Using single antigen specificity magnetic beads for the isolation of specific antibodies against HLA antigens.

The presence of multiple donor-specific antibodies (DSAs) targeting HLA antigens poses a challenge to transplantation. Various techniques, including the use of recombinant cell lines and crossmatch cells have been developed to isolate DSAs. To simplify the extraction of HLA-specific DSAs from complex sera, we introduced magnetic beads with single HLA specificity (MagSort). Sera were treated with MagSort, allowing HLA-specific antibodies to bind to the beads, and these specific antibodies were subsequently eluted. MagSort beads, coated with 59 different HLA variants, underwent testing through 1329 adsorption/elution processes, demonstrating their effectiveness and specificity in adsorbing and eluting HLA-specific antibodies. The MagSort method proves comparable to the cell method, showing similar isolated antibody binding patterns. The isolated antibody binding patterns from MagSort reveal both known eplets and unknown patterns, suggesting its utility for eplet discovery. Additionally, MagSort proved effective in extracting signals for flow cytometry cross-matching, offering a means to assess the binding capability of isolated antibodies against specific donor cells.

Engraftment and Measurable Residual Disease Monitoring after Hematopoietic Stem Cell Transplantation: Comparison of Two Chimerism Test Strategies, Next-Generation Sequencing versus a Combination of Short-Tandem Repeats and Quantitative PCR.

Chimerism testing supports the study of engraftment and measurable residual disease (MRD) in patients after allogeneic hematopoietic stem cell transplant. In chimerism MRD, relapse can be predicted by increasing mixed chimerism (IMC), recipient increase ≥0.1% in peripheral blood, and proliferating recipient cells as a surrogate of tumor activity. Conventionally, the combination of short-tandem repeat (STR) and quantitative PCR (qPCR) was needed to ensure assay sensitivity and accuracy in all chimerism status. We evaluated the use of next-generation sequencing (NGS) as an alternate technique. The median numbers of informative markers in unrelated/related cases were 124/82 (NGS; from 202 single-nucleotide polymorphism), 5/3 (qPCR), and 17/10 (STR). Assay sensitivity was 0.22% (NGS), 0.1% (qPCR), and 1% (STR). NGS batch (4 to 48 samples) required 19.60 to 24.80 hours and 1.52 to 2.42 hours of hands-on time (comparable to STR/qPCR). NGS assay cost/sample was $91 to $151, similar to qPCR ($99) but higher than STR ($27). Using 56 serial DNAs from six post-transplant patients monitored by the qPCR/STR, the correlation with NGS was strong for percentage recipient (y = 1.102x + 0.010; R2 = 0.968) and percentage recipient change (y = 0.892x + 0.041; R2 = 0.945). NGS identified all 17 IMC events detected by qPCR (100% sensitivity). The NGS chimerism provides sufficient sensitivity, accuracy, and economical/logistical feasibility in supporting engraftment and MRD monitoring.

Elimination of 15N-thymidine after oral administration in human infants.

BACKGROUND: We have developed a new clinical research approach for the quantification of cellular proliferation in human infants to address unanswered questions about tissue renewal and regeneration. The approach consists of oral 15N-thymidine administration to label cells in S-phase, followed by Multi-isotope Imaging Mass Spectrometry for detection of the incorporated label in cell nuclei. To establish the approach, we performed an observational study to examine uptake and elimination of 15N-thymidine. We compared at-home label administration with in-hospital administration in infants with tetralogy of Fallot, a form of congenital heart disease, and infants with heart failure. METHODS: We examined urine samples from 18 infants who received 15N-thymidine (50 mg/kg body weight) by mouth for five consecutive days. We used Isotope Ratio Mass Spectrometry to determine enrichment of 15N relative to 14N (%) in urine. RESULTS/FINDINGS: 15N-thymidine dose administration produced periodic rises of 15N enrichment in urine. Infants with tetralogy of Fallot had a 3.2-fold increase and infants with heart failure had a 4.3-fold increase in mean peak 15N enrichment over baseline. The mean 15N enrichment was not statistically different between the two patient populations (p = 0.103). The time to peak 15N enrichment in tetralogy of Fallot infants was 6.3 ± 1 hr and in infants with heart failure 7.5 ± 2 hr (mean ± SEM). The duration of significant 15N enrichment after a dose was 18.5 ± 1.7 hr in tetralogy of Fallot and in heart failure 18.2 ± 1.8 hr (mean ± SEM). The time to peak enrichment and duration of enrichment were also not statistically different (p = 0.617 and p = 0.887). CONCLUSIONS: The presented results support two conclusions of significance for future applications: (1) Demonstration that 15N-thymidine label administration at home is equivalent to in-hospital administration. (2) Two different types of heart disease show no differences in 15N-thymidine absorption and elimination. This enables the comparative analysis of cellular proliferation between different types of heart disease.