Cross-sensitivity was more common in patients with at least one episode of Stevens-Johnson syndrome (29%) and Stevens-Johnson/toxic epidermal necrolysis overlap (50%) than in patients with maculopapular exanthema (8%) (p=0.01). Although most cases were mild, the pattern of antiseizure medication use differed from that of controls, with a lower proportion of antiseizure medication typically associated with severe cutaneous adverse reactions (carbamazepine, phenytoin, phenobarbital, primidone, oxcarbazepine, and lamotrigine) (p<0.001). Most cases exposed to high-risk medication, however, did not develop cross-sensitivity. SIGNIFICANCE: Cutaneous adverse reaction history may influence antiseizure medication use. Cross-sensitivity is more common in severe cases and most patients are affected by mild, self-limited skin rashes. Further research should consider the relevance of mild skin rashes in lifelong epilepsy treatment.

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.

MVO Automation Platform: Addressing Unmet Needs in Clinical Laboratories with Microcontrollers, 3D Printing, and Open-Source Hardware/Software.

Laboratory automation improves test reproducibility, which is vital to patient care in clinical laboratories. Many small and specialty laboratories are excluded from the benefits of automation due to low sample number, cost, space, and/or lack of automation expertise. The Minimum Viable Option (MVO) automation platform was developed to address these hurdles and fulfill an unmet need. Consumer 3D printing enabled rapid iterative prototyping to allow for a variety of instrumentation and assay setups and procedures. Three MVO versions have been produced. MVOv1.1 successfully performed part of a clinical assay, and results were comparable to those of commercial automation. Raspberry Pi 3 Model B (RPI3) single-board computers with Sense Hardware Attached on Top (HAT) and Raspberry Pi Camera Module V2 hardware were remotely accessed and evaluated for their suitability to qualify the latest MVOv1.2 platform. Sense HAT temperature, barometric pressure, and relative humidity sensors were stable in climate-controlled environments and are useful in identifying appropriate laboratory spaces for automation placement. The RPI3 with camera plus digital dial indicator logged axis travel experiments. RPI3 with camera and Sense HAT as a light source showed promise when used for photometric dispensing tests. Individual well standard curves were necessary for well-to-well light and path length compensations.

Major Histocompatibility Mismatch and Donor Choice for Second Allogeneic Bone Marrow Transplantation.

Large alternative donor pools provide the potential for selecting a different donor for a second allogeneic (allo) bone or marrow transplant (BMT). As HLA disparity may contribute to the graft-versus-tumor effect, utilizing new mismatched haplotype donors may potentially improve the antitumor activity for relapsed hematologic malignancies despite a previous alloBMT. Data from patients who received a second alloBMT for relapsed hematologic malignancies at Johns Hopkins were analyzed. Outcomes were compared between patients who received a second allograft with the same MHC composition and those who received an allograft with a new mismatched haplotype. Loss of heterozygosity analysis was performed for patients with acute myeloid leukemia (AML) whose first allograft was haploidentical. Between 2005 and 2015, 40 patients received a second BMT for a relapsed hematologic malignancy. The median follow-up is 750 (range, 26 to 2950) days. The median overall survival (OS) in the cohort is 928 days (95% confidence interval [CI], 602 to not reached [NR]); median event-free survival (EFS) for the cohort is 500 days (95% CI, 355 to NR). The 4-year OS is 40% (95% CI, 25% to 64%), and the 4-year EFS is 36% (95% CI, 24% to 55%). The cumulative incidence of nonrelapsed mortality by 2 years was 27% (95% CI, 13% to 42%). The cumulative incidence of grade 3 to 4 acute graft-versus-host disease (GVHD) at 100 days was 15% (95% CI, 4% to 26%); the cumulative incidence of extensive chronic GVHD at 2 years was 22% (95% CI, 9% to 36%). The median survival was 552 days (95% CI, 376 to 2950+) in the group who underwent transplantation with a second allograft that did not harbor a new mismatched haplotype, while it was not reached in the group whose allograft contained a new mismatched haplotype (hazard ratio [HR], .36; 95% CI, .14 to .9; P = .02). EFS was also longer in the group who received an allograft containing a new mismatched haplotype, (NR versus 401 days; HR, .50; 95% CI, .22 to 1.14; P = .09). Although the allograft for this patient's second BMT contained a new mismatched haplotype, AML nevertheless relapsed a second time. Second BMTs are feasible and provide a reasonable chance of long-term survival. An allograft with a new mismatched haplotype may improve outcomes after second BMTs for relapsed hematologic malignancies.

Haplotype counting by next-generation sequencing for ultrasensitive human DNA detection.

Human identity testing is critical to the fields of forensics, paternity, and hematopoietic stem cell transplantation. Most bone marrow (BM) engraftment testing currently uses microsatellites or short tandem repeats that are resolved by capillary electrophoresis. Single-nucleotide polymorphisms (SNPs) are theoretically a better choice among polymorphic DNA; however, ultrasensitive detection of SNPs using next-generation sequencing is currently not possible because of its inherently high error rate. We circumvent this problem by analyzing blocks of closely spaced SNPs, or haplotypes. As proof-of-principle, we chose the HLA-A locus because it is highly polymorphic and is already genotyped to select proper donors for BM transplant recipients. We aligned common HLA-A alleles and identified a region containing 18 closely spaced SNPs, flanked by nonpolymorphic DNA for primer placement. Analysis of cell line mixtures shows that the assay is accurate and precise, and has a lower limit of detection of approximately 0.01%. The BM from a series of hematopoietic stem cell transplantation patients who tested as all donor by short tandem repeat analysis demonstrated 0% to 1.5% patient DNA. Comprehensive analysis of the human genome using the 1000 Genomes database identified many additional loci that could be used for this purpose. This assay may prove useful to identify hematopoietic stem cell transplantation patients destined to relapse, microchimerism associated with solid organ transplantation, forensic applications, and possibly patient identification.

HLA alleles and haplotypes among the Lakota Sioux: report of the ASHI minority workshops, part III.

Human leukocyte antigen (HLA) class I and II alleles were defined for 302 Lakota Sioux American Indians as part of the American Society for Histocompatibility and Immunogenetics coordinated studies on minority populations. The study group was comprised of adult volunteers from the Cheyenne River and Ogala Sioux tribes residing, respectively, on the Cheyenne River and Pine Ridge Reservations in South Dakota. Of the participants, 263 (87%) claimed full American Indian ancestry through both maternal and paternal grandparents. The study group included 25 nuclear families that were informative for genotyping. HLA phenotypes from 202 adults with no other known first-degree relative included in the study were used for calculation of allele and haplotype frequencies by maximum likelihood estimation. HLA-A, -B, and -Cw alleles were found to be in Hardy Weinberg equilibrium. Deviation from equilibrium was observed for DRB1 alleles (p=0.01), but could be attributed to the sample size and the occurrence of some genotypes with low expected frequencies. Polymorphism among the Sioux was limited with four to seven alleles comprising >80% of those observed at each locus. Several alleles were found at high frequency (0.05-0.30) among the Sioux that are also prevalent in other Native Americans and Alaska Natives, including: A*2402, *3101, and *0206; B*3501,*3901, *5101, and *2705; Cw*0702, *0404, and *03041; DRB1*0407, *0404, *1402, and *16021; and DQB1*0301, *0302, and *0402. DRB1*0811, which has been only previously described in Navajo and Tlingit Indians, was found to occur at a frequency of 0.119 among the Sioux. Two new alleles were defined among the Sioux: Cw*0204 and DRB1*040703, which were found in two and four individuals, respectively. In the haplotype analyses, significant linkage disequilibrium (p<0.00001) was seen in all pairwise comparisons of loci and numerous two and three locus haplotypes were found to have strong, positive linkage disequilibrium values. The two most common extended haplotypes among the Sioux, determined by maximum likelihood estimation and genotyping were: A*31012, B*3501, Cw*0404, DRB1*0407; and A*24021, B*3501, Cw*0404, DRB1*0404.