Oligonucleotide Probe Hybridization Research Articles

Distribution of the Prevalence of Human Leukocyte Antigen (HLA)-B*57:01 Positivity in HIV-1 Infected Individuals and Its Effects on Treatment: Türkiye Map-Buhasder Working Group

Human immunodeficiency virus (HIV)/acquired immundeficiency syndrome (AIDS) is a critical global public health problem that significantly affects both life expectancy and the overall quality of life of individuals in all age groups. The landscape of HIV infection has changed significantly in recent years due to the introduction of effective combination antiretroviral therapies (ART). A key component of first-line ART regimens for HIV treatment is abacavir, a nucleoside HIV reverse transcriptase inhibitor. Although abacavir is effective in suppressing viral replication and managing disease, its clinical utility is overshadowed by the potential for life-threatening hypersensitivity reactions in HLA-B*57:01-positive patients. In our country, local data obtained from various centers regarding the prevalence of HLA-B*57:01 in HIV-1-infected patients are available. In this study, it was aimed to determine the prevalence of the HLA-B*57:01 genotype in HIV-infected patients who were followed up and treated in many regions of our country. This retrospective study consists of the data of the patients aged 18 years and over diagnosed with HIV-1 infection between 01.01.2019 and 31.07.2022. Age, gender, place of birth, mode of transmission of the disease, death status, CD4+ T cell count and HIV RNA levels at the first clinical presentation, HLA-B*57:01 positivity, and the method used, clinical stage of the disease, virological response time with the treatment they received were recorded from the patient files. Data were collected from 16 centers and each center used different methods to detect HLA-B*57:01. These methods were sequence-specific oligonucleotide probe hybridization (SSOP), DNA sequence-based typing (SBT), single-specific primer-polymerase chain reaction (SSP-PCR), allele-specific PCR (AS-PCR) and quantitative PCR (Q-PCR). A total of 608 HIV-infected individuals, 523 males (86%) and 85 females (14%), were included in the study. The mean age of the patients was 36.9 ± 11.9 (18-73) years. The prevalence of HLA-B*57:01 allele was found to be 3.6% (22 patients). The number of CD4+ T lymphocytes in HLA-B*57:01 allele-positive patients was > 500/ mm3 in 10 patients (45.5%), while the number of CD4+ T lymphocytes in HLA-B*57:01 negative patients was > 500/mm3 in 216 patients (36.9%) (p> 0.05). Viral load at the time of diagnosis was found to be lower in patients with positive HLA-B*57:01 allele but it was not statistically significant (p> 0.05). Although different treatment algorithms were used in the centers following the patients, it was observed that the duration of virological response was shorter in HLA-B*57:01 positive patients (p= 0.006). Although the presence of the HLA-B*57:01 allele has a negative impact due to its association with hypersensitivity, it is likely to continue to attract interest due to its association with slower progression of HIV infection and reduced risk of developing AIDS. In addition, although the answer to the question of whether it is cost-effective to screen patients for HLA-B*57:01 before starting an abacavir-containing ART regimen for the treatment of HIV infection is being sought, it seems that HIV treatment guidelines will continue to recommend screening to identify patients at risk in this regard.

Highly Multiplexed and Simultaneous Characterization of Protein and RNA in Single Cells by Flow or Mass Cytometry Platforms Using Proximity Ligation Assay for RNA.

In situ hybridization of oligonucleotide probes to intracellular RNA allows quantification of predefined gene transcripts within millions of single cells using cytometry platforms. Previous methods have been hindered by the number of RNA that can be analyzed simultaneously. Here we describe a method called proximity ligation assay for RNA (PLAYR) that permits highly multiplexed RNA analysis that can be combined with antibody staining. Potentially any number of RNA combined with antigen can be analyzed together, being limited only by the number of analytes that can be measured simultaneously.

Cardiomyocyte precursors generated by direct reprogramming and molecular beacon selection attenuate ventricular remodeling after experimental myocardial infarction

BackgroundDirect cardiac reprogramming is currently being investigated for the generation of cells with a true cardiomyocyte (CM) phenotype. Based on the original approach of cardiac transcription factor-induced reprogramming of fibroblasts into CM-like cells, various modifications of that strategy have been developed. However, they uniformly suffer from poor reprogramming efficacy and a lack of translational tools for target cell expansion and purification. Therefore, our group has developed a unique approach to generate proliferative cells with a pre-CM phenotype that can be expanded in vitro to yield substantial cell doses.MethodsCardiac fibroblasts were reprogrammed toward CM fate using lentiviral transduction of cardiac transcriptions factors (GATA4, MEF2C, TBX5, and MYOCD). The resulting cellular phenotype was analyzed by RNA sequencing and immunocytology. Live target cells were purified based on intracellular CM marker expression using molecular beacon technology and fluorescence-activated cell sorting. CM commitment was assessed using 5-azacytidine-based differentiation assays and the therapeutic effect was evaluated in a mouse model of acute myocardial infarction using echocardiography and histology. The cellular secretome was analyzed using mass spectrometry.ResultsWe found that proliferative CM precursor-like cells were part of the phenotype spectrum arising during direct reprogramming of fibroblasts toward CMs. These induced CM precursors (iCMPs) expressed CPC- and CM-specific proteins and were selectable via hairpin-shaped oligonucleotide hybridization probes targeting Myh6/7-mRNA–expressing cells. After purification, iCMPs were capable of extensive expansion, with preserved phenotype when under ascorbic acid supplementation, and gave rise to CM-like cells with organized sarcomeres in differentiation assays. When transplanted into infarcted mouse hearts, iCMPs prevented CM loss, attenuated fibrotic scarring, and preserved ventricular function, which can in part be attributed to their substantial secretion of factors with documented beneficial effect on cardiac repair.ConclusionsFibroblast reprogramming combined with molecular beacon-based cell selection yields an iCMP-like cell population with cardioprotective potential. Further studies are needed to elucidate mechanism-of-action and translational potential.Graphical

Flow karyotyping of wheat-Aegilops additions facilitate dissecting the genomes of Ae. biuncialis and Ae. geniculata into individual chromosomes.

Breeding of wheat adapted to new climatic conditions and resistant to diseases and pests is hindered by a limited gene pool due to domestication and thousands of years of human selection. Annual goatgrasses (Aegilops spp.) with M and U genomes are potential sources of the missing genes and alleles. Development of alien introgression lines of wheat may be facilitated by the knowledge of DNA sequences of Aegilops chromosomes. As the Aegilops genomes are complex, sequencing relevant Aegilops chromosomes purified by flow cytometric sorting offers an attractive route forward. The present study extends the potential of chromosome genomics to allotetraploid Ae. biuncialis and Ae. geniculata by dissecting their M and U genomes into individual chromosomes. Hybridization of FITC-conjugated GAA oligonucleotide probe to chromosomes suspensions of the two species allowed the application of bivariate flow karyotyping and sorting some individual chromosomes. Bivariate flow karyotype FITC vs. DAPI of Ae. biuncialis consisted of nine chromosome-populations, but their chromosome content determined by microscopic analysis of flow sorted chromosomes indicated that only 7Mb and 1Ub could be sorted at high purity. In the case of Ae. geniculata, fourteen chromosome-populations were discriminated, allowing the separation of nine individual chromosomes (1Mg, 3Mg, 5Mg, 6Mg, 7Mg, 1Ug, 3Ug, 6Ug, and 7Ug) out of the 14. To sort the remaining chromosomes, a partial set of wheat-Ae. biuncialis and a whole set of wheat-Ae. geniculata chromosome addition lines were also flow karyotyped, revealing clear separation of the GAA-rich Aegilops chromosomes from the GAA-poor A- and D-genome chromosomes of wheat. All of the alien chromosomes represented by individual addition lines could be isolated at purities ranging from 74.5% to 96.6% and from 87.8% to 97.7%, respectively. Differences in flow karyotypes between Ae. biuncialis and Ae. geniculata were analyzed and discussed. Chromosome-specific genomic resources will facilitate gene cloning and the development of molecular tools to support alien introgression breeding of wheat.

HLA-DRB1∗1502 Is Associated With Anti-N-Methyl-D-aspartate Receptor Encephalitis in Thai Children

BackgroundAnti-N-methyl-d-aspartate receptor encephalitis (anti-NMDARE) is one of the most common types of autoimmune encephalitis. Most patients have no apparent immunologic triggers, which suggests a genetic predisposition. This study was conducted to identify human leukocyte antigen (HLA) class II alleles associated with anti-NMDARE in Thai children. MethodsThis case-control study enrolled patients younger than 18 years who were diagnosed with anti-NMDARE between January 2010 and December 2020. A “good outcome” was determined as a patient with a modified Rankin scale score of less than 2 at any follow-up visit. HLA genotypes were determined at four-digit alleles using reverse sequence-specific oligonucleotide probe hybridization. The HLA class II allele frequency in patients was compared with that in a database of 101 healthy control Thai children. ResultsThirty-four patients were enrolled with a mean age of 12.8 ± 5.6 years (females 85.3%). The HLA-DRB1∗1502 allele frequency was significantly higher in patients than in controls (odds ratio, 2.32; 95% confidence interval, 1.11-4.8, P = 0.023). A good outcome was noted in 14 of 14 (100%) HLA-DRB1∗1502-positive patients (median time to a good outcome, 6 months) and 14 of 17 (82.3%) HLA-DRB1∗1502-negative patients (median time to a good outcome, 3 months). Two (11.8%) HLA-DRB1∗1502-positive patients had one relapse each, and six (35.3%) HLA-DRB1∗1502-negative patients had one to three relapses. ConclusionsHLA-DRB1∗1502 was significantly associated with anti-NMDARE in our patients. Most patients had good outcomes. HLA-DRB1∗1502-positive patients tended to require a longer time to achieve a good outcome but had less frequent relapses than HLA-DRB1∗1502-negative patients.

A novel colorimetric aptasensor for sensitive tetracycline detection based on the peroxidase-like activity of Fe3O4@Cu nanoparticles and "sandwich" oligonucleotide hybridization.

Anovel colorimetric aptasensor has been developedfor highly sensitive tetracycline (TC) detection based on the peroxidase-like activity of Fe3O4@Cu nanoparticles and "sandwich" oligonucleotide hybridization. The Fe3O4@Cu nanoparticles with high peroxidase-like activity were successfully synthesized under mild conditions. Then, a "sandwich" oligonucleotide hybridization probe (a short amino-modified complementary sequence of a portion of the TC aptamer (cDNA1), TC aptamers, and a long complementary to 5' terminal TC aptamer sequence (cDNA2)) was created in 96-wells plates via DNA hybridization in the absence of TC from the detection system. The unique "sandwich" oligonucleotide hybridization probe adsorbed large numbers of Fe3O4@Cu nanozymes while further enhancing its peroxidase-like activity. Based on the 3,3',5,5'-tetramethylbenzidine (TMB)-hydrogen peroxide (H2O2) reporting system, the blue color of the solution decreased linearly with the increase ofTC concentration, ranging from 10-3 to 103μg/L with an ultralow limit of detection (LOD) of 0.91ng/L (2pM). Theproposed method was successfully applied to detect TC in spiked milk samples, with recoveries of 81.8 to 112%, demonstrating the excellent potential for highly sensitive TC detection in milk.

Hybridization of Oligonucleotide Probes in Aqueous Solutions: Washing in Buffers Containing Quaternary Ammonium Salts.

In this protocol, hybridization is first performed in conventional aqueous solvents at a temperature well below the melting temperature, and the hybrids are then washed at higher stringency in buffers containing quaternary alkylammonium salts. TMACl is used with probes that are 14-50 nt in length, whereas TEACl is used with oligonucleotides that are 50-200 nt in length.

HLA-A, -B, -C, -DRB1 and -DQB1 alleles and haplotypes in 271 Southeast Asia Indians from Peninsular Malaysia

A total of 271 Southeast Asia Indians from Peninsular Malaysia were genotyped for HLA-A, -B, -C, -DRB1, and -DQB1 loci using polymerase chain reaction sequence-specific oligonucleotide probe hybridization methods. In this report, HLA-B and HLA-DQB1 was in Hardy-Weinberg proportions (HWEP) (p > 0.05). We observed significant deviation from the HWEP for HLA-A (p < 0.05), HLA-C (p < 0.01) and HLA-DRB1 (p < 0.01) loci. This genotype data is available in Allele Frequencies Network Database (AFND) Dos Santos et al. (2016).

HLA-A, -B, -DRB1 and -DQB1 polymorphisms among Iraqi Arabs

In this report, HLA polymorphisms (A, B, DRB1 and DQB1 loci) were determined in 149 unrelated Iraqi Arab potential bone marrow and kidney donors. Molecular genotyping was carried out by polymerase chain reaction followed by specific oligonucleotide probe hybridizations. Data were analyzed by Arlequin software. HLA-A, -B and -DRB1 genotype frequencies were significantly deviated from Hardy-Weinberg equilibrium, while HLA-DQB1 frequencies showed no deviation. A*03, B*35, DRB1*11 and DQB1*02 were the most frequent allele groups, while A*02-B*07-DRB1*04-DQB1*03 was the most frequent haplotype. HLA data are available in the Allele Frequencies Net Database (AFND: 3680) under the population name “Iraq Arabs”.

HLA-A, -B, -C, -DRB1 and -DQB1 alleles and haplotypes in 194 Southeast Asia Chinese from Peninsular Malaysia

A total of 194 Southeast Asia Chinese from Peninsular Malaysia were genotyped for HLA-A, -B, -C -DRB1, and -DQB1 loci using polymerase chain reaction sequence-specific oligonucleotide probe hybridization methods. In this report, the HLA-B, HLA-DRB1 and HLA-DQB1 were in Hardy-Weinberg proportions (HWEP) (p > 0.05). We observed significant deviation from HWEP in HLA-A (p < 0.05) and HLA-C (p < 0.01) loci. This genotype data was available in Allele Frequencies Network Database (AFND) Dos Santos et al. (2016).

A Multicolor Fluorescence in situ Hybridization Approach Using an Extended Set of Fluorophores to Visualize Microorganisms.

Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes is a key method for the detection of (uncultured) microorganisms in environmental and medical samples. A major limitation of standard FISH protocols, however, is the small number of phylogenetically distinct target organisms that can be detected simultaneously. In this study, we introduce a multicolor FISH approach that uses eight fluorophores with distinct spectral properties, which can unambiguously be distinguished by confocal laser scanning microscopy combined with white light laser technology. Hybridization of rRNA-targeted DNA oligonucleotide probes, which were mono-labeled with these fluorophores, to Escherichia coli cultures confirmed that the fluorophores did not affect probe melting behavior. Application of the new multicolor FISH method enabled the differentiation of seven (potentially up to eight) phylogenetically distinct microbial populations in an artificial community of mixed pure cultures (five bacteria, one archaeon, and one yeast strain) and in activated sludge from a full-scale wastewater treatment plant. In contrast to previously published multicolor FISH approaches, this method does not rely on combinatorial labeling of the same microorganisms with different fluorophores, which is prone to biases. Furthermore, images acquired by this method do not require elaborate post-processing prior to analysis. We also demonstrate that the newly developed multicolor FISH method is compatible with an improved cell fixation protocol for FISH targeting Gram-negative bacterial populations. This fixation approach uses agarose embedding during formaldehyde fixation to better preserve the three-dimensional structure of spatially complex samples such as biofilms and activated sludge flocs. The new multicolor FISH approach should be highly suitable for studying structural and functional aspects of microbial communities in virtually all types of samples that can be analyzed by conventional FISH methods.

Allele and haplotype frequencies of human platelet and leukocyte antigens in platelet donors.

ABSTRACTObjective To described the allele and haplotype frequencies of human leukocyte antigen genes at the -A, -B loci and human platelet antigen genes for human platelet antigen systems 1 to 9, 11 and 15 in blood.Methods We included 867 healthy unrelated volunteer donors who donated platelets between January 2011 and December 2014. Microarray genotyping was performed using a BeadChip microarray. Medium resolution typing of the human leukocyte antigen at loci A and B was carried out using sequence-specific oligonucleotide probe hybridization. We used multivariate analysis and our human leukocyte antigen population was compared to data from the United States national bone marrow donor program. Human platelet antigen results were compared to a literature review and data from around the world.Results Our human leukocyte antigen haplotype results were more similar to those of hispanics, followed by caucasians. Likewise, our human platelet antigen sample is more similar to those of Argentina, Rio Grande do Sul and Italy.Conclusion This was the first article that discusses human platelet antigen and human leukocyte antigen data together. Rare genotypes or antibody associations can make patient management difficult. A blood bank with genotyped donors allows for optimal transfusion and can contribute to better results. Our information can serve as basis for a database of platelet antigen polymorphisms.

Toehold integrated molecular beacon system for a versatile non-enzymatic application.

A molecular beacon (MB) is an oligonucleotide hybridization probe with a hairpin-shaped structure that leads to specific and instantaneous nucleic acid hybridization, enabling a variety of applications. However, integration of additional module sequences interferes with the performance of MBs and increases the complexity of sequence design. Herein, we develop and characterize a toehold integrated molecular beacon (ToMB) strategy for nucleic acid hybridization, where the reaction rate can be flexibly regulated by a target-induced MB conformational switch. Using this basic mechanism, the ToMB is capable of identifying nucleic acids with high specificity and a wider linearity range compared with the conventional molecular beacon system. We further applied the ToMB to the construction of a hybridization chain reaction system and a basic OR logic gate VJHto explore its programmability and versatility. Our results strongly suggest that the novel ToMB can act as a powerful nano-module to construct universal and multifunctional biosensors or molecular computations. Graphical abstract Molecular beacon is employed as a flexible and switchable spacer to control the toehold-mediated strand displacement reaction.

OR28 Haploidentical donors for Hematopoietic Cell Transplantation (HCT): Changing demographics and the economics of typing this population

Aim HLA-mismatched allogeneic donors allow HCT of patients lacking an HLA identical sibling or matched unrelated donor. Most patients find a haploidentical or a partially-HLA mismatched donor among their first- and second-degree relatives or unrelated donors. We examined process improvements to reduce donor search time and cost in the new era of alternative donors and the less costly Next Generation Sequencing (NGS) technology for high resolution typing. Methods HLA class I typing was performed using reverse sequence specific oligonucleotide probe (rSSO) hybridization (One Lambda, Inc) to identify potential haploidentical donors. Select donors were typed at high resolution using Sanger sequence base typing during 2015 and 2016 (local and commercial kits) or NGS in 2017 (lllumina). Specimen (blood) collection kits were sent to donors using a third party vendor (Path-Tec). Results We performed a retrospective cost analysis of 2017 data. Currently, rSSO class I typing is performed on non-parent or child family members to rule out HLA disparate donors. Of 444 individuals, 188 appeared to be haploidentical and were reflexed to HRT. Only 3.9% of donors reflexed to NGS were disparate. If all 444 individuals had been initially typed by NGS, the cost for the program would have been 63.2% higher. As show in Table 1, the number of 2nd degree relatives has increased. As the donor pool expands beyond the patient’s nuclear family, the incidence of incorrectly reported donor relationships has increased, causing inappropriate testing and delays in reporting results. Furthermore, the donors’ geographical locations have expanded coast to coast, and even globally with increase of shipping costs. Of 413 kits sent in 2017, 20.1% were not returned. Download : Download high-res image (116KB) Download : Download full-size image Conclusions With the steady increase in the use of alternative HCT donors, the staggered class I typing schema remains cost effective even with the advent of NGS. Areas for cost reduction include obtaining correct relationships prior to testing and improving the return rate of collection kits. A.M. Jackson: 3. Speaker’s Bureau; Company/Organization; One Lambda/Thermo Fisher Scientific. M. Bettinotti: 3. Speaker’s Bureau; Company/Organization; One Lambda/Thermo Fisher Scientific.

A Lucifer-Based Environment-Sensitive Fluorescent PNA Probe for Imaging Poly(A) RNAs.

Fluorescence‐based oligonucleotide (ON) hybridization probes greatly aid the detection and profiling of RNA sequences in cells. However, certain limitations such as target accessibility and hybridization efficiency in cellular environments hamper their broad application because RNAs can form complex and stable structures. In this context, we have developed a robust hybridization probe suitable for imaging RNA in cells by combining the properties of 1) a new microenvironment‐sensitive fluorescent nucleobase analogue, obtained by attaching the Lucifer chromophore (1,8‐naphthalimide) at the 5‐position of uracil, and 2) a peptide nucleic acid (PNA) capable of forming stable hybrids with RNA. The fluorescence of the PNA base analogue labeled with the Lucifer chromophore, when incorporated into PNA oligomers and hybridized to complementary and mismatched ONs, is highly responsive to its neighboring base environment. Notably, the PNA base reports the presence of an adenine repeat in an RNA ON with reasonable enhancement in fluorescence. This feature of the emissive analogue enabled the construction of a poly(T) PNA probe for the efficient visualization of polyadenylated [poly(A)] RNAs in cells—poly(A) being an important motif that plays vital roles in the lifecycle of many types of RNA. Our results demonstrate that such responsive fluorescent nucleobase analogues, when judiciously placed in PNA oligomers, could generate useful hybridization probes to detect nucleic acid sequences in cells and also to image them.

Effect of Heterozygosity Loss in HLA Region before Transplantation on HLA Typing in Patients with Leukemia

To investigate the effect of loss of heterozygosity(LOH) in HLA region at initial diagnosis and remission of leukemia patient before transplantation on HLA typing. The HLA typing was performed in DNA extracted from peripheral blood obtained at diagnosis (Sample 1 and Sample 2) and remission (Sample 3) in one pretransplant male patient with mixedphenotype acute leukemia (MPAL). HLA typing for HLA-A, B, C, DQB1, DRB1 was performed by Sequence-based typing (SBT), Sequence-specific oligonucleotide probe hybridization (SSO) and Sequence-specific primers (SSP). To define more precisely a cutoff limit for the detection of a heterozygous DNA present in a fraction of the cells by the SBT technology, DNA mixing experiments were performed. SBT results showed that Sample 1 and Sample 2 were both homozygous HLA results at five loci (lost one haplotype) although the sequencing background of Sample 1 was a little high. Except HLA-C locus was homozygous, Sample 3 was heterozygous HLA results at four loci. Based on DNA mixing experiments, a cutoff limit for the detection of heterozygous DNA was 20% by SBT technology, and a detection threshold for HLA-A, B, C, DQB1, DRB1 heterozygosity in blood samples was <75% blasts. Because LOH may be partial, any homozygous HLA result obtained during a blast crisis, especially ≥75% blasts, would have to be confirmed by a second typing on a buccal swab or on peripheral blood from the patient in complete remission.

Detection of cystic fibrosis transmembrane conductance regulator ΔF508 gene mutation using a paper-based nucleic acid hybridization assay and a smartphone camera.

Diagnostic technology that makes use of paper platforms in conjunction with the ubiquitous availability of digital cameras in cellular telephones and personal assistive devices offers opportunities for development of bioassays that are cost effective and widely distributed. Assays that operate effectively in aqueous solution require further development for implementation in paper substrates, overcoming issues associated with surface interactions on a matrix that offers a large surface-to-volume ratio and constraints on convective mixing. This report presents and compares two related methods for determination of oligonucleotides that serve as indicators of cystic fibrosis, differentiating between the normal wild-type sequence, and a mutant-type sequence that has a 3-base replacement. The transduction strategy operates by selective hybridization of oligonucleotide probes that are conjugated to fluorescent quantum dots, where hybridization of target sequences causes a molecular fluorophore to approach the quantum dot and become emissive through fluorescence resonance energy transfer. Detection can rely on hybridization of a target that is labelled with Cy3 fluorophore, or in the presence of an unlabelled target when a sandwich assay format is implemented with a labelled reporter oligonucleotide. Selectivity to determine the presence of mismatched sequences involves appropriate selection of nucleotide sequences to set melt temperatures, in conjunction with control of stringency conditions using formamide as a chaotrope. It was determined that both direct and sandwich assays on paper substrates are able to distinguish between wild-type and mutant-type samples.

Methods of Analysis and Meta-Analysis for Identifying Differentially Expressed Genes.

Microarray approaches arewidely used high-throughput techniques to assess simultaneously the expression of thousands of genes under certain conditions and study the effects of certain treatments, diseases, and developmental stages. The traditional way to perform such experiments is to design oligonucleotide hybridization probes that correspond to specific genes and then measure the expression of the genes in order to determine which of them are up- or down-regulated compared to a condition that is used as a control. Hitherto, individual experiments cannot capture the bigger picture of how a biological system works and, therefore, data integration from multiple experimental studies and external data repositories is necessary to understand the function of genes and their expression patterns under certain conditions. Therefore, the development of methods for handling, integrating, comparing, interpreting and visualizing microarray data is necessary. The selection of an appropriate method for analysing microarray datasets is not an easy task. In this chapter, we provide an overview of the various methods developed for microarray data analysis, as well as suggestions for choosing the appropriate method for microarray meta-analysis.

P180 Moving onto plan C: Finding detours when DSA creates a roadblock to bone marrow transplantation

Aim This case study illustrates potential avenues to consider when identifying hematopoietic stem cell donor options for HLA sensitized patients. Methods HLA typing was performed using reverse sequence specific oligonucleotide probe hybridization assays (One Lambda, Inc.) and Sanger sequence base typing assays (local and commercial kits). HLA-specific antibody was assessed using class I and class II phenotypes (Lifecodes®, Immucor) and single antigen panels (One Lambda Inc.). Results The presence of donor specific antibody (DSA) can adversely impact the likelihood of bone marrow engraftment depending on the specificity and strength of the DSA. DSA assessment and monitoring is becoming crucial as centers are utilizing HLA mismatched donors. In the last 5 years, 60–70% of the overall allogeneic bone marrow transplants at our center utilized HLA mismatched donors. In a majority of our sensitized patients, the availability of multiple eligible donors allowed for either the avoidance of DSA or the minimization of DSA to levels that did not warrant desensitization. But, in 6% of the cases evaluated in 2016, DSA caused roadblocks requiring alternate pathways. The case of a 41 year old female with Acute Lymphocytic Leukemia illustrates the “Moving onto Plan C” mantra. Plan A: A related haploidentical transplant was diverted due to crossmatch positive DSA against the patient’s four eligible donors. Plan B: An unrelated matched transplant was deterred due to DRB3*01 allele specific DSA against several 10/10 HLA loci matched donors with the DRB1*13:01/DRB3*01 association instead of patient’s DRB1*13:01/DRB3*02 association. Plan C: An unrelated mismatched donor search was initiated to identify 9/10 HLA loci matched donors with a DRB1 mismatch likely to avoid the patient’s broad specificity and eliminate the potential of a DRB3*01 typed donor. Conclusion Even though there is a low incidence of patients requiring an alternate route due to DSA, the impact of the DSA is life-threatening to the individuals in this cohort. With the increase of HLA mismatched transplants, there is a greater need for initial HLA antibody assessments, continual HLA antibody monitoring, alternative back-up plans and creative solutions in order to offer all patients the best chance of survival.

Combining one-step Sanger sequencing with phasing probe hybridization for HLA class I typing yields rapid, G-group resolution predicting 99% of unique full length protein sequences.

Sanger-based DNA sequencing of exons 2+3 of HLA class I alleles from a heterozygote frequently results in two or more alternative genotypes. This study was undertaken to reduce the time and effort required to produce a single high resolution HLA genotype. Samples were typed in parallel by Sanger sequencing and oligonucleotide probe hybridization. This workflow, together with optimization of analysis software, was tested and refined during the typing of over 42,000 volunteers for an unrelated hematopoietic progenitor cell donor registry. Next generation DNA sequencing (NGS) was applied to over 1000 of these samples to identify the alleles present within the G group designations. Single genotypes at G level resolution were obtained for over 95% of the loci without additional assays. The vast majority of alleles identified (>99%) were the primary allele giving the G groups their name. Only 0.7% of the alleles identified encoded protein variants that were not detected by a focus on the antigen recognition domain (ARD)-encoding exons. Our combined method routinely provides biologically relevant typing resolution at the level of the ARD. It can be applied to both single samples or to large volume typing supporting either bone marrow or solid organ transplantation using technologies currently available in many HLA laboratories.