Transfusion Medicine and Hemotherapy最新文献

Introduction: In immunohematology case studies the knowledge about variants of the blood group gene of interest can facilitate antibody diagnosis. Known gene variants can be rapidly genotyped by specific methods, but the identification of unknown variants requires sequencing of the gene. High throughput next-generation sequencing (NGS) technologies represent important tools for DNA sequencing of many targets in larger numbers of samples but are less suitable for the analysis of one or few genes in single samples. Nanopore sequencing (Oxford Nanopore Technologies, ONT) is a fast sequencing technology that could fulfill the requirements for targeted gene sequencing in case studies. Here, we describe an optimized protocol for long-read nanopore sequencing of blood group genes that enables analysis of whole genes within less than 7 h from DNA extraction to genotype determination.

Methods: Primers for long-range PCR (LR-PCR) were designed for the blood group genes ACKR1, CD151, BCAM, KEL, SLC14A1, GYPA, GYPB, GYPE, RHD, and RHCE with amplicon sizes in the range of 2.4-15.8 kilo base pairs (kbp). For evaluation of the sequencing data, 22 samples with 25 known gene variants were selected. The optimized sequencing workflow included DNA extraction from EDTA blood, LR-PCR amplification, library preparation, nanopore sequencing on the MinION Mk1D sequencing device with FLO-MIN114 (MinION) flow cells and data analysis including variant detection and genotyping. In addition, the workflow was tested on the MinION sequencing device with FLO-FLG114 (Flongle) flow cells and the PromethION 2 Solo sequencing device with FLO-PRO114 (PromethION) flow cells.

Results: Using the outlined long-read nanopore sequencing protocol, sequencing data for reliable variant calling were obtained. All alleles were identified and the zygosity could be determined based on the read counts, except for GYPB in one sample. Besides sequencing on the MinION Mk1D sequencing device MinION flow cells, successful application of the sequencing protocol to the Flongle flow cells and the PromethION sequencing device using PromethION flow cells was demonstrated. As expected, mean coverage and mean Q scores varied between the flow cells and devices.

Conclusion: The optimized nanopore sequencing protocol enabled the generation of long-read sequence data and identification of blood group gene variants within a working day. This approach is suitable for molecular analyses of different blood group genes in immunohematology case studies under the same LR-PCR and sequencing conditions.

Introduction: Red blood cell (RBC) alloantibodies typically develop following immune sensitization through transfusion or pregnancy. Naturally occurring antibodies, in contrast, arise without such exposure and are often directed against antigens such as ABO or Lewis. Kidd system antibodies are clinically significant, usually IgG, and rarely occur naturally.

Methods: Routine pretransfusion testing was performed using automated hemagglutination (Echo® Lumena, Werfen; Norcross, GA, USA) and manual tube methods for ABO and RhD typing. Antibody screening utilized a standard three-cell panel and extended testing by solid-phase red cell adherence (SPRCA). Phenotyping was conducted manually using monoclonal anti-Jk^a and anti-Jk^b. Comparative testing employed the indirect antiglobulin test (IAT) in tube with polyethylene glycol (PEG) enhancement.

Results: Two patients were encountered with anti-Jk^a by SPRCA despite no history of transfusion, pregnancy, or immunoglobulin therapy. Both exhibited a Jk(a-, b+) phenotype. Reactivity consistent with anti-Jk^a and dosage effect was observed in SPRCA testing, while IAT with PEG enhancement failed to detect the antibody. Auto control and direct antiglobulin tests were negative, and no additional clinically significant antibodies were identified.

Conclusion: These findings provide further evidence that anti-Jk^a can arise naturally, independent of sensitizing events. Detection was possible through solid-phase testing, highlighting its ability to identify weak or developing antibodies that may be missed by conventional tube methods. Awareness of such naturally occurring Kidd antibodies is essential to ensure appropriate antibody identification and selection of compatible blood for transfusion.

Objective: The aim of the study was to compare the relationship between titer concentrations via gel microcolumn assay (GMA) and conventional tube testing (CTT) methods at a single institution that serves as a referral center for women at risk for hemolytic disease of the fetus and newborn.

Study design: Retrospective chart review on all obstetric patients treated was conducted at a single tertiary care center with RBC antibodies from November 2018 to October 2022. Patients were included in the statistical analysis if there were parallel CTT and GMA titration studies available. GMA vs. CTT titers were graphed via bubble plots, and polynomial regression analysis was used to evaluate the correlation between the two methods. Logistical regression was used to evaluate the sensitivity and specificity of GMA titers for prediction of a critical CTT titer of 16.

Results: A total of 166 GMA titers from 87 patients had corresponding CTT titers. The most common antibody was anti-D (76) followed by anti-K (29). Polynomial regression indicated a relationship between GMA titers and CTT titers (R ² = 0.5325, p < 0.001). The relationship changed significantly with removal of anti-K antibodies (R ² = 0.6733, p < 0.001). GMA titers were predictive of a critical CTT (AUC 0.97, p < 0.001) for non-anti-K antibodies but not predictive for anti-K antibodies (p = 0.134). Overall a GMA titer of 64 was predictive of a CTT titer of 16 or higher with 90.7% (95% CI: 82.5-95.9%) sensitivity and 85.0% (95% CI: 75.3-92.0%) specificity. All GMA results of 32 or higher for anti-K antibodies had CTT of at least 16, whereas the same could not be said of the non-anti-K antibodies until GMA titers reached at least 512.

Conclusion: GMA titers are consistently higher than CTT titers in all antibodies tested in our study, and the correlation between the titers is statistically significant for all antibodies except for anti-K. GMA methodology is often preferred as first-line testing by the hospital transfusion service due to cost-effectiveness and ability to be automated. A predictive table was created for provider reference if their hospital's blood bank employs GMA techniques.

Introduction: Effective therapeutic options for advanced solid tumors remain severely limited, causing high fatality rates especially after metastasis. The carbohydrate structure CD176 has been identified as a promising target for precise immunotherapy in multiple carcinomas, as it is present in about 90% of carcinomas but unavailable for binding on healthy tissue. Here, we report the development of CD176-specific 4th-generation chimeric antigen receptor T cells (CAR-Ts), also known as T cells redirected for antigen-unrestricted cytokine-initiated killing (TRUCKs). To address the immunosuppressive tumor microenvironment (TME) and the heterogeneous antigen expression of solid tumors, which limit the efficacy of CAR-Ts, they were endowed with NFAT-inducible interleukin-12 (iIL12) release to improve pro-inflammatory autocrine and paracrine effects.

Methods: The CD176-iIL12-TRUCK construct was tested for target specificity in a reporter cell assay using a JE6-1-derived reporter cell line. Afterward, CD176-iIL12-TRUCKs were manufactured using primary CD8⁺ T cells. The influence of iIL12 on functionality of CD176-iIL12-TRUCKs, including T-cell activation levels, cytotoxic capacity, and recruitment of bystander immune cells, was evaluated following cocultures with CD176⁺ cell lines from different carcinomas.

Results: Upon recognition of CD176⁺ cancer cell lines, CD176-iIL12-TRUCKs specifically released pro-inflammatory mediators (interferon-γ, tumor necrosis factor-α) and showed an increased activation marker expression (CD25, CD69). Using both a 7-AAD-based viability assay and an impedance-based cytotoxicity assay, elimination of CD176⁺ cell lines from different tumor entities by CD176-iIL12-TRUCKs was shown. Additionally, iIL12 released by CD176-iIL12-TRUCKs led to recruitment of monocyte and NK cell lines in a chemotaxis chamber assay.

Discussion/conclusion: Overall, the IL-12 release substantially improved effector functionality against CD176⁺ cells but not CD176^- cells, indicating efficacy while maintaining specificity. Thus, CD176-iIL12-TRUCKs, with their potent antitumor efficacy and TME modulation potential, are a promising treatment option for patients with a variety of advanced solid tumors.

Introduction: Impairment of autophagy may be considered a potential mechanism underlying androgenetic alopecia (AGA). The aim of this study was to assess the therapeutic efficacy of platelet-rich plasma (PRP) in AGA treatment and investigate the role of autophagy in this process.

Methods: The experiment was conducted in two phases. In phase I, an AGA mouse model was established and treated with PRP. Following the treatment period, the therapeutic effects on hair growth were evaluated. The expression levels of autophagy-related genes (LC3 and Beclin-1) were assessed using immunohistochemistry (IHC), Western blot (WB), and quantitative real-time PCR (qPCR). In phase II, based on the first phase, additional experimental groups were introduced: (1) AGA model mice treated with the autophagy activator rapamycin (RAPA) alone and (2) AGA model mice receiving combined treatment with PRP and the autophagy inhibitor 3-methyladenine (3-MA). Hair growth progression, histopathological changes in hair follicles, and the expression of autophagy markers were analyzed to elucidate the role of autophagy in PRP-mediated AGA treatment.

Results: Results demonstrated that PRP treatment significantly increased both length and weight of newly grown hair in AGA model mice. The AGA model group exhibited markedly reduced mRNA and protein expression levels of autophagy-related markers (LC3 and Beclin-1) compared to controls. PRP intervention substantially enhanced autophagy levels in treated mice. Notably, therapeutic outcomes achieved with RAPA monotherapy were comparable to those observed with PRP treatment. However, the cohort receiving combined PRP and 3-MA treatment showed significantly diminished hair growth parameters (length and weight) and attenuated expression of autophagy-related genes relative to PRP-treated mice.

Conclusion: Autophagy levels in the hair follicle cells of AGA model mice are reduced, and impaired autophagy may represent a potential pathogenic mechanism underlying AGA. PRP therapy promotes hair growth and alleviates symptoms in AGA model mice by enhancing autophagy.

Introduction: Preoperative anaemia is associated with increased morbidity and mortality in surgical patients. While iron deficiency is a well-recognized cause, the contribution of other nutritional deficiencies, such as folate and vitamin B12, remains underexplored. Therefore, this study aimed to assess the prevalence and role of folate and vitamin B12 deficiencies in preoperative anaemia among patients undergoing major surgery.

Methods: This retrospective observational study included 410 patients aged ≥18 years who underwent major surgery and were evaluated at a preoperative anaemia clinic in a large tertiary hospital between June 2016 and June 2023. Key outcome measures included the prevalence of iron, folate, and vitamin B12 deficiencies and their associations with preoperative anaemia.

Results: Anaemia was observed in 41.5% (95% confidence interval [CI]: 36.8-46.3), iron deficiency in 51.5% (95% CI: 46.6-56.3), folate deficiency in 18.0% (95% CI: 14.6-22.1), and vitamin B12 deficiency in 3.2% (95% CI: 1.9-5.4). Anaemic patients exhibited higher rates of iron (61.8%, 95% CI: 54.3-68.7 vs. 44.2%, 95% CI: 38.0-50.5) and folate deficiencies (27.1%, 95% CI: 20.9-34.2 vs. 11.7%, 95% CI: 8.2-16.3, p < 0.001) compared to non-anaemic patients. Combined deficiencies, primarily iron and folate, were more frequent in anaemic patients (17.7%, 95% CI: 12.7-24.1 vs. 7.5%, 95% CI: 4.8-11.5, p = 0.003). Substantial heterogeneity in deficiency patterns was observed across surgical subgroups, with overall prevalences ranging from 20% to 59% for iron, and from 11% to 31% for folate. Iron (odds ratio [OR] 3.27, 95% CI: 2.03-5.27, p < 0.001) and folate (OR 2.6, 95% CI: 1.45-4.59, p = 0.001) deficiencies were independently associated with anaemia and together accounted for approximately one-third of preoperative anaemia cases.

Conclusion: Iron deficiency remains the predominant contributor to preoperative anaemia, with folate deficiency playing a significant yet underrecognized role. The high occurrence of combined deficiencies and substantial heterogeneity across surgical populations support the need for population-specific diagnostic and supplementation strategies.

Introduction: Immuno-magnetic CD34 positive (CD34+) selection is generally used for stem cell boosts after allogeneic stem cell transplantation. In some cases, only cryopreserved cells are available as starting material. We present a new automatic device and technique for preparing and subsequently magnetically enriching these cells.

Methods: We used the CliniMACS Prodigy® platform in combination with the TS 320 tubing set, equipped with a large 800 mL chamber, which enables to include the DMSO wash, platelet wash, antibody incubation, and antibody wash and the subsequent CD34 enrichment on an automatic basis with only short hands-on time. We performed three validation runs using DNase and sodium citrate for cell preparation to reduce the risk of aggregates and clots.

Results: Our robust preclinical results show the feasibility and safety of the process with a mean of 53% CD34 yield after thawing and CD34 purity and viability of 93 and 97%, respectively. CD3 log depletion exceeds 5.0 in all 3 cases, which provides optimal GvHD prevention.

Conclusion: This shows that the updated technique gains unaffected CD34+ cells of high quality, not only from fresh but also from cryopreserved stem cell products.

Background: Dry eye disease (DED), as one of the most prevalent ocular surface diseases, is a multifactorial disorder disrupting tear film homeostasis and ocular surface integrity, profoundly impacts patients' quality of life. Conventional therapies such as artificial tears and anti-inflammatory agents provide transient relief but fail to address underlying pathological mechanisms and may induce complications with prolonged use.

Summary: Platelet-rich plasma (PRP), an autologous biologic agent enriched with growth factors (e.g., platelet-derived growth factor, transforming growth factor-β, epidermal growth factor), has emerged as a promising therapeutic strategy. PRP promotes corneal epithelial regeneration, reduces inflammation, and restores glandular function, offering a pathophysiologically targeted approach. Recent studies highlight synergistic benefits of combining PRP with agents like hyaluronic acid, stem cells, or nanomaterials, which enhance tear film stability and tissue repair. Despite encouraging preclinical and clinical outcomes, optimal protocols and long-term safety of PRP-based combination therapies remain under investigation.

Key messages: This review synthesizes current evidence on PRP's mechanisms, clinical efficacy, and innovative combinatorial approaches for DED, emphasizing the need for standardized trials to validate these strategies. Future integration of PRP with biologics, advanced materials, or laser therapies may revolutionize precision medicine in DED management.