Intelligent Image-Activated Cell Sorting.

A fundamental challenge of biology is to understand the vast heterogeneity of cells, particularly how cellular composition, structure, and morphology are linked to cellular physiology. Unfortunately, conventional technologies are limited in uncovering these relations. We present a machine-intelligence technology based on a radically different architecture that realizes real-time image-based intelligent cell sorting at an unprecedented rate. This technology, which we refer to as intelligent image-activated cell sorting, integrates high-throughput cell microscopy, focusing, and sorting on a hybrid software-hardware data-management infrastructure, enabling real-time automated operation for data acquisition, data processing, decision-making, and actuation. We use it to demonstrate real-time sorting of microalgal and blood cells based on intracellular protein localization and cell-cell interaction from large heterogeneous populations for studying photosynthesis and atherothrombosis, respectively. The technology is highly versatile and expected to enable machine-based scientific discovery in biological, pharmaceutical, and medical sciences.

[Novel disease entities due to antiplatelet factor 4 antibodies: HIT, aHIT, and VITT].

Antiplatelet factor 4 (PF4) antibodies, also known as anti-PF4/heparin complex antibodies, are measured to diagnose heparin-induced thrombocytopenia (HIT). In HIT, anti-PF4 antibodies induced by heparin exposure cause thrombocytopenia and thrombosis. However, in recent years, autoimmune HIT (aHIT) that develops without heparin exposure has been getting attention. In 2021, anti-PF4 antibodies were reported to cause the fatal vaccine-induced immune thrombotic thrombocytopenia (VITT) that developed after adenoviral vector vaccination for COVID-19. HIT, aHIT, and VITT are considered to be caused by anti-PF4 antibodies, and their pathological conditions are similar. However, they have different levels of severity, and the detection sensitivity of their antibodies varies depending on the assay. Herein, we review three pathologies, namely, HIT, aHIT, and VITT, associated with anti-PF4 antibodies.

High lymphocyte counts before antithymocyte globulin administration predict acute graft-versus-host disease.

Antithymocyte globulin (ATG) reduces severe acute and chronic graft-versus-host disease (GVHD) in allogeneic peripheral blood stem cell transplantation (PBSCT). However, risk factors for severe acute GVHD in PBSCT using ATG remain to be determined. We conducted a single-center, retrospective study to analyze the association of acute GVHD requiring systemic corticosteroid (SC-aGVHD) with absolute lymphocyte counts (ALC) before the administration of ATG or conditioning in 53 patients with HLA-matched PBSCT using low-dose thymoglobulin (2 mg/kg) after myeloablative conditioning. The cumulative incidence of SC-aGVHD was 17.0% and ALC before ATG were significantly higher in patients with SC-aGVHD compared to that in patients without it (median, 0.15 × 109/L vs 0.06 × 109/L, P = 0.047). The cumulative incidence of SC-aGVHD was significantly higher in patients with high ALC before ATG (≥ 0.15 × 109/L) than in those with low ALC (38.5% vs 10.0%, P = 0.016). Non-relapse mortality (NRM) was also significantly higher in the high ALC before ATG group than the low ALC before ATG group (2-year NRM: 23.9% vs 6.0%, P = 0.048), leading to worse survival (2-year overall survival: 69.2% vs 83.5%, P = 0.039). Our study suggested that high ALC before ATG is a risk factor for SC-aGVHD.

The role of anti-complement factor H antibodies in the development of atypical haemolytic uremic syndrome: a possible contribution to abnormality of platelet function.

Atypical haemolytic uremic syndrome (aHUS) is associated with complement system abnormality, such as production of complement factor H (CFH) autoantibodies. The growing evidence indicates complement overactivation on platelets is intimately involved in aHUS pathogenesis, besides endothelial injury. We here showed plasma from patients with anti-CFH antibodies induced aggregation of washed platelets, while purified anti-CFH antibodies suppressed aggregation. This suggested anti-CFH antibody itself suppressed thrombosis, while other plasma factor including complement factors could overactivate the platelets, leading to aggregation, which augmented the notion the state of complement activation influenced by anti-CFH antibodies is important in the aggregation of platelets in aHUS.

Intelligent frequency-shifted optofluidic time-stretch quantitative phase imaging.

Optofluidic time-stretch quantitative phase imaging (OTS-QPI) is a powerful tool as it enables high-throughput (>10,000 cell/s) QPI of single live cells. OTS-QPI is based on decoding temporally stretched spectral interferograms that carry the spatial profiles of cells flowing on a microfluidic chip. However, the utility of OTS-QPI is troubled by difficulties in phase retrieval from the high-frequency region of the temporal interferograms, such as phase-unwrapping errors, high instrumentation cost, and large data volume. To overcome these difficulties, we propose and experimentally demonstrate frequency-shifted OTS-QPI by bringing the phase information to the baseband region. Furthermore, to show its boosted utility, we use it to demonstrate image-based classification of leukemia cells with high accuracy over 96% and evaluation of drug-treated leukemia cells via deep learning.

Limitations of deuterium-labeled internal standards for quantitative electrospray ionization mass spectrometry analysis of fatty acid metabolites.

RATIONALE: The electrospray ionization mass spectrometry (ESI-MS) methodology often shows poor ionization reproducibility in the analysis of biological samples. Therefore, normalization of the measured peak intensities is essential. It is believed that quantitative data with high reproducibility can be obtained by adding a constant amount of an internal standard (IS) material labeled with stable isotopes to each sample, thus allowing the correction of the quantitative value of the target compound by that of the IS. We investigated whether the presence or absence of a labeled IS improves the accuracy of these quantitative values. METHODS: Triple quadrupole MS coupled with liquid chromatography was used to analyze fatty acid metabolites in biological samples as target compounds. Two independent systems were used to provide a measure of reproducibility in two different laboratories. RESULTS: Data having poor reproducibility in the raw peak areas were efficiently normalized using the IS, but, crucially, the IS method using stable isotopes was not always necessary. In some cases, the reproducibility was relatively good even without using the IS. In a contaminant matrix, the MS response behavior of the target compound and its stable isotope-labeled material was complicated. Since ion suppression by matrix contaminants was dependent on the concentration of the target compound, the added amounts of the ISs were also important, Furthermore, an equivalent normalization effect was obtained by using a pooled quality control sample as an external standard, thus obviating the need for labeled IS samples, which are often expensive and sometimes not commercially available. CONCLUSIONS: Our results raise the question as to whether the quantitative method using stable-isotope-labeled ISs is always necessary and beneficial. However, the results obtained in this study cannot be generalized because only fatty acid metabolites were examined using ESI-MS and only a highly substituted deuterium-labeled IS was used.

[Innovative activated platelet detection technology by artificial intelligence].

Although antiplatelet drugs are widely used for the prevention and treatment of atherothrombosis, clinical tests capable of evaluating their efficacy have not been established. Focusing on platelet aggregates in blood, we announced the world's first basic technology, an intelligent Image-Activated Cell Sorter (iIACS), that can exhaustively and rapidly identify cells one-by-one using image analysis with high-speed imaging and deep learning to sort specific cells according to the analysis results. This technology has even enabled the detection of single platelets with a size of 2 µm in blood samples and the quantification of the proportion of platelet aggregates by size. Furthermore, by applying this technique, we discovered different morphological features of platelet aggregates formed by different types of agonists that activate platelets. Here, we discuss this application in the early diagnosis of thrombotic microangiopathy (TMA). In the early stage of TMA, consumptive thrombocytopenia is caused by excessive platelet activation. Therefore, the detection of excessive platelet aggregates can lead to early TMA diagnosis.

Hepatectomy in patients with inherited blood coagulation disorders can be safely performed with adequate coagulation factor replacement.

BACKGROUND: Haemophilia and von Willebrand disease (VWD) are common inherited bleeding disorders. Although patients with haemophilia or VWD have a high risk of hepatitis virus infection and hepatocellular carcinoma (HCC), little is known about the safety of liver resection in these patients. METHODS: From 2006 to 2016, there were seven hepatectomies with haemophilia A and three hepatectomies with VWD for malignant liver tumours at tertiary care hospitals in Japan and Switzerland. To evaluate the safety of hepatectomy in the blood coagulation disorder group (BD group), short-term outcomes in these patients were compared with 20 hepatectomies (non-BD group) for HCC, matched to a 2:1, operative procedure, period and background liver. RESULTS: Ten liver resections were performed in patients with haemophilia or VWD with administration of recombinant FVIII or VWF concentrate. Comparison of the BD vs non-BD group revealed no significant differences in the operative time (327 vs 407 minutes, P = 0.359), estimated blood loss (730 vs 820 mL, P = 0.748), red blood cell transfusion rate (10.0% vs 5.0%, P = 0.605), major complication rate (Clavien-Dindo grade III or IV) (10.0% vs 5.0%, P = 0.605) or mortality rate (0% vs 0%, P > 0.999). Additionally, the length of the postoperative hospital stay was similar between the two groups (13 vs 14 days, P = 0.296). CONCLUSION: Liver resection for treatment of HCC in patients with haemophilia or VWD can be safely performed through an appropriate perioperative administration protocol of coagulation factors.

Optofluidic time-stretch quantitative phase microscopy.

Innovations in optical microscopy have opened new windows onto scientific research, industrial quality control, and medical practice over the last few decades. One of such innovations is optofluidic time-stretch quantitative phase microscopy - an emerging method for high-throughput quantitative phase imaging that builds on the interference between temporally stretched signal and reference pulses by using dispersive properties of light in both spatial and temporal domains in an interferometric configuration on a microfluidic platform. It achieves the continuous acquisition of both intensity and phase images with a high throughput of more than 10,000 particles or cells per second by overcoming speed limitations that exist in conventional quantitative phase imaging methods. Applications enabled by such capabilities are versatile and include characterization of cancer cells and microalgal cultures. In this paper, we review the principles and applications of optofluidic time-stretch quantitative phase microscopy and discuss its future perspective.

Disseminated fusariosis emerged from prolonged local genital infection after cord blood transplantation.

Disseminated fusariosis (DF) is a rare life threatening fungal infection in immunocompromised hosts. We herein report a case of a fatal DF mimicking varicella zoster virus (VZV) infection that was emerged from a localized genital infection during cord blood transplantation (CBT) in a patient with severe aplastic anemia (SAA). The patient developed an ulcer following small painful vesicles mimics herpes simplex virus infection (HSV) on the glans penis before CBT, but a Fusarium species was identified. Despite administration of voriconazole, liposomal amphotericin B and granulocyte transfusion, the lesion was extended to extensive skin looked like VZV infection and the patients died after CBT. Massive fusarium infiltration was detected in multiple organs at autopsy. A genetic analysis of the mold identified Fusarium solani after his death. It should be noted that in patients with fusarium infection, localized and disseminated lesions of fusarium infection sometimes mimic HSV and VZV infections, which hampers an early diagnosis.

[Serum ALP elevation early after treatment is a predictor for response in myeloma patients treated with bortezomib].

Several studies have shown the predictive value of elevated serum alkaline phosphatase (ALP) level in multiple myeloma (MM) patients treated with bortezomib (BTZ). We assessed the relationship between changes in ALP levels during treatment and response. Thirty patients treated with BTZ in our hospital were analyzed retrospectively. Of the patients analyzed, 12 were male, median age was 62 years (42-86), and 11 had a history of prior chemotherapy. Eighteen patients were treated with BTZ alone or in combination with dexamethasone, while the others were treated with a combination regimen employing an alkylating agent. Seven patients had undergone autologous stem cell transplantation following BTZ therapy. Ten of 28 patients showed ALP elevation of 25% or more from the baseline at 3 weeks, and 14 of the 28 had this finding at 6 weeks. Four of 5 patients who had achieved VGPR or more showed ALP elevation of 25% or more at 3 weeks, and all five had this finding by 6 weeks. No patient without ALP elevation achieved VGPR or a better response. ALP elevation exceeding 25% from the baseline by day 42 is significantly associated with a treatment response better than VGPR (p=0.019). In conclusion, ALP elevation during BTZ treatment is a valuable prognostic marker.

Prognosis of patients with core binding factor acute myeloid leukemia after first relapse.

Core binding factor acute myeloid leukemia is known to have a favorable prognosis, however, there have been no detailed analyses on prognostic factors after first relapse. Using a nationwide database, we retrospectively analyzed core binding factor acute myeloid leukemia patients who relapsed after being treated with chemotherapy alone during their first complete remission. Of a total of 397 patients who were diagnosed with core binding factor acute myeloid leukemia, 208 experienced a first relapse, and analyses were performed in 139 patients for whom additional data were available. In the entire cohort, the overall survival rate after relapse was 48% at 3 years. By multivariate analysis, younger age at diagnosis, a longer interval before relapse, and inv(16) were shown to be independently associated with better survival after relapse. Although there was no significant difference in survival after relapse between patients who underwent allogeneic hematopoietic cell transplantation and those who did not in the overall series of relapsed patients, we found that transplantation significantly improved survival among patients who had t(8;21) (54% versus 26% at 3 years, P=0.002). In addition, among patients with t(8;21), those who had different cytogenetics at relapse had a significantly improved survival after transplantation, while those who had same cytogenetics did not. We showed that the prognosis differs significantly and optimal treatment strategies may vary between groups of patients with core binding factor acute myeloid leukemia with different cytogenetic profiles at relapse. These findings may help to guide therapeutic decisions after first relapse.

Virtual-freezing fluorescence imaging flow cytometry with 5-aminolevulinic acid stimulation and antibody labeling for detecting all forms of circulating tumor cells.

Circulating tumor cells (CTCs) are precursors to cancer metastasis. In blood circulation, they take various forms such as single CTCs, CTC clusters, and CTC-leukocyte clusters, all of which have unique characteristics in terms of physiological function and have been a subject of extensive research in the last several years. Unfortunately, conventional methods are limited in accurately analysing the highly heterogeneous nature of CTCs. Here we present an effective strategy for simultaneously analysing all forms of CTCs in blood by virtual-freezing fluorescence imaging (VIFFI) flow cytometry with 5-aminolevulinic acid (5-ALA) stimulation and antibody labeling. VIFFI is an optomechanical imaging method that virtually freezes the motion of fast-flowing cells on an image sensor to enable high-throughput yet sensitive imaging of every single event. 5-ALA stimulates cancer cells to induce the accumulation of protoporphyrin (PpIX), a red fluorescent substance, making it possible to detect all cancer cells even if they show no expression of the epithelial cell adhesion molecule, a typical CTC biomarker. Although PpIX signals are generally weak, VIFFI flow cytometry can detect them by virtue of its high sensitivity. As a proof-of-principle demonstration of the strategy, we applied cancer cells spiked in blood to the strategy to demonstrate image-based detection and accurate classification of single cancer cells, clusters of cancer cells, and clusters of a cancer cell(s) and a leukocyte(s). To show the clinical utility of our method, we used it to evaluate blood samples of four breast cancer patients and four healthy donors and identified EpCAM-positive PpIX-positive cells in one of the patient samples. Our work paves the way toward the determination of cancer prognosis, the guidance and monitoring of treatment, and the design of antitumor strategies for cancer patients.

Time of Day of Vaccination Does Not Associate With SARS-CoV-2 Antibody Titer Following First Dose of mRNA COVID-19 Vaccine.

The immune system exhibits circadian rhythms, and its response to viral infection is influenced by the circadian clock system. Previous studies have reported associations between the time of day of vaccination against COVID-19 and production of anti-SARS-CoV-2 antibody titer. We examined the effect of vaccination time of day on anti-SARS-CoV-2 antibody titer after the first dose of vaccination with the mRNA-1273 (Moderna) COVID-19 vaccine in an adult population. A total of 332 Japanese adults participated in the present study. All participants were not infected with SARS-CoV-2 and had already received the first dose of mRNA-1273 2 to 4 weeks prior to participating in the study. The participants were asked to provide basic demographic characteristics (age, sex, medical history, allergy, medication, and mean sleep duration), the number of days after the first dose of vaccination, and the time of day of vaccination. Blood was collected from the participants, and SARS-CoV-2 antibody titers were measured. Ordinary least square regression was used for assessing the relationship between basic demographic characteristics, number of days after vaccination, time of day of vaccination, and the log10-transformed normalized antibody titer. The least square mean of antibody titers was not associated with the vaccination time and sleep durations. The least square means of antibody titers was associated with age; the antibody titers decreased in people aged 50 to 59 years and 60 to 64 years. The present findings demonstrate that the vaccination time with mRNA-1273 was not associated with the SARS-CoV-2 antibody titer in an adult population, suggesting that these results do not support restricting vaccination to a particular time of day. The present findings may be useful in optimizing SARS-CoV-2 vaccination strategies.

[Thrombosis with Thrombocytopenia Syndrome after ChAdOx1 nCoV-19 vaccination].

A 48-year-old Japanese man who had no previous medical history received his first dose of the ChAdOx1 nCoV-19 vaccine. Ten days after the vaccine administration, he developed a headache. Laboratory results indicated throm-bocytopenia and DIC. A head CT revealed microbleeding in the left parietal lobe. Contrast-enhanced CT showed thrombus in the left transverse sinus and left sigmoid sinus. A brain MRI demonstrated venous hemorrhagic infarction and subarachnoid hemorrhages in the left parietal lobe, and whole-body enhanced CT also revealed portal vein embolism and renal infarction. He was diagnosed with thrombosis with thrombocytopenia syndrome, and was treated according to the guideline. He has been recovering with the treatments. This is the first reported case of TTS associated with the ChAdOx1 nCoV-19 vaccine in Japan.

COVID-19 and Thrombosis: Clinical Aspects.

In coronavirus disease 2019 (COVID-19), thrombus formation is related to the pathogenesis of acute respiratory distress syndrome (ARDS) and the progression of clinical symptoms. Severe damage to vascular endothelial cells and the associated cytokine storm after SARS-CoV-2 infection cause thrombogenesis and contribute to the development of more severe and unique thromboses compared to other infectious diseases. Thromboses occur more often in critically ill patients. In addition to pulmonary thromboembolism (PE) and deep vein thrombosis, acute myocardial infarction, peripheral arterial thrombosis, and aortic thrombosis have also been reported. In PE, thrombi develop in both pulmonary arteries and alveolar capillaries. These, together with intraalveolar fibrin deposition, interfere with effective gaseous exchange in the lungs and exacerbate the clinical symptoms of ARDS in patients with COVID-19. Pharmacological thromboprophylaxis is recommended for all hospitalized patients to prevent both thrombosis and aggravation of ARDS, and other organ failures. Although the pediatric population is mostly asymptomatic or develops mild disease after SARS-CoV-2 infection, a new inflammatory disorder affecting the cardiovascular system, multisystem inflammatory syndrome in children (MIS-C), has been reported. Similar to Kawasaki disease, acute myocarditis, coronary vasculitis, and aneurysms are typically seen in MISC, although these two are now considered distinct entities. A similar acute myocarditis is also observed in young male adults, in which a hyperinflammatory state after SARS-CoV-2 infection seems to be involved. Several side effects following vaccination against COVID-19 have been reported, including vaccine-induced immune thrombotic thrombocytopenia and acute myocarditis. Although these could be serious and life-threatening, the cases are very rare, thus, the benefits of immunization still outweigh the risks.

Aleukemic Extramedullary Blast Crisis as an Initial Presentation of Chronic Myeloid Leukemia with E1A3 BCR-ABL1 Fusion Transcript.

Right neck swelling and pain occurred in a 49-year-old man. A Blood count showed a slight increase in platelet count without leukemoid reaction. After a biopsy of the cervical mass and bone marrow aspiration, a diagnosis of extramedullary blast crisis (EBC) of chronic myeloid leukemia (CML) was made. Fluorescence in situ hybridization (FISH) analysis showed a BCR-ABL1 fusion signal, but results of real-time polymerase chain reaction (RT-PCR) for major and minor BCR-ABL1 transcripts were negative. We identified a rare e1a3 BCR-ABL1 fusion transcript. Administration of dasatinib resulted in disappearance of the extramedullary tumor. This is the first reported case of CML-EBC with e1a3 transcript. An aleukemic extramedullary tumor can be the initial presentation of CML.

Intelligent Platelet Morphometry.

Technological advances in image-based platelet analysis or platelet morphometry are critical for a better understanding of the structure and function of platelets in biological research as well as for the development of better clinical strategies in medical practice. Recently, the advent of high-throughput optical imaging and deep learning has boosted platelet morphometry to the next level by providing a new set of capabilities beyond what is achievable with traditional platelet morphometry, shedding light on the unexplored domain of platelet analysis. This Opinion article introduces emerging opportunities in 'intelligent' platelet morphometry, which are expected to pave the way for a new class of diagnostics, pharmacometrics, and therapeutics.

Differential effects of interleukin-12 and interleukin-15 on expansion of NK cell receptor-expressing CD8+ T cells.

The cytolytic activity of cells expressing natural killer cell receptors (NKRs) depends on the balance between stimulatory and inhibitory signals. We investigated both inhibitory NK receptor (CD94/NKG2A) expression and stimulatory NKR (NKG2D) expression on T cells after stimulation with cytokines (IL-12 or IL-15). Cytolytic NKR-expressing CD8(+) T cells were expanded from normal adult peripheral blood mononuclear cells using anti-CD3 monoclonal antibody and cytokines (IL-12 or IL-15). The proportion and absolute number of CD94/NKG2A-expressing T cells expanded by IL-12 were significantly larger than those of the cells expanded by IL-15. On the other hand, the proportion and absolute number of NKG2D-expressing T cells expanded by IL-15 were significantly larger than those of the cells expanded by IL-12. The proportions of NKG2D and intracellular granzyme A expression in CD94-expressing cells were much more increased in PBMCs cultured with IL-15 than those of cells cultured with IL-12. A real-time polymerase chain reaction assay showed that there was a 1.68-fold increase in NKG2D mRNA expression level and a 1.37-fold increase in DAP10 mRNA expression level in CD94-expressing cells expanded by IL-15 compared with those of the cells expanded by IL-12. The cytolytic activity levels of purified CD94-expressing cells from 8-day culture with IL-15 tested against (51)Cr-labeled K562 cells by standard 4-h (51)Cr release assays without prior sensitization were much higher than those of cells from 8-day culture with IL-12. IL-15 appears to be able to enhance the cytolytic activity of CD94/NKG2A-expressing cells through induction of NKG2D and intracellular granzyme expression much more efficiently than does IL-12.

Establishment of a megakaryoblastic cell line for conventional assessment of platelet calcium signaling.

Platelet function tests utilizing agonists or patient serum are generally performed to assess platelet activation ex vivo. However, inter-individual differences in platelet reactivity and donor requirements make it difficult to standardize these tests. Here, we established a megakaryoblastic cell line for the conventional assessment of platelet activation. We first compared intracellular signaling pathways using CD32 crosslinking in several megakaryoblastic cell lines, including CMK, UT-7/TPO, and MEG-01 cells. We confirmed that CD32 was abundantly expressed on the cell surface, and that intracellular calcium mobilization and tyrosine phosphorylation occurred after CD32 crosslinking. We next employed GCaMP6s, a highly sensitive calcium indicator, to facilitate the detection of calcium mobilization by transducing CMK and MEG-01 cells with a plasmid harboring GCaMP6s under the control of the human elongation factor-1α promoter. Cells that stably expressed GCaMP6s emitted enhanced green fluorescent protein fluorescence in response to intracellular calcium mobilization following agonist stimulation in the absence of pretreatment. In summary, we have established megakaryoblastic cell lines that mimic platelets by mobilizing intracellular calcium in response to several agonists. These cell lines can potentially be utilized in high-throughput screening assays for the discovery of new antiplatelet drugs or diagnosis of disorders caused by platelet-activating substances.