Background: Patients suffering from hemolytic anemia, thrombocytopenia and organ damage may suffer from microangiopathic anemia or also called thrombotic microangiopathy (TMA). This condition is caused by many different pathogenic mechanism and is always a life-threatening due to vessel occlusion in vital organs. Rapid and careful workup is mandatory to identify the cause of TMA. To identify patients suffering from immune mediated thrombotic thrombocytopenic purpura (iTTP) ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) measurement is mandatory. All patients with ADAMTS13 activity below 10 IU/dL are assigned to the diagnosis iTTP and need urgent targeted treatment. Meanwhile caplacizumab – an anti-von Willebrand factor humanized single-variable-domain immunoglobulin fragment – is approved for the treatment of iTTP. Patients with TMA and ADAMTS13 activity>10 IU/dL can be assigned to other forms of TMA such as hemolytic uremic syndrome (HUS), complement mediated TMA (cmTMA) - previously assigned to the term atypical HUS (aHUS) - or TMA secondary to underlying diseases such as autoimmune disorders, cancer, or infectious diseases. Complement inhibition with C5 targeted treatment, such as eculizumab or ravulizumab, is approved for the treatment of cmTMA. Even more challenging may be the differential diagnosis in pregnancy, in cancer patients with complex medication and the need to rule out conditions imitating TMA such as Evans syndrome, intoxication, infection or severe vitamin B12 deficiency. Summary: Identifying TMA and defining the pathophysiology of TMA is urgently necessary in patients with thrombocytopenia, hemolytic anemia with or without obvious organ damage. Key message: ADAMTS13 testing is the most important specific test to classify thrombotic microangiopathy.

Background: The molecular basis of the RHD gene found in serologically D– phenotype individuals differs with race/ethnicity. Therefore, we aimed to develop a single-tube multiplex PCR-sequence specific primer (multiplex PCR-SSP) to detect RHD variant alleles commonly found in serologically D– phenotype individuals in a Thai population. Study design and methods: In total, 205 blood samples with a serologically D– phenotype were tested using a single-tube multiplex PCR-SSP targeted RHD exons 1, 4, 7, 10, and c.1227G>A in RHD exon 9 in combination with a hybrid Rhesus box, and results were confirmed by direct DNA sequencing. Results: In a single-tube multiplex PCR-SSP, three patterns of amplified RHD exons were observed: total deletion of the RHD gene, Asian-type DEL, and RHD-CE-D hybrid. The allele frequencies of RHD*01N.01, RHD*01EL.01, and RHD-CE-D hybrid were 83.4%, 12.9%, and 2%, respectively. All of the Asian-type DEL samples present the RHCE*C/E allele (predicted RhCE phenotype: C/E+). Conclusion: This study successfully established a simple and reliable molecular diagnostic platform for analyzing RHD variant alleles commonly found in serologically D– phenotype individuals in a Thai population. This technique could enable broader RHD*01EL.01 (Asian-type DEL) analyses in high-prevalence areas such as Thailand and other countries in East and Southeast Asia, serving as an example for blood bank routine settings.

Background: Chloride intracellular channel 1 (CLIC1) is expressed in platelets, but CLIC1 action in these cells has not been clarified. Methods: CLIC1 function was probed in platelets in vitro as well as in a mouse dorsal skin fold chamber model in vivo to assess thrombus formation. Results: Co-localization of CLIC1 with F-actin was detected in lamellipodia of platelets, which relocate CLIC1 to their cell surface in an integrin-dependent manner. Treatment with the CLIC1 inhibitor IAA94 hindered CLIC1 relocation to the platelet membrane, diminished platelet aggregation and reduced integrin αIIbβ3 activation. Injecting mice with IAA94 delayed vaso-occlusion in a mouse model of photo-chemical thrombus formation in vivo. The role of CLIC1 for platelet activation could be reproduced in platelet concentrates, which even after prolonged storage expressed minimal CLIC1 on the platelet surface unless platelets were exposed to an activating stimulus. Conclusion: These data show that CLIC1 is regulated by adhesive interactions with integrin ligands that cause CLIC1 to relocate to the platelet cell surface. This process in turn appears to be relevant for integrin-mediated functions involved in platelet thrombus formation in vitro and in vivo. Moreover, CLIC1 has the potential to be a useful marker for monitoring the activation state of platelet concentrates during prolonged storage.

Background: Chimeric antigen receptor (CAR)-modified T cells have shown remarkable results for the treatment of selected hematological malignancies, but recapitulating these results in solid cancers has been a major challenge. In this review, we discuss lessons learned from recent clinical trials, mechanisms of tumor immune evasion in solid cancers and strategies to alleviate these effects through advanced engineering strategies to augment the efficacy of CAR-T cell products. Summary: Despite early signs of clinical efficacy, CAR-T cells have repeatedly failed to achieve curative responses in solid cancers. While a major bottleneck remains the availability of tumor-specific antigens, recent studies suggest that conventional CAR-T cell products are not sufficiently well equipped to deal with the challenges encountered in the context of solid cancers. Various approaches to augment the potency and clinical efficacy of CAR-T cells are currently being evaluated, but the majority is yet to reach clinical trials. Moving forward, promising approaches include the use of next-generation CAR-T cell products, targeting physical barriers or cellular components within the tumor microenvironment (TME), and leveraging advanced engineering strategies to shield immune cells from the TME. These techniques aim to address current challenges and significantly improve the effectiveness of CAR-T cell therapies in treating solid tumors. Key Messages: Extensive research efforts have been made to understand the underlying mechanisms impeding curative treatment outcomes for CAR-T cell therapy in solid tumors. Early clinical trials, predominantly using second-generation CAR-T cell products, have shown promising signs of early clinical efficacy in the absence of consistent curative effects. Based on these data, it has become apparent that strategies to augment the efficacy of CAR-T cell therapy need to be implemented. Various approaches are currently being developed and are expected to enter clinical trials in the near future.

Introduction: Crystalloid and colloid solutions commonly used in intensive and perioperative care can affect haemocoagulation status. This in vitro study assessed the impact of Plasma-Lyte, albunorm 5%, and Gelaspan 4% solutions on primary and secondary haemostasis using rotational thromboelastometry and platelet function analyser. Methods: In this prospective study, we examined blood samples from 20 healthy volunteers using rotational thromboelastometry and platelet function analyser. Simultaneously, we analysed the blood samples subjected to 10% dilution using Plasma-Lyte, albunorm 5%, and Gelaspan 4% solutions. Results: Compared to controls, Plasma-Lyte shortened EXTEM-CT (p = 0.005) and reduced FIBTEM-MCF (p = 0.017). albunorm 5% prolonged EXTEM-CFT (p = 0.001), decreased EXTEM-alpha (p < 0.001) and MCF in EXTEM, INTEM, and FIBTEM tests (p < 0.001, p = 0.038, p = 0.001, respectively), along with MCE in the PLTEM test (p < 0.001). Gelaspan 4% also prolonged EXTEM-CFT (p < 0.001), decreased EXTEM-alpha (p < 0.001) and MCF in EXTEM, INTEM, and FIBTEM tests (p < 0.001, p < 0.001, p = 0.009, respectively), along with MCE in the PLTEM test (p < 0.001). Gelaspan 4% also reduced EXTEM-CT (p = 0.021). All solution prolonged CT in PFA Col/ADP (p = 0.003 for Plasma-Lyte, p < 0.001 for albunorm and Gelaspan) and albunorm 5% also prolonged CT in Col/Epi (p = 0.003). Conclusion: Plasma-Lyte had the least effect on secondary haemostasis, whereas albunorm 5% had the least effect among colloids. Gelaspan 4% adversely affected the propagation phase of coagulation, maximal strength and elasticity of the coagulum, and the level of functional fibrinogen. All solutions adversely affected platelet function in primary haemostasis, with Plasma-Lyte showing the least effect.

Introduction: Robotic-assisted surgery is increasingly performed in various surgical disciplines demonstrating improved oncological and functional outcomes compared to conventional surgery. Objective: Unclear is how robotic-assisted surgery affects perioperative anemia and the need for blood products. Methods: In this case-control study, 15,009 matched patient pairs undergoing urological, visceral, or thoracic surgery were included. Pairwise comparisons between robotic-assisted surgery, laparoscopic surgery, and open surgery were performed with propensity score matching. Results: Robotic-assisted surgery compared to open surgery was associated with a risk reduction of allogeneic red blood cell transfusion by RR: 0.32 (95% CI: 0.27–0.37) and a limited reduction of perioperative hemoglobin (perioperative hemoglobin difference of 0.40 g/dL, 95% CI: 0.31–0.49). Robotic-assisted surgery was associated with a shorter length of hospital stay by 4.29 days (95% CI: 3.74–4.84). Compared to laparoscopic surgery, robotic-assisted surgery had no significant effect on red blood cell transfusions (RR: 0.94, 95% CI: 0.75–1.18), perioperative hemoglobin (0.27 g/dL, 95% CI: 0.16–0.38), or length of hospital stay 0.53 days (95% CI: −0.14–1.19). Conclusions: Robotic-assisted and laparoscopic procedures are associated with reduced blood transfusions compared to open surgery and, thus the advancement of minimally invasive procedures constitutes an important measure to improve patient outcomes.

Background: Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with a high relapse rate and still limited therapeutic options. Natural killer (NK) cell-based immunotherapy has the potential to improve outcomes for patients with AML. Summary: Recent preclinical studies and early-stage clinical trials aim to enhance the intrinsic anti-leukemic properties of NK cells by selectively targeting AML cells with chimeric antigen receptors (CARs). Furthermore, NK and CAR-NK cells can be combined with other therapeutic modalities or engineered further to overcome the immunosuppressive microenvironment, and treatment resistance of AML blasts and leukemia-initiating cells (LIC). Key Messages: In this review, we summarize preclinical studies with cytokine-stimulated or genetically engineered NK cells derived from different cell sources for the treatment of AML and their translation into early-phase clinical trials. We also provide an overview of promising recent developments toward innovative NK cell-based therapies that may be implemented in the near future.

Background: Immunocompromised individuals are at major risk for severe infectious complications. This is particularly relevant in the context of allogeneic hematopoietic stem cell transplantation (allo-HCT) – a treatment modality that has proven curative for a range of malignant and nonmalignant hematological diseases. However, transplant-associated immune suppression leaves patients susceptible to infectious complications from viruses such as cytomegalovirus (CMV), adenovirus (AdV), Epstein-Barr virus (EBV), and BK virus (BKV). While pharmacological agents are available to prevent and/or treat some of these viruses, they can be associated with significant toxicities and are often ineffective. To circumvent these issues, several groups have explored the clinical potential of adoptively transferred virus-specific T cells (VSTs) to prevent/treat virus-associated complications after allo-HCT or solid organ transplantation (SOT) and this review will provide an overview of these endeavors. Summary: This review will focus on the progress that has been made over the past 30 years in the field of nonengineered VST manufacturing technologies and will summarize the clinical experience with VSTs, primarily in the posttransplant setting. Key Messages: Over the last 3 decades, adoptively transferred VSTs – both HCT donor and third party-derived – have been tested in numerous single and multicenter clinical trials and have unequivocally proven to be safe and associated with clinical activity.

Background: Immunotherapies in general, and cellular immunotherapies, in particular are becoming increasingly integrated into current personalized cancer treatment, though still facing some obstacles in the allogeneic hematopoietic stem cell transplantation (HSCT) setting. Summary: The concept of isolating immune effector cells, expanding their numbers, enhancing their anticancer capabilities by modifying them without increasing their alloreactive potential is the mainstay of adoptive cellular immunotherapy after allogeneic HSCT. In this context, cytokine-induced killer (CIK) cells, a polyfunctional heterogenous population of conventional T cells, natural killer (NK) cells, and T-NK cells capable of using T cell and NK cell-like cytotoxicity mechanisms against a various cancers, showed minimal alloreactivity in pediatric and adult patients allografted for hematological malignancies. Furthermore, CIK cells have already shown compatibility with chemotherapy, different kinds of immune checkpoint inhibitors, epigenetic drugs, antibody-targeted therapies, and recently with chimeric antigen receptor-engineering techniques. Key Messages: Hence, CIK cell therapy represents a unique platform for adoptive cell immunotherapies, guiding innovative treatment approaches from preclinical research to future clinical trials for cancer patients with yet unmet medical needs. In this context, the allogeneic HSCT setting provides an alternative source for safe and efficient adoptive allogeneic CIK cell strategies against a variety of cancers.

<b><i>Objectives:</i></b> This study aimed to evaluate the distribution of genotypes and iron metabolism imbalance in transfusion-dependent thalassemia patients. <b><i>Methods:</i></b> Genotype analysis was conducted on 84 thalassemia patients requiring transfusion, and retrospective analysis of iron overload was performed on 48 transfusion-dependent patients. <b><i>Results:</i></b> Among the 84 thalassemia cases requiring transfusion, six mutations of α-thalassemia were identified, including --<sup>SEA</sup>, α<sup>CS</sup>, -α<sup>3.7</sup>, -α<sup>4.2</sup>, α<sup>QS</sup>, and α<sup>WS</sup>. Nine mutations of β-thalassemia were also found, with CD41-42 being the most common. Of the 48 transfusion-dependent patients, 40 (83.3%) had iron overload with serum ferritin (SF) levels above 1,000 ng/mL. The recent SF level was lower than 3 years ago, but the overall ferritin level remains elevated. <b><i>Conclusions:</i></b> β-thalassemia was the predominant type among transfusion-dependent thalassemia patients, with CD41-42/-28, CD41-42/IVS-II-654, and CD17/IVS-II-654 being the most common genotypes. Proper blood transfusion and iron chelation therapy are essential for managing transfusion-dependent thalassemia. While some patients show a reduction in SF levels after 3 years of treatment, there are still individuals who exhibit elevated levels necessitating ongoing management.

<b><i>Background:</i></b> Autoimmune hemolytic anemia (AIHA) is a rare disease due to increased destruction of erythrocytes by autoantibodies, with or without complement activation. <b><i>Summary:</i></b> AIHA is usually classified in warm AIHA (wAIHA) and cold agglutinin disease (CAD), based on isotype and thermal amplitude of the autoantibody. The direct antiglobulin test (DAT) or Coombs test is the cornerstone of AIHA diagnosis, as it is positive with anti-IgG in wAIHA, and with anti-C3d/IgM antisera plus high titer cold agglutinins in CAD. Therapy is quite different, as steroids and rituximab are effective in the former, but have a lower response rate and duration in the latter. Splenectomy, which is still a good option for young/fit wAIHA, is contraindicated in CAD, and classic immunosuppressants are moving to further lines. Several new drugs are increasingly used or are in trials for relapsed/refractory AIHAs, including B-cell (parsaclisib, ibrutinib, rilzabrutinib), and plasma cell target therapies (bortezomib, daratumumab), bispecific agents (ianalumab, obexelimab, povetacicept), neonatal Fc receptor blockers (nipocalimab), and complement inhibitors (sutimlimab, riliprubart, pegcetacoplan, iptacopan). Clinically, AIHAs are highly heterogeneous, from mild/compensated to life-threatening/fulminant, and may be primary or associated with infections, neoplasms, autoimmune diseases, transplants, immunodeficiencies, and drugs. Along with all these variables, there are rare forms like mixed (wAIHA plus CAD), atypical (IgA or warm IgM driven), and DAT negative, where the diagnosis and clinical management are particularly challenging. <b><i>Key Messages:</i></b> This article covers the classic clinical features, diagnosis, and therapy of wAIHA and CAD, and focuses, with the support of clinical vignettes, on difficult diagnosis and refractory/relapsing cases requiring novel therapies.

<b><i>Background:</i></b> The clinical success of autologous adoptive cell therapy (ACT) is substantial but wide application is challenged by the quality and quantity of the patient’s immune cells and the need for personalized manufacturing processes. Induced pluripotent stem cells (iPSCs) can be differentiated into immune effectors and thus provide an alternative, allogeneic cell source for ACT. Here, we compare iPSC-derived immune effectors to their PBMC-derived counterparts and review iPSC-derived ACT products currently under preclinical and clinical development. <b><i>Summary:</i></b> iPSC-derived T cells, NK cells, macrophages, and neutrophils largely mimic their PBMC-derived counterparts in terms of cell-surface marker expression and cytotoxic effector functions. iPSC-derived immune effectors can be engineered with chimeric antigen receptors and other activating receptors to redirect their cytotoxic potential specifically to tumor-associated antigens (TAAs). However, several differences between iPSC- and PBMC-derived immune effectors remain and have inspired additional engineering strategies to enhance the antitumor capacity of iPSC-derived immune effectors. <b><i>Key Messages:</i></b> iPSCs can be engineered to facilitate the generation of immune effectors with homogenous specificity for TAAs and enhanced effector functions. TAA-specific and functionally enhanced iPSC-derived T and NK cells are currently undergoing clinical evaluation in phase 1 trials. Engineered iPSC-derived macrophages and neutrophils are in preclinical development.

<b><i>Introduction:</i></b> Red blood cells (RBCs) undergo progressive biochemical and morphological changes during storage, collectively called storage lesion. The quality of red cell concentrates (RCCs) is typically assessed by quantifying hemolysis. An assessment of morphological changes, associated with low quality RBCs, could give an additional indication of the safety and efficacy of the concentrates. The current standard for determining morphological changes is a manual, laborious, and subjectively biased microscopic process that limits the number of cells that can be examined. When using alternative methods like flow cells, flow and shear-induced morphologies affecting especially stomatocyte morphologies must be taken into account. We already established an automated flow morphometric RBC analysis system as an alternative to manual microscopic evaluation. The goal of the present work is to obtain a robust, automated, morphology-related signal (lesion index) quantifying RBC storage lesion in a laminar flow channel under conditions similar to stasis that is not affected by shear-induced reversible morphology changes. <b><i>Methods:</i></b> We use a convolutional neural network (CNN) for high throughput classification of RBCs. We analyzed the morphological changes of 5 RCCs over a period of 12 weeks and classified RBC morphologies, including such that are degradation-induced and reversible. We introduce a lesion index to denote the percentage of irreversible spherical morphologies, known to reduce the post-transfusion survival of erythrocytes. We further addressed shear-induced stomatocyte morphologies in laminar flow and whether these affect CNN-based RBC classification. <b><i>Results:</i></b> Our flow morphometry system achieves a high-resolution classification comprising nine morphological classes with an excellent overall accuracy of 92% and F<sub>1</sub> scores between 84% and 97%. We generate strong evidence that the morphological lesion index can predict the hemolysis level in RCCs during storage. The power of this new classification technique allowed it, for the first time, to detect and measure the lateral concentration gradient of stomatocytes in a conventional flow chamber. Importantly, we show that reversible shear rate-induced morphologies, typical for microfluidic systems, bear no influence on the lesion index. <b><i>Conclusion:</i></b> Flow morphometry combined with evaluation by a CNN allows to reliably assess RBC storage lesion and thus concentrate quality. Additionally, this method reduces the need for complex laboratory procedures.

Background: Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by intravascular hemolysis (IVH) due to diminished or absent inhibition of the complement system because of deficient expression of cell-anchored complement regulating surface proteins. IVH leads to heterogeneous symptoms such as anemia, abdominal pain, dyspnea, fatigue and increased rates of thrombophilia. Inhibitors of the terminal Complement cascade can reverse IVH leading to a significant reduction of disease burden such as thrombembolic events and also mortality. Summary: Therapeutic inhibitors of the terminal complement cascade such as eculizumab or ravulizumab significantly improve overall survival through IVH-inhibition. However, not all patients experience complete disease control with normalization of hemoglobin levels and absolute reticulocyte counts (ARC) under terminal complement inhibition as a significant part of patients develop extravascular hemolysis (EVH). EVH can be clinically relevant causing persistent anemia and fatigue. New proximal complement inhibitors (CI) mainly targeting complement component C3 or factors of the amplification pathway such as pegcetacoplan, danicopan, and iptacopan became available and are meanwhile approved for marketing. Additional complement-inhibiting strategies are under clinical development. A switch from terminal to proximal CI in patients with significant EVH can achieve hemoglobin and ARC normalization and significant improvement in quality of life (QoL). Additional approvals of proximal CI agents for the treatment of hemolytic PNH in the first line are available for pegcetacoplan and iptacopan. So far, no evidence-based algorithm is available for decision-making in first-line treatment of which type of drug should be used for individual patients. Key Messages: Terminal CIs in hemolytic PNH patients can block IVH and have led to a dramatically improved survival. Proximal CIs ameliorate anemia and improve QoL in patients with relevant EVH. However, more (real-world) data are needed to demonstrate long-term improvement in all patients with hemolytic PNH, especially those under first-line treatment with proximal CI.