Droplet Digital PCR-Based Chimerism Analysis for Primary Immunodeficiency Diseases

Objective

In the current study, we aimed to accurately evaluate donor/recipient or male/female chimerism in samples from patients who underwent hematopoietic stem cell transplantation (HSCT).

Methods

We designed the droplet digital polymerase chain reaction (ddPCR) for SRY and RPP30 to detect the male/female chimerism. We also developed mutation-specific ddPCR for four primary immunodeficiency diseases.

Results

The accuracy of the male/female chimerism analysis using ddPCR was confirmed by comparing the results with those of conventional methods (fluorescence in situ hybridization and short tandem repeat-PCR) and evaluating dilution assays. In particular, we found that this method was useful for analyzing small samples. Thus, this method could be used with patient samples, especially to sorted leukocyte subpopulations, during the early post-transplant period. Four mutation-specific ddPCR accurately detected post-transplant chimerism.

Conclusion

ddPCR-based male/female chimerism analysis and mutation-specific ddPCR were useful for all HSCT, and these simple methods contribute to following the post-transplant chimerism, especially in disease-specific small leukocyte fractions.

     

Development of multiplex short tandem repeat (STR)-PCR for chimerism analysis in patients with hematological malignancies and comparison of chimerism in different sample sources

Polymerase chain reaction analysis of short-tandem repeat (STR) markers (STR-PCR) has been used for chimerism testing to assess engraftment following hematopoietic stem cell transplantation (HSCT). We investigated the informativity of 7 STR loci (FGA, D5S818, SE33, TH01, VWF, PentaE, and D18S51) in 82 pre-HSCT DNA samples from 41 donor/recipient pairs and developed 2 multiplex STR-PCRs using VWF, SE33, and D18S51, D5S818 and FGA, respectively. The multiplex STR-PCRs could distinguish the recipients and donors in 92.7% of the cases. Dilution experiments using mixed DNA showed that the sensitivity of the multiplex STR-PCRs for detecting the minor population was 1-5%. To compare chimerism in different samples such as peripheral blood, mononuclear cells (MNC), and CD3-positive cells (CD3+), we investigated the relationship between the chimerisms at approximately day 30 post-HSCT and the interval from the day of HSCT to achievement of complete chimerism (CC) in 70 patients undergoing HSCT. CC was found in all samples of 54 patients at day 30 post-HSCT, and these samples showed CC thereafter. Eleven patients with mixed chimerism (MC) in all samples or in MNC and CD3+ showed CC at day 60-270 post-HSCT or persistent MC. The remaining 5 patients with MC in only CD3+ showed CC at day 30-60 post-HSCT. Taken together, MNC which can be separated easily may be a useful source for detecting patients who require longer time to achieve CC and those with high risk of graft failure.     

Impact of donor KIRs and recipient KIR/HLA class I combinations on GVHD in patients with acute leukemia after HLA-matched sibling HSCT

In addition to T cells, NK cells can also participate in the outcome of hematopoietic stem cell transplantation (HSCT) mainly through the interaction between donor killer cell immunoglobulin-like receptors (KIRs) and recipient human leukocyte antigen (HLA) class I molecules. There is a risk of GVHD other than leukemia relapse after allogeneic HSCT that activation of donor NK cells in the absence of appropriate inhibitory ligands will be one of the reasons. To investigate the impact of donor KIRs and recipient KIR/HLA class I combinations on GVHD and leukemia relapse in patients with acute leukemia after HSCT, 100 patients with acute leukemia who received HSCT from their HLA-matched siblings were included in this study. Genotypes of 16 KIR genes and two 2DS4 variants (full length and deleted alleles), along with HLA-A/B genotypes, were determined by PCR-SSP. HLA-C genotyping was done with the SSO-Luminex method. Chimerism analysis was done using 16 short tandem repeats (STRs) to detect early leukemia relapse. Acute (a)GVHD occurred in 38 patients, and 16 of them died during the study. None of the recipients showed any sign of leukemia relapse after HSCT. Full donor chimerism was observed in all tested patients during the first year after HSCT. Our results also indicated an increased risk of aGVHD in AA recipients with the C2/Cx, Bw4⁺ (or A-Bw4⁺) or HLA-A3⁻/A11⁻ genotypes who received HSCT from Bx donors. Our results showed that donor selection based on donor-recipient KIR genotypes and recipient HLA class I status can improve the outcome of HSCT.     

Donor bone marrow infusion in deceased and living donor renal transplantation

The infusion and persistence in a transplant recipient of donor-derived bone marrow cells (DBMC) of multi-lineage can lead to a state of permanent chimerism. In solid vascular organ transplantation, the donor bone marrow lineage cells can even be derived from the transplant organ, and these cells can be detected in very small numbers in the recipient. This has been called microchimerism. Much controversy has developed with respect to the function of chimeric cells in organ transplantation. One idea is that the occurrence of these donor cells found in microchimerism in the recipient are coincidental and have no long-term beneficial effect on engraftment. A second and opposing view, is that these donor cells have immunoregulatory function that affect both the acute and chronic phases of the recipient anti-donor responses. It follows that detecting quantitative changes in chimerism might serve as an indication of the donor-specific alloimmune or regulatory response that could occur in concert with or independent of other adaptive immune responses. The latter, including autoimmune native disease, need to be controlled in the transplant organ. The safety and immune tolerance potential of DBMC infusion with deceased and living donor renal transplants was evaluated in a non-randomized trial at this center and compared with non-infused controls given identical immunosuppression. Overall DBMC infusions were well tolerated by the recipients. There were no complications from the infusion(s), no episodes of graft-vs-host disease (GVHD) and no increase infections or other complications. In the deceased DBMC-kidney trial, actuarial graft survival at 5 years was superior especially when graft survival was censored for recipient death. Acute rejections were significant reduced in patients given two DBMC infusions, and chronic rejection was dramatically reduced in all DBMC treated patients. The most interesting finding was that the degree of microchimerism slowly increased over the years the DBMC group that had exhibited no rejection episodes. In the DBMC-living related trial, the incidence of acute rejection did not differ between groups. However, DBMC chimerism in recipient iliac crest marrow had increased more rapidly than might be predicted from results previously seen in the cadaver group, despite four times fewer DBMC infused, with the generation of T- regulartory cells in-vitro assays.     

Chimerism Patterns of Long-Term Stable Mixed Chimeras Posthematopoietic Stem Cell Transplantation in Patients with Nonmalignant Diseases: Follow-Up of Long-Term Stable Mixed Chimerism Patients

Long-term stable mixed chimerism (MC) is a rare phenomenon after hematopoietic stem cell transplantation (HSCT) characterized by 5% to 95% residual recipient hematopoietic cells. The underlying mechanisms of MC are largely unknown. In this study we compared full donor chimerism with long-lasting stable MC for a median of 9.5 years (range, 5 to 16.5) post-HSCT in patients with nonmalignant diseases. Several factors significantly associated with the likelihood of stable MC development were identified by univariate analysis, eg, younger donor age, sibling donor, and conditioning regimen. Despite a limited patient cohort, our multivariate analysis could confirm that a sibling donor was associated with stable MC development. Furthermore, development of acute-graft-versus-host disease and blood stream infection was significantly more prevalent in the full donor chimerism patient group. Additionally, significant fluctuations in the recipient-to-donor chimerism ratio decreased over time after HSCT in MC patients.     

Haploidentical In Utero Hematopoietic Cell Transplantation Improves Phenotype and Can Induce Tolerance for Postnatal Same-Donor Transplants in the Canine Leukocyte Adhesion Deficiency Model

In the murine model, in utero hematopoietic cell transplantation (IUHCT) has been shown to achieve low levels of allogeneic chimerism and associated donor-specific tolerance permitting minimal conditioning postnatal hematopoietic stem cell transplantation (HSCT). In this pilot study, we investigated IUHCT in the canine leukocyte adhesion deficiency (CLAD) model. Haploidentical IUHCT resulted in stable low-level donor cell chimerism in all dogs that could be analyzed by sensitive detection methodology (4 of 10) through 18 months of follow-up. In the 2 CLAD recipients, low-level chimerism resulted in amelioration and complete reversal of the CLAD phenotype, respectively. Six recipients of IUHCT (5 carriers and 1 CLAD) subsequently received postnatal HSCT from the same haploidentical prenatal donor after minimal conditioning with busulfan 10 mg/kg. Chimerism in 2 of 5 CLAD carriers that underwent HSCT increased from < 1% pre-HSCT to sustained levels of 35% to 45%. Control animals undergoing postnatal haploidentical HSCT without IUHCT had no detectable donor chimerism. These results demonstrate that haploidentical IUHCT in the CLAD model can result in low-level donor chimerism that can prevent the lethal phenotype in CLAD dogs, and can result in donor-specific tolerance that can facilitate postnatal minimal conditioning HSCT.     

Peripheral blood allogeneic microchimerism in lung and cardiac allograft recipients.

We have been investigating two parameters, donor antigen-specific hyporeactivity and peripheral blood allogeneic microchimerism, to determine whether these parameters will predict a chronic rejection-free state and which recipients may be candidates for steroid withdrawal. We have identified donor antigen-specific hyporeactivity for 33% (16/48) of lung and 23% (11/47) of heart recipients. For both organ groups, the hyporeactive subgroup experienced a lower incidence of chronic rejection. The probability of donor antigen-specific hyporeactivity predicting a chronic rejection-free state is 100% for lung and 91% for heart recipients. We have identified peripheral blood allogeneic microchimerism for 77% (20/26) of lung and 36% (9/25) of heart recipients tested at 12-18 months posttransplant. Donor antigen-specific hyporeactivity correlates with a critical level of donor cells in lung recipients; the probability of high peripheral blood allogeneic microchimerism levels predicting a chronic rejection-free state in lung recipients is 100%. The results in heart recipients are not as clear with a short-, but not long-term, trend of higher chimerism levels correlating with the development of donor antigen-specific hyporeactivity. These results illustrate the usefulness of immmune parameters to predict long-term graft outcome in an organ-specific manner.     

Clinical correlations of peripheral blood microchimerism after liver transplantation

The aim of this study was to evaluate microchimerism after human liver transplantation (LT). This study included 13 female recipients who received hepatic allograft from male donors at Asan Medical Center. A nested PCR specific for Y-chromosome gene (DYZ3) was used to analyze the small number of male cells in the peripheral blood mononuclear cells of the female recipients. Microchimerism was observed in 6 of 13 recipients and 16 out of 35 samples. Only 3 patients showed microchimerism 3 months after LT. There was no statistical difference between the presence of microchimerism and clinical findings such as type of donor, type of immunosuppression, episode of rejection and age of recipient. This study did not show any clinical relevance of microchimerism and further larger study are needed to confirm the results.     

Antiviral T Cells for Adenovirus in the Pretransplant Period: A Bridge Therapy for Severe Combined Immunodeficiency

Viral infections can be life threatening in patients with severe combined immunodeficiency (SCID) and other forms of profound primary immunodeficiency disorders both before and after hematopoietic stem cell transplantation (HSCT). Adoptive immunotherapy with virus-specific T cells (VSTs) has been utilized in many patients in the setting of HSCT, but has very rarely been attempted for treatment of viral infections before HSCT. Here we describe the use of VSTs in an infant with RAG1 SCID who had developed disseminated adenovirus which failed to improve on cidofovir. Adenovirus cleared following 2 doses of VSTs and marrow infusion from a matched unrelated donor, without incidence of graft versus host disease. T cell receptor-b sequencing demonstrated expansion of adenovirus-specific T cell fraction of the VSTs, suggesting that infusion facilitated viral clearance. This report suggests that VSTs are likely safe in the pre-HSCT period, and may be a useful bridge therapy for infants with SCID and persistent viral infections.     

Simultaneous administration of a low-dose mixture of donor bone marrow cells and splenocytes plus adenovirus containing the CTLA4Ig gene result in stable mixed chimerism and long-term survival of cardiac allograft in rats

T-cell costimulatory blockade combined with donor bone marrow transfusion may induce mixed chimerism, rendering robust tolerance in transplanted organs and cells. However, most protocols entail high doses of donor bone marrow cells (BMCs) or repeated administration of costly agents that block costimulatory pathways, thus delaying clinical development. To circumvent these shortcomings, we developed a strategy in which the dosage of donor BMCs was reduced but compensated by donor splenocytes (SPLCs). Furthermore, repeated administration of costly agents was replaced with a single injection of adenovirus expressing a gene of interest. In rat cardiac transplantation models, cardiac allografts from DA (RT-1a) rats were transplanted heterotopically into the abdomen of LEW (RT-11) recipient rats. Immediately after cardiac transplantation, an adenovirus vector (AdCTLA4Ig; 5 x 10(9) plaque-forming units) containing the gene for CTLA4Ig was administered to recipients (n = 6) simultaneously with a low dose of donor BMCs (1 x 10(8)/rat) and SPLCs (5 x 10(7)/rat) via the portal vein. The treated LEW recipient rats developed long-lasting mixed chimerism (>10% at >100 days) and exhibited long-term cardiac allografts (mean survival time of > 200 days) compared with control recipients. Moreover, recipients displaying long-lasting mixed chimerism accepted subsequent donor skin allografts while promptly rejecting third-party skin allografts. These results suggest that blockade of the CD28-B7 pathway, using adenovirus-mediated CTLA4Ig gene transfer, in concert with a low dosage of donor BMCs and SPLCs, may represent a feasible strategy to induce stable mixed chimerism and permit long-term survival of cardiac allografts.     

Long-Term Outcomes of Hematopoietic Stem Cell Transplantation for ZAP70 Deficiency

ZAP70 deficiency is a rare T?+?B?+?NK+ combined immunodeficiency with limited outcome data to help guide decisions around hematopoietic stem cell transplant (HSCT). We sought to understand the long-term clinical and immunologic outcomes of both conditioned and unconditioned HSCT for ZAP70 deficiency following transplant from a variety of graft sources. We performed a retrospective, single center review of all cases of HSCT for genetically confirmed ZAP70 deficiency since 1992. At a median of 13.5-year post-HSCT, 8/8 (100 %) patients are alive. Three received unconditioned bone marrow transplants from human leukocyte antigen (HLA)-matched siblings and achieved stable mixed donor-recipient T cell chimerism but low B cell (4–9 %) and absent to near-absent myeloid donor engraftment. Despite this, all three have normal immunoglobulin levels, have developed specific protective antibody responses to post-HSCT vaccinations, and have discontinued immunoglobulin replacement. Five patients received myeloablative conditioning (three T cell-depleted haploidentical and two unrelated cord blood) and have full donor chimerism for T and B cells and myeloid lineages. One patient experienced primary graft failure after serotherapy only. CD8 T cell count is normal in 5/8, high in 1/8, and low in 2/8. Infectious complications in 5/5 and autoimmune thrombocytopenia in one patient resolved post-HSCT. Mitogen proliferation to phytohemagglutinin was normal after HSCT in 8/8 patients. In total, seven have discontinued immunoglobulin replacement. In conclusion, HSCT using a variety of graft sources and approaches, including unconditioned matched sibling donor transplant, is a life-saving therapy for ZAP70 deficiency, providing excellent long-term immune function and resolution of clinical problems.     

Post-transplant lymphoproliferative disorders

Post-transplant lymphoproliferative disorders (PTLD) are the second most frequent neoplasia in transplant patients after skin carcinomas. They occur following both solid organ transplants (SOT) and haematopoietic stem cell transplants (HSCT). Most PTLD in solid organ recipients are of host origin, whereas in HSCT recipients they are most often of donor origin. The EBV status of the recipient and the donor, the type of transplant, the type of immunosuppressive therapy used, and the time after transplant are all important parameters that have been associated with the incidence and the type of PTLD. Although most PTLD are B-cell lesions up to 15% may be T-cell or NK-cell type. Most PTLD are associated with EBV, but EBV-negative PTLD are also clearly recognized. In the 2008 WHO classification of lymphoid neoplasms (ref) PTLD are subclassified according to their histological and immunophenotypic characteristics into early lesions, polymorphic type, monomorphic type, and classical Hodgkin lymphoma-type. Overall PTLD mortality rates are around 50%, but new therapies that include early treatment with rituximab and novel anti-EBV therapies promise better outcomes.     

Establishment and Operation of a Third-Party Virus-Specific T Cell Bank within an Allogeneic Stem Cell Transplant Program

Hematopoietic stem cell transplantation (HSCT) donor–generated virus-specific T cells (VSTs) can provide effective treatment for viral infection post-HSCT but are not readily accessible to all patients. Off-the-shelf cryopreserved VSTs suitable for treatment of multiple patients are an attractive alternative. We generated a bank of 17 cytomegalovirus (CMV)–, 14 Epstein-Barr virus (EBV)–, and 15 adenovirus (AdV)–specific T cell products from 30 third-party donors. Donors were selected for expression of 6 core HLA antigens expressed at high frequency in the local transplant population. T cells were generated by co-culturing venous blood or mobilized hematopoietic stem cell (HSC)–derived mononuclear cells with monocyte-derived dendritic cells pulsed with overlapping peptides covering CMV pp65, AdV5 hexon, or EBV BZLF1/LMP2A/EBNA1 proteins. Addition of a CD14⁺ selection step instead of plate adherence to isolate monocytes before culture initiation significantly improved expansion in cultures from HSC material. Phenotyping showed the CD8⁺ subset to have significantly higher numbers of terminal effector T cells (CD45RA⁺62L^?) and lower numbers of effector memory T cells (CD45RA^?62L^?) when compared with the CD4⁺ subset. Increased expression of the immunoinhibitory markers PD-1 and TIM-3 was noted on CD4⁺ but not CD8⁺ cells when compared with the control group. VST showed antiviral activity restricted through a variety of common HLAs, and modelling suggested a suitably HLA-matched product would be available for >90% of HSCT patients. Only a small number of carefully selected third-party donors are required to generate a VST bank of broad coverage, indicating the feasibility of local banking integrated into existing allogeneic HSCT programs.     

Allele-specific in situ analysis of microchimerism by fluorescence resonance energy transfer (FRET) in nonhuman primate tissues

Rhesus monkeys are relevant models for tolerance induction. Hematopoetic chimerism is believed to be one of these strategies. The purpose of this study was to detect donor class I A locus allele specific mRNA in Rhesus monkey kidney recipient. We report here for the first time the results of frequency resonance energy transfer (FRET) hybridization technology in frozen tissues. Frequency resonance energy transfer hybridization was performed by using two Mamu-A*05 allele specific oligonucleotides: a donor probe labeled with FITC and acceptor probe conjugated to Texas Red. The PCR-SSP microchimerism analysis method produced 0.05% and 0.5% of donor DNA for Mamu-DRB1*1002 and Mamu-DRBw301/3 alleles, respectively. The donor cells were detected in mesenteric and/or inguinal lymph nodes, spleen, and liver, where the signal was the strongest. The results of FRET hybridization demonstrated the identical staining pattern in the recipient frozen tissues to that determined by PCR-SSP. Following FRET hybridization, the sections underwent immunohistochemical analysis, which revealed that donor cells had CD8+ phenotype. We demonstrate here for the first time that FRET in situ hybridization technique can be utilized for microchimerism analysis in frozen tissues. We conclude that using two donor mRNA specific oligonucleotide probes, rather than one, produce higher specificity.     

Epstein-Barr Virus-Associated Post-Transplantation Lymphoproliferative Disease in Patients Who Received Anti-CD20 after Hematopoietic Stem Cell Transplantation

Post-transplantation lymphoproliferative disease (PTLD) is a serious complication associated with Epstein-Barr virus (EBV) infection after hematopoietic stem cell transplantation (HSCT). Although anti-CD-20 therapy is now used as a preemptive strategy for EBV reactivation, PTLD still occurs in some patients. Here we analyzed outcomes and risk factors associated with PTLD transformation in 208 HSCT recipients who were diagnosed with EBV-DNAemia and received at least 1 course of rituximab. The median patient age was 42.52 years (range, 8.35 to 74.77 years), and the median duration of follow-up was 47.33 months (range, 3.18 to 126.20 months). The 2-year overall survival of the entire cohort was 62.8 (95% confidence interval [CI], 56.4 to 69.9), and the 2-year cumulative incidence function of PTLD was 6.3% (95% CI, 3.5% to 10.1%), for a median follow-up of patients diagnosed with PTLD of 37.85 months. Multivariable analysis identified 4 risk factors associated with PTLD: HSCT from an unrelated donor, recipient HLA-DRB1*11:01, fever at diagnosis of EBV infection, and donor-recipient sex-mismatched HSCT. The presence of more than 2 of these risk factors was associated with an increased risk of developing PTLD. This retrospective study identifies risk factors associated with PTLD in EBV-infected patients after HSCT and defines patient subgroups that may benefit from intensified preemptive strategies.     

Donor Chimerism Early after Reduced-Intensity Conditioning Hematopoietic Stem Cell Transplantation Predicts Relapse and Survival

The impact of early donor cell chimerism on outcomes of T cell–replete reduced-intensity conditioning (RIC) hematopoietic stem cell transplantation (HSCT) is ill defined. We evaluated day 30 (D30) and 100 (D100) total donor cell chimerism after RIC HSCT undertaken between 2002 and 2010 at our institution, excluding patients who died or relapsed before D30. When available, donor T cell chimerism was also assessed. The primary outcome was overall survival (OS). Secondary outcomes included progression-free survival (PFS), relapse, and nonrelapse mortality (NRM). We evaluated 688 patients with hematologic malignancies (48% myeloid and 52% lymphoid) and a median age of 57 years (range, 18 to 74) undergoing RIC HSCT with T cell–replete donor grafts (97% peripheral blood; 92% HLA-matched), with a median follow-up of 58.2 months (range, 12.6 to 120.7). In multivariable analysis, total donor cell and T cell chimerism at D30 and D100 each predicted RIC HSCT outcomes, with D100 total donor cell chimerism most predictive. D100 total donor cell chimerism <90% was associated with increased relapse (hazard ratio [HR], 2.54; 95% confidence interval [CI], 1.83 to 3.51; P < .0001), impaired PFS (HR, 2.01; 95% CI, 1.53 to 2.65; P < .0001), and worse OS (HR, 1.50; 95% CI, 1.11 to 2.04, P = .009), but not with NRM (HR, .76; 95% CI, .44 to 2.27; P = .33). There was no additional utility of incorporating sustained D30 to D100 total donor cell chimerism or T cell chimerism. Low donor chimerism early after RIC HSCT is an independent risk factor for relapse and impaired survival. Donor chimerism assessment early after RIC HSCT can prognosticate for long-term outcomes and help identify high-risk patient cohorts who may benefit from additional therapeutic interventions.     

配偶供肾移植后微嵌合体发生与急性排斥反应

背景：微嵌合作为移植物与受者之间的双相细胞移动的标志,在移植免疫耐受中的作用日益受到重视。 目的：探讨夫妻生活与嵌合体的发生,与肾移植后急性排斥反应及其他相关性的研究。 方法：将接受肾脏移植的女性受者(有过生育史的女性除外)分为丈夫活体供肾组、无关男性尸体供肾组,并设立接受妻子活体供肾的对照组。STR方法检测女性受者体内男性供者来源的Y染色体反映微嵌合体的存在,与急性排斥反应发生的关系,并比较配偶间供肾效果的差异。 结果与结论：尽管配偶间供肾移植存在供者年龄偏大以及人类白细胞抗原错配率较高的因素,但与接受无关男性尸肾移植的女性受者相比,接受丈夫活体供肾移植的女性更易检测出微嵌合体,而且肾移植后恢复情况好,急性排斥反应发生率低。而与接受妻子供肾的丈夫相比,接受丈夫供肾的妻子肾移植效果好。说明夫妻间长期相处导致女性接受丈夫体液的机会多,由此产生免疫耐受对于肾移植后人/肾的相容性好,急性排斥反应小。     

De novo infection of hepatitis B virus in patients with orthotopic liver transplantation: analysis by determining complete sequence of the genome

De novo infection of hepatitis B virus (HBV) occurs after liver transplantation from donors with HBV markers that suggest past infection. In the present study, the complete nucleotide sequences of HBV derived from a donor and recipients were determined to determine the clinical and virological characteristics. A total of 57 donor-recipient pairs, which underwent living-related orthotopic liver transplantation, were enrolled in the present study; all were negative for HBsAg before transplantation. HBV DNA was tested in serum, liver tissue, and peripheral blood mononuclear cells (PBMCs) by the polymerase chain reaction (PCR). The nucleotide sequence of HBV was determined based on PCR products and the phylogenetic analysis. De novo infection of HBV was found in 3 of the 57 recipients. Anti-HBc was positive in all donors of 3 recipients with the de novo infection but was positive only in 4 donors of the remaining 54 recipients (P=0.001). HBV DNA was detected in the liver but not in the serum or PBMCs in donor 3 whose recipient developed de novo HBV infection. The nucleotide sequence covering entire genome of HBV (3,215 bases) derived from the liver of donor 3 had a homology of 99.8-100% with that derived from the serum of corresponding recipient 3. The strain of recipient 3 showed the closest association with that of the donor 3 by phylogenetic analysis. Complete sequences from two recipients with de novo HBV infection including recipient 3 conserved the basic organisation of HBV genome. Analysis of the entire nucleotide sequence of HBV genome proved that HBV existed in the liver of the donor with anti-HBc, and it caused de novo infection in the corresponding recipient.     

Long-Term Recipient Health-Related Quality of Life and Donor-Recipient Relationship following Sibling Pediatric Hematopoietic Stem Cell Transplantation

Despite the fact that the choice of donors and the number of sources of hematopoietic stem cells have increased, a sibling remains a preferred donor for allogeneic hematopoietic stem cell transplantation (HSCT). Transplant donation between siblings is a unique life experience that may have an impact on their future relationship. The aim of the study was to quantitatively measure the quality of life (QoL) in patients who underwent transplant and to describe the relationship between a recipient and a sibling donor after HSCT. We identified and invited 82 adults aged 18.0 to 38.7 years (median, 23.6) who underwent HSCT in our center and their sibling donors to participate in this survey. Forty-five patients (54.9%) and their siblings consented to take part in the study. The studied group consisted of 45 matched siblings donor (MSD)-HSCT recipients (19 women and 26 men) aged 18.0 to 36.2 (median, 28.5) years, who underwent MSD-HSCT at the age of 5.8 to 16.3 (median, 11.9) years, and their sibling donors aged 21.0 to 36.0 (median, 31.0) years, who were aged 11.2 to 20.2 (median, 15.5) years at bone marrow harvesting. For QoL and sibling relationship assessment, we used the Functional Assessment of Cancer Therapy–Bone Marrow Transplantation (FACT-BMT) and the Adult Sibling Relationship Questionnaire (ASQR). Higher scores indicate better quality of life in each scale of the FACT-BMT and the more significant is the factor in a sibling relationship measured by the ASQR. The questionnaires were given to both subgroups, HSCT recipients and donors, and the results were compared with each other. The overall result of the FACT-BMT questionnaire was 117 ± 35.0. The highest QoL was found in the functional (25.0 ± 3.5) and social well-being (25.0 ± 3.5) subscales, whereas the worst was in the emotional well-being (18.0 ± 9.5) subscale. Statistically, the QoL score was not influenced by current age (P = .378), age at the moment of HSCT (P = .256), and sex (P = .117). Being a recipient or a donor of HSCT was not a significant factor associated with warmth (2.6 ± 0.5 versus 3.1 ± 0.5; P = .830) and conflict (2.0 ± 0.7 versus 2.1 ± 1.2; P = .886) within the sibling relationship, whereas recipients scored significantly lower in rivalry within the sibling relationship compared with HSCT donors (0.8 ± 0.3 versus 1.2 ± 0.2; P = .012). The FACT Treatment Outcome Index remained the only significant predictor of warmth in the sibling relationship between HSCT recipient and donor. QoL in adult patients after HSCT in childhood was good. Sibling donor-recipient relationship is unbalanced, with a higher level of rivalry presented among donors. Further multicenter studies based on a larger cohort of patients are necessary to assess all aspects of the sibling relationship after transplantation experience.     

Post-Transplant CD34+ Selected Stem Cell “Boost” for Mixed Chimerism after Reduced-Intensity Conditioning Hematopoietic Stem Cell Transplantation in Children and Young Adults with Primary Immune Deficiencies

Mixed chimerism and eventual graft loss occurs in a proportion of children with primary immune deficiencies receiving alemtuzumab, fludarabine, and melphalan reduced-intensity conditioning (RIC) regimens before allogeneic hematopoietic stem cell transplantation (HSCT). We investigated the usefulness of a CD34⁺ selected stem cell “boost” without conditioning to treat mixed chimerism in children and young adults who received predominantly an alemtuzumab, fludarabine, and melphalan RIC regimen for primary immune deficiencies and reported the outcomes. Patients with a primary immune deficiency disorder who were either enrolled on a prospective CD34⁺ boost study for treatment of mixed chimerism from 2011 to 2014 (n = 9) or treated with a CD34⁺ boost on a clinical basis from 2014 to 2016 (n = 3) were included in this analysis. Response to a CD34⁺ boost was defined as a rise in donor chimerism by ≥15% with donor chimerism of at least 20%, stabilization was defined as a rise in chimerism by <15% with donor chimerism ≥ 20%, and no response was defined as any decline in donor chimerism or need for a second HSCT after a CD34⁺ boost. Twelve patients received alemtuzumab, fludarabine, and melphalan. Median age was 4.5 years (range, .9 to 20.6), and median whole blood donor chimerism before the boost was 25% (range, 3% to 61%). Three patients (25%) met criteria for response, 1 patient (8%) was considered to have stabilization, and 8 patients (67%) had no response 12 months after the boost. None of the patients developed any complications from a CD34⁺ boost, including no acute graft-versus-host disease (GVHD). All patients are alive with a median follow-up of 32 months (range, 8 to 79). We conclude that a CD34⁺ selected stem cell boost can be considered for treatment of mixed chimerism after alemtuzumab, fludarabine, and melphalan RIC HSCT in children and young adults with primary immune deficiencies. Approximately one-third of patients can be expected to benefit from a CD34⁺ selected stem cell boost and may avoid the need for a second HSCT. Lack of any GVHD or toxicity makes a stem cell boost an attractive option compared with donor lymphocyte infusions for treatment of mixed chimerism.