Digital PCR Improves Sensitivity and Quantification in Monitoring CAR-T Cells in B Cell Lymphoma Patients.

Chimeric antigen receptor T cells (CAR-T) has emerged as a promising therapy, over 60% of patients fail to sustain a long-term response. The underlying factors that leads to the effectiveness of this therapy are not completely understood, CAR-T cell persistence and monitoring seems to be pivotal for ensuring a successful response. Various monitoring methods such as multiparametric flow cytometry (MFC) or quantitative PCR (qPCR) have been applied. Our objective is to develop digital PCR (dPCR) assays for detection and quantification of CAR-T cells, comparing them with MFC and qPCR. Samples taken at different follow-up times from 45 patients treated with CAR-T therapy were analyzed to assess the correlation between the different methodologies. dPCR presented a high correlation with MFC and qPCR (r = 0.97 and r = 0.87, respectively), while offering a higher sensitivity (0.01%) compared to MFC (0.1%) and qPCR (1%). dPCR emerged as an alternative and highly sensitivity method for monitoring CAR-T cell dynamics. This technique is well-suited for implementation in clinical practice as a complementary technique to MFC.

Advantages of high cell concentration prior to cryopreservation of initial leukapheresis in CAR-T cell therapy.

BACKGROUND: Chimeric antigen receptor (CAR) T-cell therapy is increasingly used in patients affected by B-cell lymphoma and acute lymphoblastic leukemia. For logistical reasons, initial apheresis products may be cryopreserved for shipment to manufacturing centers. Due to the characteristics of these patients, cells are often collected in large volumes, meaning more bags must be cryopreserved. This requires increased storage, time and monetary costs. In this context, we aimed to evaluate a high cell concentration cryopreservation protocol by centrifugation to standardize the initial CAR-T manufacturing procedure. MATERIALS AND METHODS: Sixty-eight processes of leukapheresis of 57 patients affected by refractory/relapsed B cell lymphoma and 9 patients affected by acute lymphoblastic leukemia who were eligible for anti-CD19 CAR-T cell treatment performed between June 2019 and October 2022 were analyzed. Whole blood count, percentage and number of T cells were assessed on the apheresis final product. The apheresis product, which was alternatively stored overnight at 4°C, was centrifuged, adjusting the volume to approximately 40 mL. The product was immediately cryopreserved to achieve a final cell concentration of 50-200×106 cells/ml for cryopreservation. RESULTS: Leukapheresis volume was reduced by almost fivefold (median: 185 to 40 mL), resulting in a higher product concentration in one bag. In addition, the number of non-target cells (monocytes, platelets and erythrocytes) was also reduced during the development of CAR T cell therapy, thereby maintaining T lymphocyte levels and providing a purer starting material. DISCUSSION: The advantages of the protocol include reducing economic costs, saving storage space, simplifying the manufacturing process, and facilitating shipping logistics. In conclusion, we present a validated, simple, and cost-effective cell enrichment processing protocol that provides high-quality cryopreserved products as starting material for the CAR-T cell manufacturing process.

Red Blood Cell Transfusion after Postpartum Hemorrhage: Clinical Variables Associated with Lack of Postpartum Hemorrhage Etiology Identification.

Postpartum hemorrhage (PPH) remains a significant obstetric emergency worldwide and a leading cause of maternal death. However, it is commonly underreported, which can represent a major concern for maternal morbidity and mortality. This retrospective case series study analyzed patients with red blood cell transfusion (RBCt) in the postpartum period over a four-year interval at a specific center. A total of 18,674 patients delivered between January 2018 and December 2021. Patients with postpartum RBCt were classified into two groups: those with identified PPH (i-PPH) and those without (non-i-PPH). Clinical variables, delivery details, blood loss data, and treatment information were collected. Statistical analysis involved a comparison of variables between the i-PPH and non-i-PPH groups. Univariate and multivariate analyses were performed, aiming to identify significant associations between the clinical variables and a lack of PPH identification. The incidence of RBCt was 1.26% (236 cases). Patients receiving RBCt had higher rates of cesarean delivery, twin pregnancy, labor induction, and previous cesarean section. Among patients with postpartum RBCt, 34.3% lacked an identified PPH. The rarity of postpartum RBCt contrasts with the increasing rates of PPH, highlighting the importance of diagnosing PPH and postpartum anemia. A strategy of systematic quantification of blood loss during delivery could help detect PPH and anemia before adverse consequences occur.

Use of intraoperative ultrasound to improve tension-free vaginal tape-obturator placement: A pilot study.

OBJECTIVE: To evaluate the use of intraoperative ultrasound (IUS) to achieve a postoperative optimal placement of the tension-free vaginal tape-obturator (TVT-O). METHODS: A cohort study was performed among women who underwent TVT-O placement. In 25 women, ultrasound was used for the placement, and based on current evidence, the optimal intraoperative and postoperative ultrasound pattern was selected. They were compared with 25 women where IUS was not used. RESULTS: IUS increased operative time (by 7.5 min), and in 36% (9/25) of cases it was necessary to perform some corrections based on the ultrasound findings. After 1 month, in patients of the IUS group, the tapes were more frequently placed at a distance of 3-5 mm (P = 0.01), and it was more common for it to be placed between 40% and 70% relative to the urethral length (P = 0.011). Of tapes placed with IUS, 76% (met the optimal postoperative ultrasound pattern, as opposed to only 48% placed without IUS (P = 0.041). No differences were found in the complications or the functional results at 1 month post-surgery. CONCLUSION: The use of IUS for the placement of TVT-O allows us to position them optimally and avoid erroneous placements, although IUS increases the operative time without improving the functional results and the rate of complications.

Role of long non-coding RNAs in adipose tissue metabolism and associated pathologies.

The incidence of obesity and its related disorders has increased dramatically in recent years and has become a pandemic. Adipose tissue is a crucial regulator of these diseases due to its endocrine capacity. Thus, understanding adipose tissue metabolism is essential to finding new effective therapeutic approaches. The "omic" revolution has identified new concepts about the complexity of the signaling pathways involved in the pathophysiology of adipose tissue-associated disorders. Specifically, advances in transcriptomics have allowed its application in clinical practice and primary or secondary prevention. Long non-coding RNAs (lncRNAs) have emerged as critical regulators of adipose tissue since they can modulate gene expression at the epigenetic, transcriptional, and post-transcriptional levels. They interact with DNA, RNA, protein complexes, other non-coding RNAs, and microRNAs to regulate a wide range of physiological and pathological processes. Here, we review the emerging field of lncRNAs, including how they regulate adipose tissue biology, and discuss circulating lncRNAs, which may represent a turning point in the diagnosis and treatment of adipose tissue-associated disorders. We also highlight potential biomarkers of obesity and diabetes that could be considered as therapeutic targets.

Allogeneic CD34-selected stem cell boost as salvage treatment of life-threatening infection and severe cytopenias after CAR-T cell therapy.

BACKGROUND: A variable incidence of profound cytopenia has been described in patients receiving chimeric antigen receptor T-cell (CAR-T) therapy for relapsed or refractory (R/R) B-cell acute lymphoblastic leukemia (ALL). This complication leads to severe infection in some cases, especially those who present additional risk factors including prior hematopoietic stem cell transplantation (HSCT). STUDY DESIGN AND METHODS: We report a case of breakthrough invasive fungal infection in a patient with prolonged neutropenia after CAR-T cell therapy administered for relapsed B-cell ALL after allogeneic haploidentical HSCT. RESULTS: After disease progression was discarded, therapy with antifungal agents, G-CSF and thrombopoietin analogue was started. However, no sign of haematological recovery or infection improvement was observed. A fresh mobilized selected CD34-stem cell boost from her haploidentical transplant donor was infused without further conditioning. Within 15 days of mobilized CD34-boost administration the patient showed complete resolution of both the aplasia and fungal infection. DISCUSSION: This case illustrates as proof-of-concept the efficacy and safety of selected CD34-stem cell boost from prior donor as salvage treatment of prolonged cytopenias after CAR-T cell therapy.

An integrative skeletal and paleogenomic analysis of stature variation suggests relatively reduced health for early European farmers.

Human culture, biology, and health were shaped dramatically by the onset of agriculture ∼12,000 y B.P. This shift is hypothesized to have resulted in increased individual fitness and population growth as evidenced by archaeological and population genomic data alongside a decline in physiological health as inferred from skeletal remains. Here, we consider osteological and ancient DNA data from the same prehistoric individuals to study human stature variation as a proxy for health across a transition to agriculture. Specifically, we compared "predicted" genetic contributions to height from paleogenomic data and "achieved" adult osteological height estimated from long bone measurements for 167 individuals across Europe spanning the Upper Paleolithic to Iron Age (∼38,000 to 2,400 B.P.). We found that individuals from the Neolithic were shorter than expected (given their individual polygenic height scores) by an average of −3.82 cm relative to individuals from the Upper Paleolithic and Mesolithic (P = 0.040) and −2.21 cm shorter relative to post-Neolithic individuals (P = 0.068), with osteological vs. expected stature steadily increasing across the Copper (+1.95 cm relative to the Neolithic), Bronze (+2.70 cm), and Iron (+3.27 cm) Ages. These results were attenuated when we additionally accounted for genome-wide genetic ancestry variation: for example, with Neolithic individuals −2.82 cm shorter than expected on average relative to pre-Neolithic individuals (P = 0.120). We also incorporated observations of paleopathological indicators of nonspecific stress that can persist from childhood to adulthood in skeletal remains into our model. Overall, our work highlights the potential of integrating disparate datasets to explore proxies of health in prehistory.

Maternal mirror syndrome with foetal hydrops due to isoimunization by anti-KPa antibodies: A case report and narrative literature review.

We present a rare case of mirror syndrome due to anti-Kpa antibodies, which can be difficult to identify with routine screening tests.

Hemophagocytic lymphohistiocytosis/macrophage activation syndrome (HLH/MAS) following treatment with tisagenlecleucel.

Chimeric antigen receptor (CAR) T cell-related HLH/MAS is an unusual manifestation of severe cytokine release syndrome (CRS) with poor prognosis and a challenging diagnosis. The establishment of specific diagnosis criteria is essential, and the combination of several techniques for CAR T-cell follow-up, allows a more precise management of this complication.

Clinical grade production of IL-15 stimulated NK cells for early infusion in adult AML patients undergoing haploidentical stem cell transplantation with post-transplant cyclophosphamide.

BACKGROUND: Allogeneic stem cell transplantation is the treatment of choice for acute myeloid leukemia (AML) patients. Unmanipulated haploidentical transplantation (Haplo-HSCT) is commonly used for those AML patients who need a timely transplant and do not have a suitable matched donor, but relapse rates are still high, and improvements are needed. Adoptive immunotherapy using natural killer cells (NK cells) could be a promising tool to improved Haplo-HSCT but, to date, no optimal infusion and manufacturing protocols have been developed. STUDY DESIGN AND METHODS: In this study, we describe a quick and reproducible protocol for clinical-grade production of haploidentical donor NK cells using double immunomagnetic depletion and enrichment protocol and overnight IL-15 stimulation. RESULTS: Thus, we have obtained 8 viable and functional NK cell products that have been safely infused to five AML patients undergoing unmanipulated Haplo-HSCT. DISCUSSION: Our results demonstrate the safety and feasibility of manufactured NK IL15 cells obtained from an adult allogeneic donor in the setting of haploidentical transplantation for AML patients.

Application of the French TMA Reference Center Score and the mortality in TTP Score in de novo and relapsed episodes of acquired thrombotic thrombocytopenic purpura at a tertiary care facility in Spain.

Acquired thrombotic thrombocytopenic purpura (aTTP) is still associated with a 10% to 20% death rate and its clinical course is characterized by recurrent episodes in up to 50% of cases. Over the last decade, mortality predicting models like the French TMA Reference Center Score and the Mortality In TTP Score (MITS) have been developed in an attempt to personalize treatment. The objective of the present study was to compare the results in both scores of de novo and relapsed aTTP episodes. For such purpose, a total of 29 episodes of aTTP (16 de novo and 13 relapses) were analyzed. All patients were homogeneously diagnosed and treated. First episodes had a higher score in both models in comparison with relapsed aTTP, (MITS median, 1 r: 1-4 vs 0 r: 1-2, P = .038 and French TMA Reference Center Score median, 2 r: 1-3 vs 1 r: 0-1, P = .006). The prevalence of neurological symptoms was significantly higher in the first episodes (P = .001) and patients >60 years old were more common in this group (P = .013), which may have been related to the results. Platelet count at presentation was higher in recurrences than in the first disease episode (P = .016) and ADAMTS13 activity <5% was more frequent in the last group (P = .016). There was no significant difference in the rate of refractoriness or exacerbations. In conclusion, first aTTP episodes had a higher probability of short-term mortality compared to relapsed aTTP episodes according to the MITS and French TMA Reference Center Score.

Exploring the expression and functionality of the rsm sRNAs in Pseudomonas syringae pv. tomato DC3000.

The Gac-rsm pathway is a global regulatory network that governs mayor lifestyle and metabolic changes in gamma-proteobacteria. In a previous study, we uncovered the role of CsrA proteins promoting growth and repressing motility, alginate production and virulence in the model phytopathogen Pseudomonas syringae pv. tomato (Pto) DC3000. Here, we focus on the expression and regulation of the rsm regulatory sRNAs, since Pto DC3000 exceptionally has seven variants (rsmX1-5, rsmY and rsmZ). The presented results offer further insights into the functioning of the complex Gac-rsm pathway and the interplay among its components. Overall, rsm expressions reach maximum levels at high cell densities, are unaffected by surface detection, and require GacA for full expression. The rsm levels of expression and GacA-dependence are determined by the sequences found in their -35/-10 promoter regions and GacA binding boxes, respectively. rsmX5 stands out for being the only rsm in Pto DC3000 whose high expression does not require GacA, constituting the main component of the total rsm pool in a gacA mutant. The deletion of rsmY and rsmZ had minor effects on Pto DC3000 motility and virulence phenotypes, indicating that rsmX1-5 can functionally replace them. On the other hand, rsmY or rsmZ overexpression in a gacA mutant did not revert its phenotype. Additionally, a negative feedback regulatory loop in which the CsrA3 protein promotes its own titration by increasing the levels of several rsm RNAs in a GacA-dependent manner has been disclosed as part of this work.

Complete RH and Kell matching related to low alloimmunisation risk in sickle cell disease: prevalence and risk factors of alloimmunisation in a Spanish Tertiary Care National Reference Centre.

BACKGROUND: Red blood cell (RBC) transfusion remains an essential part of sickle cell disease (SCD) management but it can lead to alloimmunisation, with an increased incidence in this population. Prevention is based on RBC antigen phenotype matching, with complete RH and Kell matching being a standard of care. MATERIALS AND METHODS: We performed a retrospective, single-centre study analysing alloimmunisation prevalence and risk factors in a cohort of transfused SCD patients. RESULTS: Eighty-seven patients (96.5% of paediatric age) received 1,781 RBC units (RBCu). Complete RH and Kell matched RBCu represented a median of 100% among total transfusions per patient. Of the 87 patients, 52 (59.8%) underwent chronic transfusion therapy, whereas 35 (40.2%) were only episodically transfused. Seven patients were alloimmunised (8.4%) and eleven antibodies were detected (alloimmunisation rate: 0.62/100 units transfused). 54.6% of these antibodies corresponded to RH-Kell despite the high accomplishment of the RH-Kell matching transfusion protocol. Alloimmunised patients had a median of 90.9% RH-Kell matched transfusions vs 100% in non-alloimmunised patients, but no statistical differences were observed (p=0.127). Number of transfused RBCu (19 vs 7; p=0.023), number of episodic RBCu (8 vs 2; p=0.006), episodic to chronic RBCu ratio (0.57 vs 0.09; p=0.045), number of vaso-occlusive crises (VOC) (4 vs 2; p=0.011), and autoantibody presence (57.1 vs 0%; p<0.001) were all statistically related to alloimmunisation. DISCUSSION: We report a low alloimmunisation prevalence (8.4%) related to a high grade of RH-Kell matching. However, deviation from 100% translates into alloimmunisation, with >50% of alloantibodies corresponding to RH-Kell. Alloimmunisation risk increases with transfusion burden, particularly during acute complications, and in patients with a higher number of VOC, probably reflecting underlying inflammation and disease severity. Further studies will be needed to elucidate additional risk factors and help prevent alloimmunisation in these patients.

Active or Passive Commuter? Discrepancies in Cut-off Criteria among Adolescents.

Active commuting to school has health implications for young people. Previous research has shown the need to consistently define the concept of "active commuter", given that assessment as well as comparison between studies may be hindered by current discrepancies in frequency criteria. Using a sample of 158 Spanish students (12th-13th grade, 60.8% girls), the current study aimed to compare several cut-off criteria to rigorously identify the frequency of weekly active trips to school in order to categorize adolescents as active or passive commuters, and to analyze whether the threshold living distance to school is associated with the different trip cut-off criteria. Percentages of active commuters ranged from 75% to 88.6%, varying significantly depending on the cut-off criteria (5-10 active trips/week) used. The results also support the need to be stricter in the selection of a cut-off criterion when the distance to the school becomes shorter. Our findings highlight the importance of following a standard criterion to classify individuals as active or passive commuters, considering the characteristics of the context in which each study is conducted.

Factors predicting peripheral blood progenitor cell mobilization in healthy donors in the era of related alternative donors: Experience from a single center.

BACKGROUND: Poor mobilization results are unexpected after G-CSF-induced peripheral blood stem cell collection in healthy donors. However, 2%-5% of the donors are poor mobilizers. Factors predicting CD34+-cell yield after mobilization in related alternative donors are still poorly known. PATIENTS AND METHODS: Baseline characteristics and efficacy results of G-CSF induced mobilization of 159 adult healthy donors in our institution from 2008 to 2016 were retrospectively analyzed. All donors received 10 µg/kg of G-CSF once a day subcutaneously for 4 days. Leukapheresis started on the 5th day of G-CSF treatment. Donors were classified as poor mobilizers if they had less than 20 000 CD34 + cell/mL peripheral blood count in the 5th day of G-CSF treatment or if they needed three or more leukapheresis for graft collection. RESULTS: Age, weight, and platelet count before and after mobilization were significantly different between poor and good mobilizers. Poor mobilizers (n = 16) were older (50.6 vs 41.7 years, P = 0.002), weight lower (64 vs 75 kg, P = 0.00) and showed a lower platelet count before (199.5 vs 219.0 × 109 /L, P = 0.03) and after (192.5 vs 206 × 109 /L, P = 0.019) mobilization. In the multivariate analysis only the 30% of the variability of mobilization was explained by the model (sensitivity 80%, specificity 70%). CONCLUSION: In this cohort of healthy donors in a single institution, older age, less weight, and lower platelet count was associated with poorer mobilization. With clinical and analytic factors it is not possible to predict more than 30% of the variability. Further studies are needed to investigate new variables.

Mononuclear cell collection for extracorporeal photopheresis by using the "off-line" system: A comparative study between COBE Spectra and Spectra Optia devices.

BACKGROUND: Extracorporeal photopheresis (ECP) is an efficient and established therapy to treat acute and chronic graft vs host disease (GVHD). Using an "off-line" method, the first step (mononuclear cell [MNC] collection) is decisive, as long as a high MNC yield and purity in the collected product is desirable. Two "off-line" devices were compared: the COBE Spectra and the Spectra Optia (Terumo BCT), using both continuous and intermittent protocols. PATIENTS AND METHODS: Twelve patients with GvHD (7 acute/5 chronic) were enrolled between June 2014 and May 2015 and were alternatively assigned for each procedure to either the COBE Spectra or the Spectra Optia cell separator. Patients characteristics and procedure/product parameters were analyzed. RESULTS: Two hundred procedures (100 per device) were included. The Spectra Optia system showed higher total nucleated cells and MNC collection efficiencies (18.6(10.2-29.7) vs 7.9(4.1-14.8)% and 43.6(20.3-59.5) vs 23.3(11.4-37.1)%, P < .001) and monocyte and lymphocyte collection efficiencies (55.2(17.7-83.2) vs 22.8(9-38.9)% and 38.3(26.7-53.4) vs 22.2(9-38.9)%, respectively, P < .001). Absolute platelet loss (PL) and PL per liter of blood processed were significantly lower in the Spectra Optia group (22.9(18.3-28.1) vs 33.6(26.5-41.1)%, P < .001 and 3.7(3.1-4.5) vs 4.3(3.5-4.2)%, P = .01, respectively). However, granulocyte contamination was higher (4.5(1.3-36) vs 1.2(0.4-5.7)%, P < .001) and a higher product haematocrit was obtained with the Spectra Optia (1(0.5-1.6) vs 0.3(0.2-0.5)%, P < .001), without an impact on irradiation time. CONCLUSIONS: In our study, Spectra Optia proved to be safe and effective in collecting MNC with high yield and purity for ECP in GvHD.

Busulfan-based myeloablative conditioning regimens for haploidentical transplantation in high-risk acute leukemias and myelodysplastic syndromes.

BACKGROUND: High-risk acute leukemia (AL) and myelodysplastic syndrome (MDS) remain a therapeutic challenge. Unmanipulated haploidentical-related donor transplantation based on a myeloablative conditioning regimen (HAPLO-MAC) and post-transplant cyclophosphamide (PT-Cy) as prophylaxis against graft vs host disease (GvHD) is now a promising rescue strategy that could become universally available. OBJECTIVE: To evaluate the results of HAPLO-MAC with PT-Cy in patients with AL and MDS reported to the Haploidentical Transplantation Subcommittee of the Spanish Group for Hematopoietic Transplantation (GETH). PATIENTS AND METHODS: We report our multicenter experience using an IV busulfan-based HAPLO-MAC regimen and PT-Cy for treatment of 65 adults with high-risk AL and MDS. RESULTS: Engraftment was recorded in 64 patients (98.5%), with a median time to neutrophil and platelet recovery of 16 and 27 days, respectively. The cumulative incidence of grade II-IV acute GvHD and chronic GvHD was 28.6% and 27.5%, respectively. After a median follow-up of 31 months for survivors, the cumulative incidence of non-relapse mortality and relapse at 2 years was 18.8% and 25%, respectively. Estimated 30-month event-free survival and overall survival were 56% and 54.5%, respectively. CONCLUSION: HAPLO-MAC comprising an IV busulfan-based conditioning regimen enabled long-term disease control with acceptable toxicity in high-risk AL and MDS.