Annals of Laboratory Medicine最新文献

The safety and efficacy of both cell and gene therapies have been demonstrated in numerous preclinical and clinical trials. Chimeric antigen receptor T (CAR-T) cell therapy, which leverages the technologies of both cell and gene therapies, has also shown great promise for treating various cancers. Advancements in pertinent fields have also highlighted challenges faced while manufacturing cell and gene therapy products. Potential problems and obstacles must be addressed to ease the clinical translation of individual therapies. Literature reviews of representative cell-based, gene-based, and cell-based gene therapies with regard to their general manufacturing processes, the challenges faced during manufacturing, and QC specifications are limited. We review the general manufacturing processes of cell and gene therapies, including those involving mesenchymal stem cells, viral vectors, and CAR-T cells. The complexities associated with the manufacturing processes and subsequent QC/validation processes may present challenges that could impede the clinical progression of the products. This article addresses these potential challenges. Further, we discuss the use of the manufacturing model and its impact on cell and gene therapy.

Measurable residual disease (MRD) testing, a standard procedure in B-lymphoblastic leukemia (B-ALL) diagnostics, is assessed using multiparametric flow cytometry (MFC) and next-generation sequencing (NGS) analysis of immunoglobulin gene rearrangements. We evaluated the concordance between eight-color, two-tube MFC-MRD the LymphoTrack NGS-MRD assays using 139 follow-up samples from 54 pediatric patients with B-ALL. We also assessed the effect of hemodilution in MFC-MRD assays. The MRD-concordance rate was 79.9% (N=111), with 25 (18.0%) and 3 (2.2%) samples testing positive only by NGS-MRD (MFC^-NGS⁺MRD) and MFC-MRD (MFC⁺NGS^-MRD), respectively. We found a significant correlation in MRD values from total nucleated cells between the two methods (r=0.736 [0.647-0.806], P<0.001). The median MRD value of MFC^-NGS⁺MRD samples was estimated to be 0.0012% (0.0001%-0.0263%) using the NGS-MRD assays. Notably, 14.3% of MFC^-NGS⁺MRD samples showed NGS-MRD values below the limit of detection in the MFC-MRD assays. The percentages of hematogones detected in MFC-MRD assays significantly differed between the discordant and concordant cases (P<0.001). MFC and NGS-MRD assays showed relatively high concordance and correlation in MRD assessment, whereas the NGS-MRD assay detected MRD more frequently than the MFC-MRD assay in pediatric B-ALL. Evaluating the hematogone percentages can aid in assessing the impact of sample hemodilution.

Background: Flow cytometric immunophenotyping of hematolymphoid neoplasms (FCI-HLN) is essential for diagnosis, classification, and minimal residual disease (MRD) monitoring. FCI-HLN is typically performed using in-house protocols, raising the need for standardization. Therefore, we surveyed the current status of FCI-HLN in Korea to obtain fundamental data for quality improvement and standardization.

Methods: Eight university hospitals actively conducting FCI-HLN participated in our survey. We analyzed responses to a questionnaire that included inquiries regarding test items, reagent antibodies (RAs), fluorophores, sample amounts (SAs), reagent antibody amounts (RAAs), acquisition cell number (ACN), isotype control (IC) usage, positive/negative criteria, and reporting.

Results: Most hospitals used acute HLN, chronic HLN, plasma cell neoplasm (PCN), and MRD panels. The numbers of RAs were heterogeneous, with a maximum of 32, 26, 12, 14, and 10 antibodies used for acute HLN, chronic HLN, PCN, ALL-MRD, and multiple myeloma-MRD, respectively. The number of fluorophores ranged from 4 to 10. RAs, SAs, RAAs, and ACN were diverse. Most hospitals used a positive criterion of 20%, whereas one used 10% for acute and chronic HLN panels. Five hospitals used ICs for the negative criterion. Positive/negative assignments, percentages, and general opinions were commonly reported. In MRD reporting, the limit of detection and lower limit of quantification were included.

Conclusions: This is the first comprehensive study on the current status of FCI-HLN in Korea, confirming the high heterogeneity and complexity of FCI-HLN practices. Standardization of FCI-HLN is urgently needed. The findings provide a reference for establishing standard FCI-HLN guidelines.

Background: Plasma oxalate measurements can be used for the screening and therapeutic monitoring of primary hyperoxaluria. We developed a gas chromatography-mass spectrometry (GC-MS) assay for plasma oxalate measurements with high sensitivity and suitable testing volumes for pediatric populations.

Methods: Plasma oxalate was extracted, derivatized, and analyzed by GC-MS. We measured the ion at m/z 261.10 to quantify oxalate and the ¹³C₂-oxalate ion (m/z: 263.15) as the internal standard. Method validation included determination of the linear range, limit of blank, limit of detection, lower limit of quantification, precision, recovery, carryover, interference, and dilution effect. The cut-off value between primary and non-primary hyperoxaluria in a pediatric population was analyzed.

Results: The detection limit was 0.78 μmol/L, and the linear range was up to 80.0 μmol/L. The between-day precision was 5.7% at 41.3 μmol/L and 13.1% at 1.6 μmol/L. The carryover was <0.2%. The recovery rate ranged from 90% to 110%. Interference analysis showed that Hb did not interfere with plasma oxalate quantification, whereas intralipids and bilirubin caused false elevation of oxalate concentrations. A cut-off of 13.9 μmol/L showed 63% specificity and 77% sensitivity, whereas a cut-off of 4.15 μmol/L showed 100% specificity and 20% sensitivity. The minimum required sample volume was 250 μL. The detected oxalate concentrations showed interference from instrument conditioning, sample preparation procedures, medications, and various clinical conditions.

Conclusions: GC-MS is a sensitive assay for quantifying plasma oxalate and is suitable for pediatric patients. Plasma oxalate concentrations should be interpreted in a clinical context.