The role of next-generation sequencing in hematologic malignancies.

Next-generation sequencing (NGS) allows high-throughput detection of molecular changes in tumors. Over the past 15 years, NGS has rapidly evolved from a promising research tool to a core component of the clinical laboratory. Sequencing of tumor cells provides an important step in detecting somatic driver mutations that not only characterize the disease but also influence treatment decisions. For patients with hematologic malignancies, NGS has been used for accurate classification and diagnosis based on genetic alterations. The recently revised World Health Organization classification and the European LeukemiaNet recommendations for acute myeloid leukemia consider genetic abnormalities as a top priority for diagnosis, prognostication, monitoring of measurable residual disease, and treatment choice. This review aims to present the role and utility of various NGS approaches for the diagnosis, treatment, and follow-up of hemato-oncology patients.

Integration of in vivo electrophysiology and optogenetics in rodents with PEDOT:PSS neural electrode array.

Here, we present a protocol for the fabrication of transparent implantable electrode arrays for integrating optogenetics and electrophysiology. We describe steps for fabricating microelectrodes using the conductive polymer poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate). We then detail procedures for analyzing performance of the electrodes and recording light-evoked neural activities from the transgenic mouse. This protocol utilizes photolithography rather than conventional electrodeposition. For complete details on the use and execution of this protocol, please refer to Cho et al. (2022).1.

A wireless, solar-powered, optoelectronic system for spatial restriction-free long-term optogenetic neuromodulations.

Numerous wireless optogenetic systems have been reported for practical tether-free optogenetics in freely moving animals. However, most devices rely on battery-powered or coil-powered systems requiring periodic battery replacement or bulky, high-cost charging equipment with delicate antenna design. This leads to spatiotemporal constraints, such as limited experimental duration due to battery life or animals' restricted movement within specific areas to maintain wireless power transmission. In this study, we present a wireless, solar-powered, flexible optoelectronic device for neuromodulation of the complete freely behaving subject. This device provides chronic operation without battery replacement or other external settings including impedance matching technique and radio frequency generators. Our device uses high-efficiency, thin InGaP/GaAs tandem flexible photovoltaics to harvest energy from various light sources, which powers Bluetooth system to facilitate long-term, on-demand use. Observation of sustained locomotion behaviors for a month in mice via secondary motor cortex area stimulation demonstrates the notable capabilities of our device, highlighting its potential for space-free neuromodulating applications.

Validation of High-sensitivity Flow Cytometry for Reliable Immune Cell Analysis in Real-world Laboratory Settings.

The adoption of high-sensitivity flow cytometry (HSFC) in routine laboratory settings has been slow owing to concerns regarding the reliability and reproducibility of results. Validation is an essential prerequisite for conducting assays, and implementing the CLSI guidelines has been confusing, primarily because many aspects are not yet established. We aimed to validate an HSFC protocol for detecting follicular helper T (Tfh) cells in a real-world laboratory environment. The analytical validity of the Tfh cell panel was ensured through rigorous testing, including evaluations of precision, stability, carryover, and sensitivity, following the CLSI H62 guidelines. We found that Tfh cells, present in very small numbers in the blood, could be sufficiently detected through HSFC, and concerns about the reliability and reproducibility of the results in real-world laboratories could be solved through systematic validation. Establishing the lower limit of quantification (LLOQ) is a critical step in HSFC evaluations. By selecting an appropriate sample, for example, collecting residual cells from CD4 isolation in our experiment and using them as low-level samples, the LLOQ could be accurately established. The strategic validation of flow cytometry panels can facilitate the adoption of HSFC in clinical laboratories, even with limited resources.

Increased PD-1 expression of bone marrow T-cells in acute myeloid leukaemia patients after stem cell transplantation, and its association with overall survival.

BACKGROUND: Immune checkpoints are involved in mechanisms by which tumours escape from the host immune system. Our aim was to evaluate acute myeloid leukaemia (AML) patients to determine expression levels of checkpoint molecules according to diagnosis and treatments, and to identify optimal candidates for checkpoint blockade. METHODS: Bone marrow (BM) samples were obtained from 279 AML patients at different disease status and from 23 controls. Flow cytometric analyses of PD-1 and PD-L1/PD-L2 expression were performed. RESULTS: Programmed death-1 (PD-1) expression levels on CD8+ T-cells at AML diagnosis were increased compared to controls. PD-L1 and PD-L2 expression levels on leukaemic cells at diagnosis were significantly higher in secondary AML than in de novo AML. PD-1 levels on CD8+ and CD4+ T-cells after allo-SCT were significantly higher than those at diagnosis and after CTx. PD-1 expression on CD8+ T-cells increased in the acute GVHD group than in the non-GVHD group. The overall survival of patients with high PD-1 expression on CD8+ T-cells was significantly shorter than that of patients with low PD-1 expression. CONCLUSIONS: In conclusion, patients who underwent allo-SCT exhibited high PD-1 expression, suggesting that allo-SCT increases PD-1 expression on T-cells, and the patients with high PD-1 expression on CD8+ T-cells after allo-SCT showed the poor prognosis. For these patients, PD-1 blockade could be an immunotherapeutic strategy.

Ultrathin Crystalline Silicon Nano and Micro Membranes with High Areal Density for Low-Cost Flexible Electronics.

Ultrathin crystalline silicon is widely used as an active material for high-performance, flexible, and stretchable electronics, from simple passive and active components to complex integrated circuits, due to its excellent electrical and mechanical properties. However, in contrast to conventional silicon wafer-based devices, ultrathin crystalline silicon-based electronics require an expensive and rather complicated fabrication process. Although silicon-on-insulator (SOI) wafers are commonly used to obtain a single layer of crystalline silicon, they are costly and difficult to process. Therefore, as an alternative to SOI wafers-based thin layers, here, a simple transfer method is proposed for printing ultrathin multiple crystalline silicon sheets with thicknesses between 300 nm to 13 µm and high areal density (>90%) from a single mother wafer. Theoretically, the silicon nano/micro membrane can be generated until the mother wafer is completely consumed. In addition, the electronic applications of silicon membranes are successfully demonstrated through the fabrication of a flexible solar cell and flexible NMOS transistor arrays.

Effective and Practical Complete Blood Count Delta Check Method and Criteria for the Quality Control of Automated Hematology Analyzers.

Background: Delta checks increase patient safety by identifying automated hematology analyzer errors. International standards and guidelines for the complete blood count (CBC) delta check method have not been established. We established an effective, practical CBC delta check method and criteria. Methods: We assessed five delta check methods for nine CBC items (Hb, mean corpuscular volume, platelet count, white blood cell [WBC] count, and five-part WBC differential counts) using 219,804 blood samples from outpatients and inpatients collected over nine months. We adopted the best method and criteria and evaluated them using 42,652 CBC samples collected over two weeks with a new workflow algorithm for identifying test errors and corrections for Hb and platelet count. Results: The median delta check time interval was 1 and 21 days for inpatients and outpatients (range, 1-20 and 1-222 days), respectively. We used delta values at 99.5% as delta check criteria; the criteria varied among the five methods and between outpatients and inpatients. The delta percent change (DPC)/reference range (RR) rate performed best as the delta check for CBC items. Using the new DPC/RR rate method, 1.7% of total test results exceeded the delta check criteria; the retesting and resampling rates were 0.5% and 0.001%, respectively. Conclusions: We developed an effective, practical delta check method, including RRs and delta check time intervals, and delta check criteria for nine CBC items. The criteria differ between outpatients and inpatients. Using the new workflow algorithm, we can identify the causes of criterion exceedance and report correct test results.

Evaluation of laboratory diagnostic tests for light-chain clonality and bone marrow findings in AL amyloidosis.

Background: Light-chain amyloidosis (AL) is the most common form of systemic amyloidosis. This study aimed to evaluate the usefulness of laboratory tests for light-chain clonality and bone marrow (BM) findings in AL amyloidosis. Methods: We retrospectively enrolled patients newly diagnosed with AL amyloidosis on pathological examination who underwent a BM biopsy. Laboratory test data for light-chain clonality were collected and compared. Amyloid deposits were identified with H&E, Congo red, and PAS stains. Results: We reviewed 98 patients with AL amyloidosis. Light chain clonality (λ, 64 cases; κ, 34 cases) was detected by serum immunofixation electrophoresis (IFE) (63.3%), urine IFE (70.8%), serum protein electrophoresis (PEP) (44.9%), urine PEP (44.8%), serum free light chain (SFLC) ratio (79.5%), and BM immunohistochemistry (IHC) (85.7%). Flow cytometric (FCM) assay identified aberrant BM plasma cells in 92.9% of cases. BM amyloid deposits were identified in 35 of the 98 cases (35.7%); 71.4% (25/35) were Congo red-positive, and 100.0% (35/35) were PAS-positive. Conclusion: Laboratory tests for detecting light-chain clonality in AL amyloidosis in order of sensitivity include FCM assay for aberrant plasma cells, IHC for light chains on BM biopsy or clot section, SFLC ratio, and serum and urine IFE. Congo red staining of BM samples remains an important tool for identifying amyloid deposits in BM. Periodic acid-Schiff (PAS) staining can be useful in diagnosing some cases of Congo red-negative amyloidosis.

Natural Killer Cell Activity Test Helps to Suspect Aggressive Natural Killer Cell Leukemia - Diagnostic Challenge.

Aggressive natural killer cell leukemia (ANKL) is a rare disease with an aggressive clinical course. We aimed to assess the clinicopathological characteristics of the difficult to diagnose ANKL. During ten years, nine patients with ANKL were diagnosed. All the patients exhibited aggressive clinical course and underwent the BM study to rule out lymphoma and hemophagocytic lymphohistiocytosis (HLH). BM examination showed varying degrees of infiltration of neoplastic cells, which were mainly positive for CD2, CD56, cytoplasmic CD3 and EBV in situ hybridization. Five BM aspirates showed histiocytic proliferation with active heomphagocytosis. Normal or increased NK cell activity test results were obtained from 3 patients who were available for testing. Four had multiple BM studies until diagnosis. An aggressive clinical course and positive EBV in situ hybridization, often with associated secondary HLH, should raise the suspicion of an ANKL. Conducting additional supplementary tests such as NK cell activity and NK cell proportion would be helpful for the diagnosis of ANKL.

Diagnostic spectrum of hypereosinophilia based on bone marrow pathology: 10 years' experience at a tertiary care hospital.

BACKGROUND: Hypereosinophilia (HE) is defined as peripheral blood (PB) eosinophil count exceeding 1.5 × 109 /L. As the causes of HE can be diverse, the work-up of patients was complicated. In this study, we aimed to categorize the underlying diseases associated with HE and demonstrate minimum diagnostic approach. METHODS: Cases presenting with HE within 7 days of bone marrow (BM) examination conducted between 2008 and 2019 were selected. Cases were classified by the revised 2022 WHO and ICC classification. We also assessed morphologic features of unclassified persisting HE (>4 weeks) patients according to the morphologic criteria suggested a previous study by Wang et al. RESULTS: A total of 364 patients were included. The work-up confirmed primary HE in 38.7%, secondary HE in 48.9%, HE patients with insufficient evaluation in 13.7%. When conducted a slide review of HE patients with sustained HE more than 4 weeks among HE patients with insufficient evaluation, the morphological features showed abnormal eosinophils in PB/BM (69.0%/81.0%), hypercellularity (26.2%), myelofibrosis (7.1%), increased M:E ratio (5.3%), and dysmegakaryopoiesis (4.8%). Of these patients, 14 patients who met all morphologic criteria were suspected of CEL. CONCLUSIONS: This study demonstrates that HE is associated with variable conditions. BM morphological assessment based on a robust criterion can help to confirm a MN irrespective of the presence of clonal markers. The work-up of patients in whom ruled out the common secondary causes of HE requires a systematic but sufficient approach including at a minimum BM karyotyping, PDGFRA testing, lymphocyte immunophenotyping and TCR gene rearrangement.

Lupus anticoagulants as a prospective independent predictor in COVID-19 patients.

OBJECTIVE: Lupus anticoagulant (LA) are commonly detected during SARS-CoV-2 infection. However, the relationship between LA and clinical significance is still unclear. METHODS: A retrospective chart analysis was performed on COVID-19 patients who were tested for LA at our hospital from March 2020 to November 2021. We analyzed the patient's characteristics based on the result of the LA test. In addition, subgroup analysis performed the LA-positive group who had undergone serial LA tests. RESULTS: A total of 219 COVID-19 patients were enrolled in the study, 148 patients (67.6%) were positive for LA test. The LA-positive group received more treatment of high flow nasal cannula (LA-positive 73.0%, LA-negative 57.7%, p = 0.024). The LA-positive group showed prolonged aPTT, higher levels of CRP and fibrinogen (all p's < 0.05). Among 148 LA-positive patients, 127 patients (86.5%) were found to be LA-positive within 10 days of SARS-CoV-2 positive, and LA-positive group confirmed a median time to LA loss of 10 days. However, there was a group that was negative for LA in the early stages of infection and became positive about 13 days later. A subgroup analysis showed that these patients had different characteristics due to their longer hospital stays and higher D-dimer levels. CONCLUSIONS: In COVID-19 patients, LA is expected to be associated to disease severity. Since the clinical significance of LA is different depending on the onset time of LA positivity, the LA test is suggested to be done at diagnosis of SARS-CoV-2 infection, even if LA is negative, follow-up test should be considered within 10 days.

Effect of changes in lymphocyte subsets at diagnosis in acute myeloid leukemia on prognosis: association with complete remission rates and relapse free survivals.

We prospectively investigated whether the characteristics of lymphocyte subsets at diagnosis in acute myeloid leukemia (AML) patients are different from healthy controls and affect treatment outcomes. A total of 91 AML patients classified into 3 genetic risk subgroups (favorable/intermediate/poor) according to 2022 NCCN guidelines were enrolled. We measured lymphocyte subsets by flow cytometry with peripheral blood samples at diagnosis and compared results with healthy controls. Influences of lymphocyte subsets on complete remission (CR) rates and survivals were also evaluated. AML patients had significantly lower numbers and proportions of CD56dimCD16+ natural killer (NK) cells, central memory T cells, and regulatory T cells than healthy controls. Higher proportion of helper/inducer T cells, CD4+CD31+ naïve T cells, and decreased proportion of NK cells significantly increased CR rates in 65 non-promyelocytic leukemia patients (P = 0.034, 0.027, and 0.019, respectively), and it was also significant in multivariable analysis with age/risk adjusted (P = 0.014, 0.016, and 0.045, respectively). NK cells < 4.8% of lymphocytes demonstrated significantly shorter relapse free survivals (RFS) in both univariate and multivariate analyses with risk adjusted (P = 0.006 and 0.037, respectively). AML patients showed significant lower numbers of CD56dimCD16+ NK cells, central memory T cells, and regulatory T cells than healthy controls at diagnosis. Higher proportion of helper/inducer T cells and CD4+CD31+ naïve T cells and decreased proportion of NK cells at diagnosis were independent factor of increasing probability of CR, and proportion of NK cells < 4.8% at diagnosis had adverse impact in RFS.

Establishing Reference Range and Assessing Precision Performance of the TEG6s System; Verification of TEG6s to Introduce to Clinical Laboratory.

BACKGROUND: Thromboelastography (TEG) provides assessment of global coagulation. The TEG6s (Haemonetics Corporation) is a newly developed cartridge-based system, fully automated and the first true point-of-care TEG. In this study, we evaluated the precision and established the reference intervals (RIs) for TEG6s. METHODS: TEG assays were performed on the blood of healthy donors to determine RIs and on the QC materials for precision testing. The study design was developed in accordance with Clinical and Laboratory Standards Institute Guidelines. RESULTS: TEG6s precision testing yielded low variability except R, due to a low value for the mean. The newly established RIs of R, MA, and LY30 were similar to the manufacturer's RIs. Some were different, showing short K and increased α. CONCLUSIONS: This study has shown that TEG6s has high precision and each institution should verify the manufacturer's RIs before adopting TEG6s and establish RIs if necessary.

Prognostic implication of bronchoalveolar lavage fluid analysis in patients with Pneumocystis jirovecii pneumonia without human immunodeficiency virus infection.

BACKGROUND: The prognostic value of bronchoalveolar lavage (BAL) fluid analysis in non-human immunodeficiency virus (HIV)-infected patients with Pneumocystis jirovecii pneumonia (PJP) has not been well elucidated. We aimed to investigate the prognostic implication of BAL fluid analysis in non-HIV patients with PJP. METHODS: The data of 178 non-HIV patients diagnosed with PJP based on the results of the polymerase chain reaction assay of BAL fluid specimens between April 2018 and December 2020 were retrospectively reviewed. The clinical characteristics, laboratory findings, and BAL fluid analysis results of patients who died within 90 days after hospital admission were compared. RESULTS: Twenty patients (11.2%) died within 90 days from admission. The neutrophil count in BAL fluid was significantly higher (median 22.0%, interquartile range [IQR] 2.0-46.0% vs. median 6.0%, IQR 2.0-18.0%, P = 0.044), while the lymphocyte count was significantly lower (median 24.0%, IQR 7.0-37.0% vs. median 41.0%, IQR 22.5-60.5%, P = 0.001) in the non-survivor group compared with that in the survivor group. In the multivariate analysis, the C-reactive protein level (odds ratio [OR] 1.093, 95% confidence interval [CI] 1.020-1.170, P = 0.011) and a BAL fluid lymphocyte count of ≤ 30% (OR 3.353, 95% CI 1.101-10.216, P = 0.033) were independently associated with mortality after adjusting for albumin and lactate dehydrogenase levels. CONCLUSION: A low lymphocyte count in BAL fluid may be a predictor of mortality in non-HIV patients with PJP.

Clinical implications and genetic features of clonal cytopenia of undetermined significance compared to lower-risk myelodysplastic syndrome.

Clonal cytopenia of undetermined significance (CCUS) is characterized by persistent cytopenias with genetic aberrations, which do not meet the diagnostic criteria for myelodysplastic syndrome (MDS). We aimed to compare the clinical and genetic characteristics of CCUS with lower-risk MDS and identify patients with CCUS with a high risk of progression. We performed targeted sequencing of bone marrow (BM) samples from patients with idiopathic cytopenia of undetermined significance (ICUS) (n = 139) and MDS (n = 226). Overall survival (OS) of patients with CCUS (n = 78) was worse than non-clonal ICUS (n = 61) and superior to lower-risk MDS (n = 99). Patients with CCUS showed similar characteristics to those with lower-risk MDS, except for higher haemoglobin, lower BM cellularity, and less frequent SF3B1 mutations. Lower haemoglobin, DDX41 (biallelic germline and somatic), ETV6, and RUNX1 mutations were independent prognostic factors for worse OS. Lower haemoglobin and DDX41 mutations were also associated with lower progression-free survival. Patients with CCUS with high-risk features showed similar or worse OS than patients with lower-risk MDS. Our findings suggest that patients with CCUS having certain clinical or genetic features should be regarded and treated as lower-risk MDS despite lacking significant dysplasia or MDS-associated chromosomal abnormalities.

Clinical Application for Diagnosis of Myelodysplatic/Myeloproliferative Neoplasm with Ring Sideroblasts and Thrombocytosis.

BACKGROUND: Myelodysplastic/myeloproliferative neoplasm with ring sideroblasts and thrombocytosis (MDS/ MPN-RS-T) was newly introduced as a full entity in the 2016 revision of the WHO classification. In this study, we investigated the morphologic, laboratory, and clinical features of MDS/MPN-RS-T. METHODS: We reviewed the bone marrow and genetic studies of patients whose diagnoses were coded as "refractory anemia with ring sideroblasts (RARS)" or "MDS/MPN, unclassifiable" between January 2008 and April 2018. RESULTS: A total of 8 cases fulfilled the criteria for a diagnosis of MDS/MPN-RS-T. All of them had no specific symptoms. Half of the cases had less than 450 × 109/L platelet counts by an automated hematology analyzer; however, all platelet counts exceeded 450 × 109/L when performed manually. JAK2 mutation tests were performed in 7 cases, and a heterozygous mutation was detected in 1 case. SF3B1 mutations were present in 3 of the 4 cases tested. CONCLUSIONS: When RARS is suspected in patients without thrombocytopenia, manual platelet counts should be performed. For patients with suspected essential thrombocythemia, RS evaluation through careful observation of an iron-stained slide is crucial. Since the independent evaluation of RS was reflected in the revised classification, the ambiguous disease classification becomes clearer and more consistent.