Value of a flow cytometry cross-match in the setting of a negative complement-dependent cytotoxicity cross-match in heart transplant recipients.

Complement-dependent cytotoxicity cross-match (CDCXM) is used for evaluation of preformed HLA-specific antibodies in patients undergoing heart transplantation. Flow cytometry cross-match (FCXM) is a more sensitive assay and used with increasing frequency. To determine the clinical relevance of a positive FCXM in the context of negative CDCXM in heart transplantation, the United Network for Organ Sharing (UNOS) database was analyzed. Kaplan-Meier analysis and Cox proportional hazard modeling were used to assess graft survival for three different patient cohorts defined by cross-match results: T-cell and B-cell CDCXM+ ("CDCXM+" cohort), CDCXM- but T-cell and/or B-cell FCXM+ ("FCXM+" cohort), and T-cell/B-cell CDCXM- and FCXM- ("XM-" cohort). During the study period, 2558 patients met inclusion criteria (10.7% CDCXM+, 18.8% FCXM+, 65.5% XM-). CDCXM+ patients had significantly decreased graft survival compared to FCXM+ and XM- cohorts (P = .003 and <.001, respectively). CDCXM- and FCXM+ patients did not have decreased graft survival compared to XM- patients (P = .09). In multivariate analysis, only CDCXM+ was associated with decreased graft survival (HR 1.22, 95% CI 1.01-1.49). In conclusion, positive FCXM in the context of negative CDCXM does not confer increased risk of graft failure. Further study is needed to understand implications of CDCXM and FCXM testing in heart transplant recipients.

Assessing a single targeted next generation sequencing for human leukocyte antigen typing protocol for interoperability, as performed by users with variable experience.

BACKGROUND: A simplified protocol for HLA-typing -by NGS, developed for use with the Illumina MiSeq, was performed by technologists with varying NGS experience to assess accuracy and reproducibility. METHODS: Technologists from six laboratories typed the same 16 samples at HLA-A, B, C, DRB1, and DQB1. The protocol includes long range PCR, library preparation, and paired-end 250bp sequencing. Two indexing strategies were employed: locus-specific indexing whereby each locus was tagged uniquely and sample-specific indexing whereby all 5 loci for a sample were pooled prior to library preparation. Sequence analysis was performed with two software packages, Target HLA (Omixon) and NGSengine (GenDx). RESULTS: The average number of sequence reads per library was 387,813; however, analysis was limited to 40,000 reads for locus-indexed libraries and 200,000 reads for sample-indexed libraries resulting in an average depth of coverage of 1444 reads per locus. Sufficient reads for genotype analysis were obtained for 98.4% of libraries. Genotype accuracy was >97% in pooled amplicons and >99% in individual amplicons by both software analysis. Inter-laboratory reproducibility was 99.7% and only cause of discordance was cross-contamination of a single amplicon. CONCLUSIONS: This NGS HLA-typing protocol is simple, reproducible, scalable, highly accurate and amenable to clinical testing.

Clonal evolution and clinical significance of copy number neutral loss of heterozygosity of chromosome arm 6p in acquired aplastic anemia.

Acquired aplastic anemia (aAA) results from the T cell-mediated autoimmune destruction of hematopoietic stem cells. Factors predicting response to immune suppression therapy (IST) or development of myelodysplastic syndrome (MDS) are beginning to be elucidated. Our recent data suggest most patients with aAA treated with IST develop clonal somatic genetic alterations in hematopoietic cells. One frequent acquired abnormality is copy-number neutral loss of heterozygosity on chromosome 6p (6p CN-LOH) involving the human leukocyte antigen (HLA) locus. We hypothesized that because 6p CN-LOH clones may arise from selective pressure to escape immune surveillance through deletion of HLA alleles, the development of 6p CN-LOH may affect response to IST. We used single nucleotide polymorphism array genotyping and targeted next-generation sequencing of HLA alleles to assess frequency of 6p CN-LOH, identity of HLA alleles lost through 6p CN-LOH, and impact of 6p CN-LOH on response to IST. 6p CN-LOH clones were present in 11.3% of patients, remained stable over time, and were not associated with development of MDS-defining cytogenetic abnormalities. Notably, no patient with 6p CN-LOH treated with IST achieved a complete response. In summary, clonal 6p CN-LOH in aAA defines a unique subgroup of patients that may provide insights into hematopoietic clonal evolution.

Changes in the methodology of pre-heart transplant human leukocyte antibody assessment: an analysis of the United Network for Organ Sharing database.

BACKGROUND: We sought to investigate temporal trends in the methodology of human leukocyte antibody assessment in heart transplantation. METHODS: The United Network for Organ Sharing database was queried from June 2004 to March 2013 to obtain pre-heart transplantation human leukocyte antibody results. The % panel reactive antibody for class I and II antibodies was recorded along with the methodology of assessment. Allosensitization was defined as class I and/or II panel reactive antibody of ≥ 10%. The primary outcome measure was graft survival. RESULTS: During the study period, 12,858 patients with available data underwent heart transplantation. The prevalence of allosensitization increased, with 16.8% in 2005-2006 sensitized at the time of transplantation compared to 23.1% in 2010-2011 (p < 0.001); this occurred in conjunction with an increase in the utilization of flow cytometry (77.2% in 2005-2006; 97.0% in 2010-2011, p < 0.001). Using multivariable analysis, a positive pre-heart transplantation panel reactive antibody by flow cytometry independently predicted graft loss. CONCLUSIONS: There has been a recent increase in flow cytometric assessment of human leukocyte antibodies prior to heart transplantation, which may be associated with an increase in the prevalence of pre-transplant patients being characterized as allosensitized. Flow cytometry may identify patients with the highest likelihood of graft loss.

Emergence of clonal hematopoiesis in the majority of patients with acquired aplastic anemia.

Acquired aplastic anemia (aAA) is a nonmalignant disease caused by autoimmune destruction of early hematopoietic cells. Clonal hematopoiesis is a late complication, seen in 20-25% of older patients. We hypothesized that clonal hematopoiesis in aAA is a more general phenomenon, which can arise early in disease, even in younger patients. To evaluate clonal hematopoiesis in aAA, we used comparative whole exome sequencing of paired bone marrow and skin samples in 22 patients. We found somatic mutations in 16 patients (72.7%) with a median disease duration of 1 year; of these, 12 (66.7%) were patients with pediatric-onset aAA. Fifty-eight mutations in 51 unique genes were found primarily in pathways of immunity and transcriptional regulation. Most frequently mutated was PIGA, with seven mutations. Only two mutations were in genes recurrently mutated in myelodysplastic syndrome. Two patients had oligoclonal loss of the HLA alleles, linking immune escape to clone emergence. Two patients had activating mutations in key signaling pathways (STAT5B (p.N642H) and CAMK2G (p.T306M)). Our results suggest that clonal hematopoiesis in aAA is common, with two mechanisms emerging-immune escape and increased proliferation. Our findings expand conceptual understanding of this nonneoplastic blood disorder. Future prospective studies of clonal hematopoiesis in aAA will be critical for understanding outcomes and for designing personalized treatment strategies.

Persistence of anti-human leukocyte antibodies in congenital heart disease late after surgery using allografts and whole blood.

BACKGROUND: Allografts are used for vascular reconstruction in many forms of congenital heart disease. Although allografts induce anti-human leukocyte antibody (HLA) formation, much about this response is unknown. METHODS: Three groups of patients aged 8 to 18 years old underwent analysis for class I and II anti-HLA antibodies using Luminex. Groups were defined by timing of allograft exposure and diagnosis at Norwood for hypoplastic left heart syndrome (neonatal group), at Glenn for single-ventricle lesions not requiring arch reconstruction (infant group), and cardiac defects repaired during infancy without allografts (controls). Patients had significant anti-HLA (sensitization) if mean fluorescence intensity was ≥ 1500. RESULTS: The study enrolled 29 patients (median age, 10.1 years). Significant class I anti-HLA antibodies were seen in 44% (8 of 18) of the neonatal group, 25% (1 of 4) of the infant group, and 14% (1 of 7) of controls; class II anti-HLA antibodies were seen in 44% (8 of 18) of the neonatal group, 25% (1 of 4) of the infant group, and 29% (2 of 7) of controls. All patients received fresh whole blood, but the neonatal group had greater exposure (p = 0.001). There was less sensitization with increasing time from last receipt of allograft(s) or blood transfusion (p = 0.05). CONCLUSIONS: Exposure to allograft at the Norwood procedure is associated with long-term sensitization to anti-HLA antibodies in 56% of patients. Sensitization also occurs in those without prior exposure to allografts, may decrease over time, and appears related to whole blood. These findings have implications for those in whom heart transplant is considered late in the clinical course.

A strategy for single nucleotide polymorphism analysis of chimerism for somatic cell therapy.

BACKGROUND AIMS: Chimerism is an important outcome measure in hematopoietic cell transplantation as well as somatic cell therapy. Commonly used methods to estimate chimerism are restricted by either gender or inefficient sensitivity. In principle, real-time polymerase chain reaction (PCR)-based assays can be used to assess single nucleotide polymorphisms (SNP), which are a vast resource of molecular markers, and such assays demonstrate a substantially higher sensitivity (0.001%), but the specificity is unclear because of a low-level signal from mismatched sequences. METHODS: In this study, we cloned 14 pairs of SNP selected from the SNP HapMap database and examined the specificity and sensitivity of their detection by real-time PCR using two primer/fluorescent probe pairs to allow genotyping of the two possible variant alleles. Clinical donor-recipient pairs from 18 families were used to explore the efficacy of using SNP assays to measure chimerism. RESULTS: We found that the polymorphic nucleotide influences the ability to distinguish the signal generated by the target and mismatched sequences. Moreover, the specific fluorescent reporter probe can affect the difference in signal intensity between the target and mismatched sequences. Real-time PCR SNP assays can attain a sensitivity of 0.1-0.5% with 100% specificity. When comparing possible clinical donor-recipient pairs, we found an average 3.3 out of 14 SNP were informative. CONCLUSIONS: By optimal selection of the polymorphic sequences and fluorescent reporter, the real-time PCR SNP assay is superior to the short-tandem repeat chimerism assay and broadly applicable. This strategy may be applied in future clinical trials of bone marrow cell therapy.

Ventricular assist device-associated anti-human leukocyte antigen antibody sensitization in pediatric patients bridged to heart transplantation.

BACKGROUND: Ventricular assist devices (VAD) are associated with the formation of antibodies to anti-human leukocyte antigens (HLA) or sensitization. The incidence and effects of VAD-associated anti-HLA sensitization have not been well studied in the pediatric population. METHODS: A retrospective review of all patients undergoing VAD implant at our institution from 1998 to 2008 was performed. Panel reactive antibody (PRA) results before VAD implant, after VAD implant, and after orthotopic heart transplantation (OHT) were recorded. Patients who became sensitized (PRA for class I and/or II immunoglobulin G antibodies >or= 10%) on VAD support were compared with non-sensitized patients with regard to demographics, diagnosis, device type, and blood product exposure on VAD support. Outcomes after OHT were also compared between groups. RESULTS: VAD support was initiated in 20 patients (median age, 14.4 years), with 75% survival to OHT or recovery. PRA data before and after VAD implant were available for 17 patients. VAD-associated sensitization developed in 35% of recipients. There were no differences between those sensitized in association with VAD support and non-sensitized patients with regard to age, gender, diagnosis, device type, extracorporeal membrane oxygenation use, or blood product exposure on VAD support. Black race predicted sensitization on VAD (p = 0.02). There were no differences in survival or rejection between groups. CONCLUSIONS: VAD therapy was associated with the development of anti-HLA sensitization in 35% of recipients. Black race predicted sensitization, but there were no differences in overall survival or outcomes after OHT.

Successful deceased donor renal transplant in a sensitized pediatric recipient with the use of plasmapheresis.

Sensitization following renal transplant is a significant barrier to repeat transplantation in children. We report a successful DD renal transplant, with the use of PP, in an 11-yr-old girl who became highly sensitized following a prior failed transplant. She received PP treatments after failure of high-dose IVIg (Gamimune). We established the effectiveness of PP by attaining a 0% PRA and negative cross-matches after five PP treatments. Subsequently, our patient underwent a second round of scheduled PP. When the PRA was 0%, unacceptable antigens were removed from the UNOS wait list, PP was continued, and a kidney became available within 10 days. The final flow cytometry cross-match with the eventual donor was negative. This success demonstrates that coordination of desensitization by PP and advanced laboratory monitoring techniques with recent policies regarding allocation of organs to pediatric patients provides new opportunities for children awaiting transplantation. Since the transplant, our patient sustained a low-titer increase of anti-HLA antibodies. However, she has had no episodes of acute rejection and has maintained excellent graft function more than 17 months later.