AI Approach for Enhanced Thalassemia Diagnosis Using Blood Smear Images.

This paper aims to propose an approach leveraging Artificial Intelligence (AI) to diagnose thalassemia through medical imaging. The idea is to employ a U-net neural network architecture for precise erythrocyte morphology detection and classification in thalassemia diagnosis. This accomplishment was realized by developing and assessing a supervised semantic segmentation model of blood smear images, coupled with the deployment of various data engineering techniques. This methodology enables new applications in tailored medical interventions and contributes to the evolution of AI within precision healthcare, establishing new benchmarks in personalized treatment planning and disease management.

Enhancing Thrombophilia Risk Prediction Through AI-Based Methodologies.

Thrombophilia, a predisposition to thrombosis, poses significant diagnostic challenges due to its multi-factorial nature, encompassing genetic and acquired factors. Current diagnostic paradigms, primarily relying on a combination of clinical assessment and targeted laboratory tests, often fail to capture the complex interplay of factors contributing to thrombophilia risk. This paper proposes an innovative artificial intelligence (AI)-based methodology aimed to enhance the prediction of thrombophilia risk. The designed multidimensional risk assessment model integrates and elaborates through AI a comprehensive collection of patient data types, including genetic markers, clinical parameters, patient history, and lifestyle factors, in order to obtain advanced and personalized explainable diagnoses.

miRNAs in the Box: Potential Diagnostic Role for Extracellular Vesicle-Packaged miRNA-27a and miRNA-128 in Breast Cancer.

Circulating extracellular vesicle (EV)-derived microRNAs (miRNAs) are now considered the next generation of cancer "theranostic" tools, with strong clinical relevance. Although their potential in breast cancer diagnosis has been widely reported, further studies are still required to address this challenging issue. The present study examined the expression profiles of EV-packaged miRNAs to identify novel miRNA signatures in breast cancer and verified their diagnostic accuracy. Circulating EVs were isolated from healthy controls and breast cancer patients and characterized following the MISEV 2018 guidelines. RNA-sequencing and real-time PCR showed that miRNA-27a and miRNA-128 were significantly down-regulated in patient-derived EVs compared to controls in screening and validation cohorts. Bioinformatics analyses of miRNA-target genes indicated several enriched biological processes/pathways related to breast cancer. Receiver operating characteristic (ROC) curves highlighted the ability of these EV-miRNAs to distinguish breast cancer patients from non-cancer controls. According to other reports, the levels of EV-miRNA-27a and EV-miRNA-128 are not associated with their circulating ones. Finally, evidence from the studies included in our systematic review underscores how the expression of these miRNAs in biofluids is still underinvestigated. Our findings unraveled the role of serum EV-derived miRNA-27a and miRNA-128 in breast cancer, encouraging further investigation of these two miRNAs within EVs towards improved breast cancer detection.

Safe and Effective Administration of Caplacizumab in COVID-19-Associated Thrombotic Thrombocytopenic Purpura.

Thrombotic thrombocytopenic purpura (TTP) is a potentially life-threatening, rare acute thrombotic microangiopathy (TMA), caused by a severe ADAMTS13 deficiency. As the COVID-19 pandemic rapidly spread around the globe, much data about the pathogenicity of this virus were published. Soon after the detection of the first cases of COVID-19, it was clear that there was a wide range of COVID coagulopathy manifestations, such as deep venous thrombosis, pulmonary thromboembolism, and thrombotic microangiopathies. In the literature, little data have been reported about the association between TTP and COVID-19, and the treatment of COVID-19-associated TTP is still under debate. Here we present the case of a 46-year-old woman who developed a COVID-associated TTP, successfully treated with plasma exchange (PEX), steroids, and caplacizumab.

Safety and efficacy of caplacizumab in a case of thrombotic thrombocytopenic purpura in the postpartum period.

Thrombotic thrombocytopenic purpura (TTP) is a rare and life-threatening disease for which pregnancy and the postpartum period represent risk factors. Here, we present the case of a 39-year-old woman at the 31st week of gestation, who presented with cutaneous haemorrhagic symptoms. The complete blood count showed anaemia, thrombocytopenia, increase in haemolysis indices and undetectable ADAMTS13 activity. Acquired TTP was diagnosed, and she started daily plasma exchange (PEX) and methylprednisolone. After 5 days, an emergency caesarean section was performed with success because of pathologic cardiotocographic findings. After 7 days of PEX, the patient showed an initial laboratoristic improvement; unfortunately, 3 days later, she had a recurrence of disease and started daily PEX, caplacizumab and steroid, obtaining a haematological improvement. No literature data about caplacizumab use in pregnant or breastfeeding patients are available. In the present study, we describe that caplacizumab in the postpartum period could be well tolerated and effective.

Single and Synergistic Effects of Fenbendazole and Metronidazole Against Subclinical Infection by Giardia duodenalis in Non-Human Primates in a Zoological Garden in Southern Italy.

The aim of this study was to assess the single and synergistic effects of fenbendazole (Fenb) and metronidazole (Metro) for the treatment of Giardia duodenalis infection in different species of non-human primates (NHPs) housed in a zoological garden of southern Italy. Moreover, the study also aimed to better define the circulation of G. duodenalis zoonotic assemblages in NHP and the potential occurrence of zoonotic transmission between the staff from the zoo and NHP. Briefly, six species that belonged to four families (Lemuridae, Cercopithecidae, Atelidae, and Hylobatidae) of NHP and housed in six cages (CG) were identified as Giardia positive and divided into two groups. Group F (N = 16 animals) was treated with Fenb (50 mg/kg, every 24 h for 5 consecutive days) and Group M (N = 7 animals) was treated with Metro (25 mg/kg, two times a day for 5 consecutive days). After the first round of therapy, all the animals were retreated for 5 days by inverting the drugs in each group. On each sampling day [study days (SDs) 3-24], the samples were tested for the presence of Giardia cysts using the FLOTAC technique. Multiple fecal tests for the antigen detection of Giardia, such as rapid ELISA and direct immunofluorescence (IFA), were performed at each sampling point only on samples that resulted in positive for Giardia cysts with FLOTAC. The efficacy of Fenb ranged from 30 to 67% and for Metro ranged from 82 to 96%. The results showed the synergistic effects of Metro and Fenb (98-100%) over the combination of Fenb and Metro (52-90%) against the infection by Giardia in NHPs. The overall k agreement between FLOTAC and IFA was reached 0.858 (p = 0.0001). In contrast, all the samples had a negative antigen result when using ELISA. At molecular analysis, six samples were confirmed positive for Giardia by nested PCR. Only two positive samples were successfully sequenced that showed 100% of identity with assemblage B. All the samples from the humans included in the study resulted in negative for Giardia cysts. Overall, the study emphasizes the need for regular monitoring of Giardia infections in NHP housed in zoos by traditional diagnostic tools combined with molecular characterization of the parasite.

[The biological resources and molecule archives organization: turn a need into an opportunity for the Smart Specialization Strategy of the Lazio region.] / Organizzare risorse biologiche e archivi di molecole: trasformare una necessità in opportunità per la Smart Specialisation Strategy della Regione Lazio.

Smart Specialization Strategy (S3) of Lazio defines smart specialization strategies to bring out the excellence of the territory with prospects of success on the global market. Chemical-pharmaceutical, biomedical and biotechnological field is one of the 7 sectors considered of greatest interest for the S3. Key engine of biotechnology development are biological materials and associated data, stored in biobanks. However, to ensure that the research and product development carried out with that resources gives statistically significant and reproducible results, it is essential that they are collected, manipulated and stored using standardized and traced methods. Implementation of the recent published standard ISO 20387- "Biotechnology-Biobanking-General requirements for biobanking" is bridging biobanks toward to storage and distribution of qualified biological material only. Human biobanks are also an essential part of the assistance and care of the citizen and constitute an unavoidable cost of the regional health system. However, biobanks organization, rationalization of their territorial distribution, completion of the process of recognition and regional accreditation, parallel to the implementation of the offer of remunerated services for biobanking, can turn the cost of the necessary preservation of the samples, into an opportunity of territorial development. The paper describes the necessity, shared by a working group represented by several Lazio biobanks, of including biobank activities in the virtuous circle designed by the S3,concretizing the framework prefigured by the S3 document on infrastructures for research, innovation and technology transfer. To allow inclusion of biobank activities in the virtuous circle, we underline the need to quickly start the process of recognition of the territorial research biobanks, to implement at regional level the process of optimization and rationalization of the management of biological samples, in accordance with the international harmonization standards and with the territorial indications of sustainability.

Efficacy of eculizumab in severe ADAMTS13-deficient thrombotic thrombocytopenic purpura (TTP) refractory to standard therapies.

Thrombotic thrombocytopenic purpura (TTP) is a rare microangiopathic hemolytic anemia (MAHA) defined by mechanical hemolytic anemia, severe thrombocytopenia, and systemic visceral ischemia due to systemic platelet-rich microthrombi. Forty percent of patients with autoimmune TTP experience one or multiple relapses. Patients with refractory TTP are currently managed by corticosteroids, twice-daily PEX, and the anti-CD20 monoclonal antibody rituximab. Herein, we report two cases of severe TTP, refractory to those standard agents. On the basis of the fact that in cases of severe TTP the classical complement pathway is activated, and that the alternative pathway is also involved, both patients underwent eculizumab (anti-C5 monoclonal antibody) therapy. We observed prompt hematological and organ system responses to the eculizumab and the recovery of plasma ADAMTS-13 activity in both cases. Moreover, the fact that both patients discontinued eculizumab, maintaining the response, emphasizes the possibility of its usefulness for limited treatment periods. In conclusion, the diagnostic and therapeutic algorithm in TTP appears complicated by increasing evidence of complement involvement and the eculizumab seems to be a potential agent for refractory patients.

A new system for quality control in hematopoietic progenitor cell units before reinfusion in autologous transplant.

BACKGROUND: In our Center, the cell viability, the integrity of the bag, and the clonogenic assay were evaluated before the reinfusion of hematopoietic progenitor cells-apheresis (HPC-A). This quality control (QC) should be made 14 days before the reinfusion to the patient to have the result of the functional test on the proliferative capacity of hematopoietic progenitors. STUDY DESIGN AND METHODS: This study was designed to assess the potential of an automatic cell counting system (NucleoCounter NC-3000, ChemoMetec) in our clinical routine as a support of the clonogenic assay and the cytofluorimetric analysis for the QC of the cryopreserved HPC-A. The cell viability was evaluated by flow cytometry using the modified International Society of Hematotherapy and Graft Engineering protocol. The proliferative potential was assessed by specific clonogenic tests using a commercial medium. Furthermore, we evaluated the cellular functionality with NucleoCounter NC-3000, by using two protocols: "vitality assay" and "mitochondrial potential assay." RESULTS: The evaluation of the total nucleated cells in preapoptosis measured by 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazol-carbocyanine iodide (JC-1) assay showed a negative correlation (r=-0.43) with the total number of colonies (colony-forming unit [CFU]-granulocyte-macrophage progenitors plus burst-forming unit-erythroid progenitors plus CFU-granulocyte, erythroid, macrophage, megakaryocyte progenitors) obtained after seeding of 50 × 10(6) /L viable total nucleated cells. We observed a significant difference (p<0.0001) comparing the median number of colonies (166.70; SD, ± 136.36) obtained with a value of JC-1 less than 30% to the number of colonies (61.75; SD, ± 59.76) obtained with a value of JC-1 more than 30%. CONCLUSION: The evaluation of cell functionality by the use of the NucleoCounter NC-3000 is in agreement with results from clonogenic assay and can be considered an effective alternative in the routine laboratory.

Optimization of the immunomagnetic selection in microcythemic donors enrolled for haploidentical transplantation.

BACKGROUND: Immunomagnetic cell selection (ICS) cells is increasingly used in allogeneic hematopoietic transplantation in order to reduce the T cells quantity. The aim of this study was to evaluate an protocol based on Ficoll method before ICS. STUDY DESIGN AND METHODS: The automated procedure was compared with the standard method. In the group 1 the cell processing involves the extraction of the buffy-coat by Ficoll before incubation with antibodies. This procedure was performed with the Sepax S-100 device. The efficacy of this automated procedure was compared with the group 2. In this group, the cell washing and the incubation were performed through the standard method. The CD34+ cells collected by apheresis (HPC-A) were selected with ICS. RESULTS: The results obtained after Ficoll procedure, showed a total nucleated cells (TNCs) and CD34+ cells recovery of 85.73% (75.90-90.63; SD 4.25) and 79.31% (51.77-112.31; SD 18.40), respectively. The TNC and CD34+ cells recovery after the pre-incubation washing performed through the standard method, was 75.54% (38.36-97.76; SD 22.5) and 61.51% (30.87-81.79; SD 19.3), respectively. The CD34+ cells recovery after ICS was 79% (51.77-100; SD 18.40) and 44% (15.57-88.24; SD 25.91) in the group 1 and the group 2, respectively. This difference was statistically significant (p=0.001). CONCLUSION: The efficacy of the ICS which resulted to be higher in the group 1 compared to the group 2. Overall, our data suggest that the Ficoll procedure before incubation is suitable for the clinical routine in the ICS for haploidentical transplantation in patients affected by thalassemia.

Cell processing for haplo-identical hematopoietic stem cell transplantation: automated washing and immunomagnetic-positive selection.

BACKGROUND AIMS: Immunomagnetic cell selection (ICS) of CD34(+) cells is being used increasingly in allogeneic transplantation in order to reduce T-cell quantity. The aim of this study was to evaluate an automated washing protocol before immunomagnetic selection. METHODS: The automated method was compared with a conventional washing procedure. In the study group the cell processing using the automated procedure, both before and after antibody incubation, was performed with a Sepax S-100 device. The efficacy of the automated procedure was compared with the control group, where washing were performed using a standard method. RESULTS: The results obtained after pre-incubation washing performed using the automated system showed a total nucleated cell (NC) and CD34(+) cell recovery of 84.87% (71.80-105, SD 8.62; range, standard deviation) and 83.45% (47-109, SD 16.12), respectively. The NC and CD34(+) cell recovery after the pre-incubation washing cycle was performed using the standard method was 75.54% (38.36-97.76, SD 22.5) and 61.51% (30.87-81.79, SD 19.3), respectively. The CD34(+) cell recovery after ICS was 51.27% (13.77-98.82, SD 24.97) and 48.89% (15.57-88.24, SD 25.91) for group 1 and group 2, respectively. The average purity in group 1 was 86.46% (67.4-96.10, SD 13.07) and in group 2 84.97% (58.1-97.8, SD 15.58). CONCLUSIONS: The efficacy of the ICS led to an optimal purity without affecting cell recovery, which was higher in group 1. Overall, our data suggest that the automated method is suitable for washing hematopoietic progenitor cell apheresis (HPC-A) concentrates before immunomagnetic cell selection in daily clinical routines.

Transplantation in the onco-hematology field: focus on the manipulation of αß and γδ T cells.

γ/δ T cells represent a subset of T cells expressing a T cell receptor (TCR) variant composed of gamma and delta chains. The γ/δ TCR is expressed by 2-10% of all T cells in human peripheral blood, whereas the majority of T cells express α/ß TCRs. γ/δ T cells display a range of innate effector functions including rapid secretion of chemokines and cytokines, as well as target cell lysis. Recent interest has focused on the function of γ/δ T lymphocytes in allogeneic transplantation in the onco-hematology field. Several studies, in vitro and in vivo, suggest that γ/δ T lymphocytes are potential beneficial effector cells in the context of hematopoietic stem cell transplantation (HSCT). In addition, in this review, we discuss the depletion of α/ß T lymphocytes in the graft for allogeneic transplantation. In fact, an efficient TCR α/ß cell depletion potentially reduces the risk of GvHD. Furthermore, TCR α/ß T cell depletion, especially with immunomagnetic negative selection, retains other potential beneficial effector cells in the graft, such as γ/δ T cells, NK cells, and stem cells. These "facilitating" cells might facilitate engraftment, exert GvL effects, and reduce the risk for infections.

Evaluation of cell death after treatment with extracorporeal photopheresis.

The aim of our study is to assess the mortality of leukocytes during extracorporeal photopheresis. Sixty-three photopheresis performed on 13 patients affected by chronic GvHD were evaluated. Samples were analyzed using a FACSCalibur flow cytometer. Apoptosis and necrosis of limphomononuclear cells dramatically increased after the apheretic procedure. We found a further increase of apoptotic and necrotic limphomononuclear cells after treatment with 8-MOP and UVA (p≤0.05). Our data suggested that the immunomodulatory effects of extracorporeal photopheresis, triggered by circulating apoptotic or necrotic cells, could play an important role in the treatment of GvHD with this procedure.

Processing of hematopoietic stem cells from peripheral blood before cryopreservation: use of a closed automated system.

BACKGROUND: Hematopoietic stem cell transplantation is commonly used to treat several oncohematologic diseases. The autologous hematopoietic progenitor cells collected through apheresis (HPC-A) must be cryopreserved and stored before use in vivo. Cell processing that precedes cryopreservation of HPC-A includes volume reduction aimed at reducing the amount of dimethyl sulfoxide used, as well as storage space. STUDY DESIGN AND METHODS: The aim of our study was to assess the effectiveness of volume reduction performed with an automated closed system, namely, the Sepax S100 cell separation device (Biosafe SA). A total of 165 procedures were carried out on concentrates collected from 104 adult and pediatric patients. As a control group, 30 HPC-A units processed according to the standard method (i.e., centrifugation at a speed of 850 × g for 10 minutes, followed by manual plasma reduction) were evaluated. RESULTS: The volume reduction obtained was 59% (range, 20.54%-84.21%; standard deviation [SD], ± 12.19%), going from 236 mL (range, 100-443 mL; SD, ± 80.41 mL) to 97 mL (range, 33.00-263.00 mL; SD, ± 47.41 mL); recovery of nucleated cells was 90% (range, 64.84%-105.93%; SD, ± 8.76%), while that of CD34+ cells was 91% (range, 59.30%-119.37%; SD, ± 13.30%). These values did not differ from those obtained using the standard method. Automated processing required 20 minutes versus 40 minutes of manual processing. DISCUSSION: Our data demonstrate that volume reduction carried out with the Sepax S100 automated system was particularly effective; cell recovery was excellent and the time spent was short. Moreover, the closed system allows cell processing to be carried out in a contamination-controlled environment, in accordance with good manufacturing practice guidelines.

T(reg) cells: collection, processing, storage and clinical use.

T regulatory cells are fundamental in the maintenance of immune homeostasis and self-tolerance. Experimental models suggest the existence of two functional types of T(reg) cells designated naturally occurring and induced. Interest in T(reg) cells increased with evidence from experimental mouse and human models demonstrating that the immunosuppressive potential of these cells can be utilized in the treatment of various pathological conditions. The existence of a subpopulation of suppressive T cells was the subject of significant controversy among immunologists for many years. T regulatory cells limit immune activation through a variety of direct and indirect interactions, many of which are yet to be determined. Fully understanding T(reg) cells biology will lead us to harnessing the capacity of these cells in order to develop strategies to prevent autoimmune disorders and tolerance to transplantation. Efficient isolation, expansion and cryopreservation strategies that comply with Good Manufacturing Practice (GMP) guidelines are prerequisites for the clinical application of human CD4+ CD25+ CD127(low) FOXP3+ regulatory T cells.

Pre-transplant manipulation processing of umbilical cord blood units: Efficacy of Rubinstein's thawing technique used in 40 transplantation procedures.

BACKGROUND: Umbilical cord blood (UCB) is a valid alternative to be used in transplanted patients. Limitations of the use of stem cells depends on the small number of cells available; this is the reason why UCB can be used only in very low-weight patients. In this study we have evaluated the efficacy of cellular manipulation before transplant and in particular, before thawing the units through the Rubinstein method. METHODS: We have evaluated the results obtained after thawing 40 UCB to be used for as many patients affected by several pathologies (21 ALL, 6 AML, 3 MDS, 2 LNH, 2 histiocytosis, 2 ß-thalassemia, 1 Chédiak-Higashi syndrome, 1 Fanconi anemia, 1 Wiskott-Aldrich syndrome and 1 Omenn syndrome). RESULTS: After thawing, nucleated cells (NC) mean recovery was 76.81% (SD±15.41). The quantity of NC obtained was 124.29×107 (SD±43.18) and in only 5 cases the number of NC after the procedure was lower than the requested graft dose. Among the last ones, in two cases only we did not achieve the target after manipulation. The post-manipulation cellular viability was 83.48% (SD±10.6). For all the units shipment complied with all the necessary procedures; in fact the temperature never rose above -120°C. CONCLUSION: In our study we highlighted the efficacy of UCB thawing technique, with the same method defined in 1995 at the New York Blood Centre that guarantees an excellent NC recovery and maintains a high level of cell viability.

Immunohematologic reconstitution in pediatric patients after T cell-depleted HLA-haploidentical stem cell transplantation for thalassemia.

To analyze immunohematologic reconstitution, particularly of natural killer (NK) cells, we evaluated 13 ß-thalassemia patients after 20 and 60 days and 1 year posttransplantation with T cell-depleted HLA-haploidentical stem cells. We assessed lymphocyte and bone marrow (BM) progenitor cell phenotype and differentiation capacity, spontaneous BM cytokine production, stromal cells, and stromal cell interleukin (IL)-7 production. A reduced clonogenic capability manifested at day +20. Patients had significantly lower CD4(+) T cells versus controls, mainly in the CD45RA(+)CD62L(+) subset. NKs were among the first lymphocytes to repopulate the peripheral blood. At day +60, an increase in primitive BM progenitor cells paralleled small increases in CD4(+), naïve CD4(+), and thymic naïve Th cells. A significant increase in CD4(+) and CD8(+) markers paralleled an increase in CD3⁻CD16(+) NKs, especially with full engraftment. In patients with stable mixed chimerism we observed very low levels of CD3(+) donor chimerism early after transplant that increased over time, but a stable population of high donor NK cells, suggesting a role of these cells on donor engraftment. Stromal cells secreted less IL-7 and displayed "macrophage-like" morphology. Patients initially manifested impaired stem/progenitor cell growth and differentiation capacity in parallel with altered T cell homeostasis and a reduced T cell naïve compartment. We hypothesize that T cell compartment damage partly arises from altered new T cell production from the hematopoietic stem/progenitor cells under stromal cytokine influence. NNK subset analysis might be useful for determining transplant outcome.