Minor Donors Research Articles

No Conflicting Loyalties in Parents When Their Healthy Child Donates Stem Cells to a Severely Ill Sibling: An Interview Study.

Background: When a potential stem cell donor to a seriously ill child is a healthy sibling below 18 years, Swedish parents have the legal right and obligation to decide on behalf of the donor child. However, there are potentially conflicting loyalties when parents have one severely ill child in need for a cure and one healthy child who will be subjected to medical procedures. This study explored parents' experiences related to their decision on stem cell donation, as well as ethical considerations in the donation process where outcomes are uncertain. Method: Individual interviews were performed with 18 parents of 13 minor donors after successful hematopoietic stem cell transplantations. Interviews were analyzed using inductive Reflexive Thematic Analysis. Results: The parents were living with the threat of losing a child, and in this context, the main theme No conflicting loyalties was found and included four subthemes; Focus on the ill child, Sibling as the preferred donor, Obvious that the healthy child should donate, and Keep on keeping on. Conclusion: When a healthy child is a potential donor to an ill sibling, their parents' main focus is on the cure for the ill child. The lack of obvious conflicting loyalties among parents highlights the need to secure an ethical process for healthy minor donors and the importance of a separate donor advocate for these minor donors.

Validation of Probabilistic Genotyping Software for Single Cell STR Analysis

Probabilistic genotyping (PG) and its associated software has greatly aided in forensic DNA mixture analysis, with it primarily being applied to mixed DNA profiles obtained from bulk cellular extracts. However, these software applications do not always result in probative information about the identity of all donors to said mixtures/extracts. This is primarily due to mixture complexity caused by overlapping alleles and the presence of artifacts and minor donors. One way of reducing mixture complexity is to perform direct single cell subsampling of the bulk mixture prior to genotyping and interpretation. The analysis of low template DNA samples, including from single or few cells, has also benefited from the application of PG methods. With the application of PG, multiple cell subsamples originating from the same donor can be combined into a single analysis using the software replicate analysis function often resulting in full DNA profile donor information. In the present work, we demonstrate how two PG software systems, STRmixTM and EuroForMix, were successfully validated for single or few cell applications.

Cell Subsampling Recovers Probative DNA Profile Information from Unresolvable/Undetectable Minor Donors in Mixtures.

When a minor DNA component to a binary mixture is present at a weight ratio of approximately 1:50 or less, the presence of this minor donor is undetectable (or barely detectable) by standard mixture deconvolution approaches. In an attempt to retrieve probative minor donor DNA profile information, multiple quintuple cell subsamples were collected from a 1:50 DNA mixture using direct single cell subsampling (DSCS) paired with probabilistic genotyping (PG), the latter validated for use with single or few cells. DSCS employs a simplified micromanipulation technique paired with an enhanced DNA profiling approach, involving direct cell lysis and a sensitive PCR process, to genotype individual cells. Multiple five-cell subsamples were used to interrogate sufficient cells from the mixture such that some of the created 5-cell “mini-mixture” subsamples contained a cell from the minor donor. The latter mini-mixture subsamples, which now comprised weight ratios of 1:4 as opposed to the bulk mixture 1:50, were analyzed with the PG systems STRmixTM and EuroForMix resulting in a significant probative gain of information, (LR ≅ 1011, compared to standard bulk mixture PG methods, LR ≅ 101–102).

Identification of missing persons through kinship analysis by microhaplotype sequencing of single-source DNA and two-person DNA mixtures

In forensic applications, there is an increasing demand for the analysis of DNA profiles arising from missing person identification (MPI) cases. A specific DNA profile may originate from a single source or more than one contributor (i.e., a DNA mixture). When direct references are not available, indirect relative references can be used to identify missing persons by kinship analysis. As a novel kind of multiallelic marker, microhaplotypes have proven promising for relatedness determination and mixture deconvolution. Herein, we developed a large panel of 185 microhaplotype markers and demonstrated its application in different scenarios of relationship inference through a simulation study and real pedigree analysis, combined with probabilistic genotyping models for data interpretation. Based on single-source profiles, it was shown that the present microhaplotype panel was sufficient for pairwise close relative testing (parent/child, full-sibling and 2nd-degree relative). For more distant relatives (3rd-degree relatives), there was a clear improvement when data from one well-chosen extra relative were available. We further sought to evaluate the theoretical systematic effectiveness and actual performance of microhaplotype markers in identifying the contribution of a missing pedigree member to a two-person mixture (as a minor donor). It was observed that 100% correct assignments were made in the balanced mixtures (with no dropout) when referenced to close relatives. When the mixture profiles suffered from dropout, incorrect assignments of minor donors were markedly associated with relatedness and the dropout level. Meanwhile, the studied scenarios generally exhibited zero or very low false-positive rates, indicating a low probability of incorrectly assigning an unrelated contributor as a close relative of the reference. Our results indicate that microhaplotype data can be reliably interpreted for identifying missing persons through kinship analysis based on DNA profiles of single-source samples or two-person mixtures. Furthermore, this study could be extended to more complex scenarios, such as determining the relatedness of contributors in (or among) mixed DNA profiles, if combined with different statistical frameworks.

Corpuscular Fragility and Metabolic Aspects of Freshly Drawn Beta-Thalassemia Minor RBCs Impact Their Physiology and Performance Post Transfusion: A Triangular Correlation Analysis In Vitro and In Vivo.

The clarification of donor variation effects upon red blood cell (RBC) storage lesion and transfusion efficacy may open new ways for donor–recipient matching optimization. We hereby propose a “triangular” strategy for studying the links comprising the transfusion chain—donor, blood product, recipient—as exemplified in two cohorts of control and beta-thalassemia minor (βThal+) donors (n = 18 each). It was unraveled that RBC osmotic fragility and caspase-like proteasomal activity can link both donor cohorts to post-storage states. In the case of heterozygotes, the geometry, size and intrinsic low RBC fragility might be lying behind their higher post-storage resistance to lysis and recovery in mice. Moreover, energy-related molecules (e.g., phosphocreatine) and purine metabolism factors (IMP, hypoxanthine) were specifically linked to lower post-storage hemolysis and phosphatidylserine exposure. The latter was also ameliorated by antioxidants, such as urate. Finally, higher proteasomal conservation across the transfusion chain was observed in heterozygotes compared to control donors. The proposed “triangularity model” can be (a) expanded to additional donor/recipient backgrounds, (b) enriched by big data, especially in the post-transfusion state and (c) fuel targeted experiments in order to discover new quality biomarkers and design more personalized transfusion medicine schemes.

Use of minor donors for living donor liver transplantation and associated ethical issues

Use of minor donors for living donor liver transplantation and associated ethical issues

Use of minor donors for living donor liver transplantation and associated ethical issues.

BackgroundLiving liver donation by minors is regarded as justifiable only if minors possess the capacity to consent to donation and the procedure is in their best interests. This study analyzed the incidence of and reasons for living donor liver transplantation (LDLT) by minor donors in Korea, and discussed ethical issues regarding liver donation by minors.MethodsThe databases of the Korean Network for Organ Sharing (KONOS) and Asan Medical Center (AMC) from 2010 to 2019 were retrospectively reviewed to determine the incidence of LDLT by minor donors.ResultsFrom 2010 to 2019, 590 (4.1%) of 14,243 liver donors in the KONOS database and 276 (7.5%) of 3,401 liver donors in the AMC database were minors. The proportions of minor donors in the KONOS and AMC databases were highest in 2012, at 4.1% and 12.6%, respectively, and lowest in 2019, at 1.1% and 3.0%, respectively. Because most LDLT recipients had relatively low model for end-stage liver disease scores and hepatocellular carcinoma, they were unlikely candidates for deceased-donor liver transplantation and were highly likely to drop out of LDLT if they waited for 1–2 years. The donor-recipient relationship of minor donors in the AMC database was first-degree in 256 (92.8%) and second- or third-degree in 20 (7.2%).ConclusionsLiver donation by minors is limitedly acceptable only when the minor proves informed, well‐considered, and autonomous consent to the procedure and the procedure is in the minor's best interests. We suggest that minors be allowed to donate only to first-degree family members.

Protecting Minor Donors of Hematopoietic Stem Cells: A Survey in EBMT Transplant Centers

IntroductionHematopoietic stem cell (HSC) donation by minors is a well-established practice. According to the literature, up to one-third of pediatric HSC transplants involves donation by minors, most often siblings. Even though HSC donation involves a medical intervention without direct physical benefit to the donor, a broad consensus exists about its acceptability, as it is potentially life-saving for the recipient and carries no more than small risks but conceivably significant psychosocial benefits for the donor. However, because it concerns an intervention on a patient who is considered vulnerable due to age and from which that person will not physically benefit, protective measures have been proposed in ethical guidelines and position papers published by national ethics committees and professional medical organizations. However, no studies have been conducted to assess whether, and, if so, how, these safeguards are implemented in the practices of transplant centers.MethodsA cross-sectional descriptive survey on practices regarding HSC donation by minors was prepared and distributed via email through the office of the European Society for Blood and Marrow Transplantation (EBMT) to 262 EBMT member centers. The survey included closed-ended as well as open-ended questions that assessed: (i) the conditions that have to be met before HSC donation by a minor is accepted; (ii) the approval process; (iii) how consent is obtained from the minor; and (iv) potential psychological support and follow-up. Answers were collected between March 2016 and June 2017. Descriptive statistics for closed ended questions and qualitative content analysis for open questions were used for data analysis. The study was approved by the EBMT Pediatric Diseases Working Party.ResultsForty out of 262 transplant centers from twenty countries completed the questionnaire, constituting a response rate of 15.3%. Altogether, these centers have reported 5,337 HSC donations since 2010, of which 1,651 donations were made by minors (30.9%). The proportion of HSC donation by minors ranged from 0.5% to 100% among the included transplant centers.Twenty-seven centers (67.5%) reported having in place specific guidelines or protocols with regard to HSC donation by minors. In 80% of centers minors can be evaluated for HLA compatibility simultaneously with adult family members, while in the remaining 20% of centers minors are screened only after all adult family members are found to be medically unsuitable to donate. Nearly half of transplant centers (47.5%) only allow minors to serve as donors for siblings, whereas the others also allowed donation to other close relatives, such as parents, grandparents and cousins.Whereas all of the participating centers described efforts to include minor donors in the decision-making process in a way that is in accordance with their age and maturity, significant differences exist with regard to final approval of the donation. After obtaining consent from the donor and his/her parents, fifteen centers (37.5%) do not require any additional approval, whilst the other centers seek permission from a local or national ethics committee (40%), an independent physician (5%), social worker or donor advocate (5%), or the court (7.5%).An independent donor advocate, whose purpose is to promote the wellbeing of the donor and is not involved in direct care of the recipient, was appointed in about one-third (35%) of the centers. Psychological support to the donor and his family after the donation is routinely provided by 70% of the centers.ConclusionsThis is the first study to examine HSC transplant center practices relating to donation by minors. Although all of the participating centers are members of the EBMT network, our research findings reveal significant differences with regard to: (i) the use of specific guidelines and protocols for HSC donation by minors; (ii) the screening of potential minor donors when a compatible adult minor may be available; (iii) the categories of recipients that may benefit from HSC donation by minors; (iv) the need for, and type of, final approval by an independent body; (v) the availability of a dedicated donor advocate; and (vi) the provision of psychological support to and follow-up of minor HSC donors. On the basis of these findings, we suggest that further efforts to harmonize practices involving HSC donation by minors should be undertaken. DisclosuresNo relevant conflicts of interest to declare.

Memory T Cells from Minor Histocompatibility Antigen-Vaccinated Donors Improve Engraftment with Low Intensity Conditioning

While allogeneic hematopoietic stem cell transplantation (alloSCT) is largely employed to treat malignant disorders, it can also cure inherited and acquired nonmalignant disorders. For example, inherited diseases such as sickle cell anemia, thalassemias, and varied immunodeficiencies can be cured with alloSCT. Autoimmune diseases can also be cured via the abilities of both the conditioning regimen and alloimmune T cells to eradicate autoimmune T and B cells. And importantly, tolerance to solid organ grafts can be created via transplantation of hematopoietic cells from the organ donor. However, the utility of alloSCT in these settings is limited by the morbidity and mortality of the procedure, including graft-vs-host disease (GVHD). Some progress has been made in lowering the toxicities in these settings. However, these approaches frequently result in mixed chimerism, which may not be sufficient to treat autoimmune diseases, and may lead to unreliable engraftment overall. Previously we reported that in mouse models of alloSCT memory CD8+ T cells from mice vaccinated against the single minor histocompatibility antigen (miHA) H60, which is mostly expressed on hematopoietic cells, dramatically improves graft-vs-leukemia (GVL) against H60-expressing leukemias, without causing GVHD. In the present work we have tested the utility of memory CD8 cells reactive to H60 (TMH60) in promoting engraftment. Following 6 Gy or 8Gy of total body irradiation (TBI), B6.H60 (H-2b, Ly9.1-, H60+) mice were transplanted with C3H.SW (H-2b, Ly9.1+, H60-) bone marrow (BM), with memory T cells from H60 vaccinated mice (TMH60) containing 3500 tetramer+ cells (TetH60) or with TM from unvaccinated mice as negative control. At TBI doses of 8 Gy and 6 Gy, the addition of TM from H60 vaccinated mice (TMH60), as compared to TM from unvaccinated mice, modestly improved engraftment. We next lowered the dose of TBI further. At 5 Gy, recipients of TMH60 had multilineage engraftment of donor-derived hematopoietic cells in bone marrow (BM), spleen and lymph nodes (LN) nine weeks after transplantation. In contrast, mice with TM failed to engraft. Importantly, mice did not develop clinical GVHD. At a TBI dose of 4 Gy, recipients with TMH60 had poor donor engraftment, while TMH60 recipients treated with an anti-CD40 agonist antibody (FGK45) on day 0 achieved full multilineage engraftment in spleen, LN and BM. In addition we achieved mixed chimerism in some TMH60 and anti-CD40 treated mice conditioned with 3 Gy. We also tested the utility of TM targeting the Kb-restricted SIINFEKL peptide derived from OVA (TM-OVA) in a mouse model wherein OVA expression is restricted to hematopoietic cells. We used B6.OVA mice, which express OVA ubiquitously, as BM donors and created B6.OVA–>B6 BM chimeras (H-2b, Ly9.1-, OVA+). We retransplanted these chimeras with C3H.SW (H-2b, Ly9.1+, OVA-) BM with C3H.SW TM-OVA, containing 3500 tetramer+ cells, or TM from unvaccinated mice following 5 Gy or 4 Gy of TBI. TM-OVA, but not TM promoted full multilineage engraftment in mice conditioned with 5 Gy in blood of mice analyzed two weeks after transplantation. At dose of 4 Gy, TM-OVA induced full donor engraftment, but only with the addition of anti-CD40. This experiment is ongoing. In sum, our data demonstrate that TM reactive against a miHA expressed on hematopoietic cells can dramatically promote donor multilineage engraftment without GVHD, and that the effectiveness of these TM can be augmented by stimulating CD40. Future experiments will test miHA-reactive TM with chemotherapy-based conditioning, in haploidentical models and the utility of blocking PD-1, which we have found greatly augments GVL mediated by miHA-reactive TM. This approach holds the promise of achieving consistent donor engraftment without GVHD, a prerequisite for the widespread use of alloSCT in treatment of autoimmune diseases and for tolerance induction in tandem with organ transplantation. DisclosuresNo relevant conflicts of interest to declare.

A NHS Foundation Trust v MC [2020] EWCOP 33: Revisiting Best Interests and 'Altruistic' Incapacitous Stem Cell Donation.

In A NHS Foundation Trust v MC, the Court of Protection revisits the question of whether adults should be allowed to act as bone marrow or peripheral blood stem cell donors if they lack decision-making capacity. This case note explores the positive and problematic implications of the case based on points that were raised in the judicial reasoning that specifically relate to i) practical implications concerning the key players in this environment, ii) the risk analysis within the best interest determination, iii) altruism and iv) the wider context as it relates to minor donors who lack capacity.

A sequence-based 74plex microhaplotype assay for analysis of forensic DNA mixtures

Microhaplotypes are emerging biomarkers for forensic applications. In this study, a sequence-based multiplex assay of 74 microhaplotypes (230 SNPs) was developed on the Ion Torrent S5™ (Thermo Fisher Scientific) system and the potential for its application to mixture deconvolution was explored. The 74 loci are distributed across the autosomal human genome and have Ae (i.e., effective number of alleles) values ranging from 1.307 to 6.010 (median = 2.706) and In (i.e., informativeness) values ranging from 0.096 to 0.660 (median = 0.251); the amplicon sizes range between 157 and 325 bp. The typing performance of the panel was evaluated on a series of in-silico two to five-person DNA mixtures and results were compared to fragment and sequence-based STRs. The 74plex-locus assay was found sensitive down to 0.05 ng of input DNA and effective for the analysis of mixtures at different contributor ratios and input DNA amounts. As expected, none or very partial minor CE-STR profile(s) were reported for highly imbalanced two-person and high-order DNA mixtures while sequencing of STRs enabled the detection of more individual minor alleles. For microhaplotypes, a full minor profile was detected down to a 20:1 ratio at 10 ng and minimal allele dropout at 1 ng of input DNA. A higher rate of allele dropout from the minor donor(s) was reported at 1 ng than 10 ng for three-person mixtures while for four- and five-person mixtures, the same number of dropouts was observed for almost all minor donors. Overall this microhaplotype panel is a powerful tool that can complement and enhance size- and sequence-based STR analysis of forensic DNA mixtures.

Solid Organ Transplantation between Family Members and Protection for Living Minor Donors

Solid Organ Transplantation between Family Members and Protection for Living Minor Donors

Solid Organ Transplantation between Family Members and Protection for Living Minor Donors

Solid Organ Transplantation between Family Members and Protection for Living Minor Donors

Impact of ABO incompatibility on outcomes after haploidentical hematopoietic stem cell transplantation for severe aplastic anemia.

The impact of ABO incompatibility on transplantation outcomes in severe aplastic anemia (SAA) patients receiving haploidentical hematopoietic stem cell transplantation (HSCT) remains controversial without published data. A total of 199 SAA patients receiving haploidentical HSCT from ABO-matched (n = 114), minor ABO-incompatible (n = 47), or major ABO-incompatible donors (n = 38) were included in this study. The median time and cumulative incidences of both myeloid and platelet engraftment in the ABO-compatible and ABO-incompatible groups were similar, and pure red cell aplasia was absent. Minor ABO incompatibility increased the rate of grade III-IV acute graft-versus-host disease (aGVHD) (ABO compatible: 6.14 ± 0.05%, minor incompatible: 19.15 ± 0.34%, and major incompatible: 10.53 ± 0.25%; P = 0.051), but did not influence the rates of grade II-IV aGVHD or chronic GVHD (cGVHD). Minor ABO-incompatibility was identified as an independent risk factor for grade III-IV aGVHD by multivariate analysis (hazard ration (HR) = 4.00 (1.48-10.80), P = 0.006). Chronic GVHD, mortality, and treatment failure were not increased in the minor ABO-incompatible group. For SAA patients receiving haploidentical HSCT, ABO compatible donors are better than ABO minor incompatible donors if several haploidentical donors are available.

Analysis of nondegraded and degraded DNA mixtures of close relatives using massively parallel sequencing.

Identification of the minor contributor in DNA mixture of close relatives remains a dilemma in forensic genetics. Massively parallel sequencing (MPS) can analyze multiple short tandem repeats (STRs) and single nucleotide polymorphism (SNPs) concurrently and detect non-overlapping alleles of the minor contributors in DNA mixtures. A commercial kit for MPS of 59 identity informative STRs (iiSTRs) and 94 autosomal identity-informative SNPs (iiSNPs) was used to analyzed 34 nondegraded and 33 highly degraded two-person artificial DNA mixtures of close relatives with various minor to major ratios (1:9, 1:19, 1:29, 1:39, 1:79, 1:99). EuroForMix software was used to determine the minor contributors in the mixtures based on the likelihood ratios calculated from the MPS data, and relMix software was used to perform kinship analysis of the contributors. The STRs and SNPs of the 34 nondegraded and 33 degraded DNA mixtures were genotyped using MPS. Using EuroForMix based on the genotypes of autosomal iiSTRs and autosomal iiSNPs, 82.4% (28/34) and 54.5% (18/33) of minor donors could be accurately assigned for the nondegraded and degraded DNA mixtures, respectively. The relMix software correctly inferred the relationship between contributors in 97.1% (33/34) of nondegraded mixtures and in 97.0% (32/33) of degraded mixtures. In conclusion, combined EuroForMix and MPS data of STRs and SNPs can assist in the assignment of minor donors in nondegraded DNA mixtures of close relatives, and relMix can be used to infer relationship among contributors.

A massively parallel sequencing assay of microhaplotypes for mixture deconvolution

Microhaplotypes (microhaps or MHs) are markers characterized by a set of single nucleotide polymorphisms (SNPs) within an expanse of 300 bp exhibiting multiple allelic combinations. Microhap alleles within a locus have all the same size, do not generate stutter artifacts, and have low mutation rates. These features complement short tandem repeats (STRs) for human identification and mixture deconvolution. The analysis of microhaps is enhanced by massively parallel sequencing (MPS) that enables the determination of the parental SNP haplotypes by clonal sequencing of each strand. This study aims to evaluate the mixture performance of a 74 microhap locus-assay on the Ion S5™ and compare the results to size-based and sequence-based STR analysis. The detection limit of the panel was tested and the panel’s performance evaluated in parallel with the GlobalFiler™ and Precision ID GlobalFiler™ NGS STR Panel v2 kits. An set of two-to-five person mixtures was simulated at different contribution ratios and amounts of DNA (1 ng and 10 ng) to mimic casework-like samples. The 74-locus panel was sensitive down to 50 pg of input DNA. Size-based STR mixture analysis was challenging, but was improved by MPS when the allele sequence of the minor contributor was different from the overlapping stutter of the major. For two-person mixtures, full microhap profile of the minor donor could be reported at 1:10 ratio, with minimal allele/locus dropout at 20:1, and more significant dropout at higher mixture ratios. For three-to-five person mixtures, full microhap profiles were obtained for all minor donors with the exception of few loci that underperformed at imbalanced mixture ratios. These findings suggest that microhaps can improve mixture deconvolution and supplement STR typing analysis.

Mixture deconvolution by massively parallel sequencing of microhaplotypes

Short tandem repeat polymorphisms (STRs) are the standard markers for forensic human identification. STRs are highly polymorphic loci analyzed using a direct PCR-to-CE (capillary electrophoresis) approach. However, STRs have limitations particularly when dealing with complex mixtures. These include slippage of the polymerase during amplification causing stutter fragments that can be indistinguishable from minor contributor alleles, preferential amplification of shorter alleles, and limited number of loci that can be effectively co-amplified with CE. Massively parallel sequencing (MPS), by enabling a higher level of multiplexing and actual sequencing of the DNA, provides forensic practitioners an increased power of discrimination offered by the sequence of STR alleles and access to new sequence-based markers. Microhaplotypes (i.e., microhaps or MHs) are emerging multi-allelic loci of two or more SNPs within < 300bp that are highly polymorphic, have alleles all of the same length, and do not generate stutter fragments. The growing number of loci described in the literature along with initial mixture investigations supports the potential for microhaps to aid in mixture interpretation and the purpose of this study was to demonstrate that practically. A panel of 36 microhaplotypes, selected from a set of over 130 loci, was tested with the Ion S5™ MPS platform (Thermo Fisher Scientific) on single-source samples, synthetic two-to-six person mixtures at different concentrations/contributor ratios, and on crime scene-like samples. The panel was tested both in multiplex with STRs and SNPs and individually. The analysis of single-source samples showed that the allele coverage ratio across all loci was 0.88 ± 0.08 which is in line with the peak height ratio of STR alleles in CE. In mixture studies, results showed that the input DNA can be much higher than with conventional CE, without the risk of oversaturating the detection system, enabling an increased sensitivity for the minor contributor in imbalanced mixtures with abundant amounts of DNA. Furthermore, the absence of stutter fragments simplifies the interpretation. On casework-like samples, MPS of MHs enabled the detection of a higher number of alleles from minor donors than MPS and CE of STRs. These results demonstrated that MPS of microhaplotypes can complement STRs and enhance human identification practices when dealing with complex imbalanced mixtures.

Ethical considerations of using a single minor donor for three bone marrow harvests for three HLA-matched siblings with primary immunodeficiency.

Allogeneic hematopoietic stem cell transplantation is curative for primary immunodeficiencies. Bone marrow from an unaffected human leukocyte antigen (HLA)-identical sibling donor is the ideal graft source. For minor donors, meaningful consent or assent may not be feasible, and permission from parents or legal guardians is considered acceptable. Adverse events, albeit extremely small, can be associated with bone marrow harvest in pediatric donors. Donor safety concerns potentially increase with multiple bone marrow harvests. Very little is known about multiple bone marrow harvests from pediatric donors. We describe the ethical considerations and clinical decision-making in an unusual clinical situation where three patients with the same primary immunodeficiency were HLA identical to one another and their younger sibling, who underwent bone marrow harvests three times between 1.3 and 4 years of age, resulting in successful transplantation for all three patients. We hope that this experience will provide guidance to providers and families in a similar situation.

Massively parallel sequencing analysis of nondegraded and degraded DNA mixtures using the ForenSeq™ system in combination with EuroForMix software.

Massively parallel sequencing (MPS) technologies enable the simultaneous analysis of short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs). MPS also enables the detection of alleles of the minor contributors in imbalanced DNA mixtures. In this study, 59 STRs (amelogenin, 27 autosomal STRs, 7 X-STRs, and 24 Y-STRs) and 94 identity-informative SNPs of 119 unrelated Taiwanese (50 men, 69 women) were sequenced using a commercial MPS kit. Forty-eight nondegraded and 44 highly degraded two-person artificial DNA mixtures with various minor to major ratios (1:9, 1:19, 1:29, 1:39, 1:79, and 1:99) were analyzed to examine the performance of this system for detecting the alleles of the minor contributors in DNA mixtures. Likelihood ratios based on continuous model were calculated using the EuroForMix for DNA mixture interpretation. The STR and SNP genotypes of these 119 Taiwanese were obtained. Several sequence variants of STRs were observed. Using EuroForMix software based on the sequence data of autosomal STRs and autosomal SNPs, 97.9% (47/48) and 97.7% (42/43) of minor donors were accurately inferred among the successfully analyzed nondegraded and degraded DNA mixtures, respectively. In conclusion, combined with EuroForMix software, this commercial kit is effective for assignment of the minor contributors in nondegraded and degraded DNA mixtures.

The information gain from peak height data in DNA mixtures

We present the results of a comparison of likelihood ratios obtained from DNA mixture data, obtained either by a peak height model (EuroForMix) and by a discrete model, using probabilities of dropout and integrating over them (MixKin). We use the mixture data that have been published in [1] and were made publicly available. We show that, for mixtures for which replicate analyses were available in this set, there is almost no difference in weight of evidence, suggesting that the additional information in the peak heights is limited. On the other hand, for mixtures for which only a single replicate is available, the weight of evidence for true donors can be substantially higher with the peak height model, especially so for major donors and also for minor donors provided they are the only minor contributor to the mixture. False positive rates were very low with both methods, but for related non-contributors the risk of a false positive is much higher with the discrete method unless kinship is taken into account in the likelihood ratio calculation.