Enhancing thalassemia gene carrier identification in non-anemic populations using artificial intelligence erythrocyte morphology analysis and machine learning.

BACKGROUND: Non-anemic thalassemia trait (TT) accounted for a high proportion of TT cases in South China. OBJECTIVE: To use artificial intelligence (AI) analysis of erythrocyte morphology and machine learning (ML) to identify TT gene carriers in a non-anemic population. METHODS: Digital morphological data from 76 TT gene carriers and 97 controls were collected. The AI technology-based Mindray MC-100i was used to quantitatively analyze the percentage of abnormal erythrocytes. Further, ML was used to construct a prediction model. RESULTS: Non-anemic TT carriers accounted for over 60% of the TT cases. Random Forest was selected as the prediction model and named TT@Normal. The TT@Normal algorithm showed outstanding performance in the training, validation, and external validation sets and could efficiently identify TT carriers in the non-anemic population. The top three weights in the TT@Normal model were the target cells, microcytes, and teardrop cells. Elevated percentages of abnormal erythrocytes should raise a strong suspicion of being a TT gene carrier. TT@Normal could be promoted and used as a visualization and sharing tool. It is accessible through a URL link and can be used by medical staff online to predict the possibility of TT gene carriage in a non-anemic population. CONCLUSIONS: The ML-based model TT@Normal could efficiently identify TT carriers in non-anemic people. Elevated percentages of target cells, microcytes, and teardrop cells should raise a strong suspicion of being a TT gene carrier.

Circulating Regulatory T Cells: A Novel Marker Associated with Liver Metastasis and the Treatment Response of Transarterial Embolization in Gastroenteropancreatic Neuroendocrine Tumors.

INTRODUCTION: To investigate the role of circulating regulatory T cells (Tregs) as a novel marker associated with liver metastases and treatment response to transarterial embolization (TAE) in patients with gastroenteropancreatic neuroendocrine tumors (GEP-NETs). METHODS: Circulating Tregs, defined as the CD4+CD25+CD127low/- population, were examined by flow cytometry in peripheral blood mononuclear cells (PBMCs) from patients with GEP-NETs. Clinicopathological parameters, radiologic response, and hepatic progression-free survival (hPFS) data were collected. RESULTS: The association between circulating Tregs and clinicopathological parameters was analyzed in 139 GEP-NET patients. Higher Treg levels were significantly associated with more progressive clinical features, including a higher WHO grade, more advanced TNM stage, and the presence of liver metastases. A Treg level ≥ 8.015% distinguished between patients with and without liver metastases. Among a cohort of 51 GEP-NET patients who were subjected to TAE for reducing liver metastasis burden, patients with higher Treg levels depicted unfavorable responses and significantly reduced hPFS after TAE treatment. We also revealed that patients with Treghigh (≥8.975%) displayed significantly shorter median hPFS than patients with Treglow (< 8.975%). Additionally, after adjusting for other confounding clinical parameters, the association between Tregs and treatment response as well as hPFS remained significant, suggesting that Tregs may have a strong and independent prognostic impact in GEP-NETs. CONCLUSIONS: Our data suggest that circulating Tregs are a novel immunological marker associated with liver metastases and treatment response to TAE in patients with GEP-NETs.

Decrease in peripheral natural killer cell level during early pregnancy predicts live birth among women with unexplained recurrent pregnancy loss: a prospective cohort study.

BACKGROUND: Previous studies have suggested that trophoblast cells inhibit the proliferation of peripheral natural killer cells and that the level of peripheral natural killer cells decrease in the middle and late pregnancy stage among healthy women. The change in peripheral natural killer cell level during early pregnancy and the relationship between the change in peripheral natural killer cell level and pregnancy outcomes among women with unexplained recurrent pregnancy loss have not been sufficiently explored. OBJECTIVE: This study aimed to characterize the level of prepregnancy peripheral natural killer cells in comparison with those in early pregnancy among women with unexplained recurrent pregnancy loss and to determine if the change in the level of peripheral natural killer cells from prepregnancy to early pregnancy can predict pregnancy outcomes. STUDY DESIGN: In this prospective cohort study, 1758 women with recurrent pregnancy loss were recruited between January 2017 and December 2021 among whom 252 women with unexplained recurrent pregnancy loss had prepregnancy and early pregnancy (4-6 weeks gestation) peripheral natural killer cell measurements. These 252 women were divided into 2 groups, namely those with a lower gestational peripheral natural killer cell level (group 1) when compared with prepregnancy levels and those who did not (group 2). The respective outcomes of these groups in terms of live birth and pregnancy loss were comparatively analyzed using chi-square and Student's t tests. Candidate factors that could influence live birth were selected using the Akaike information criterion. The participates were then randomly divided into training and testing groups. A multivariable logistic regression analysis was performed and a nomogram was created to assess the possibility of live birth. The predictive accuracy was determined by the area under the receiver operating characteristic curve and validated by plotting the predicted probabilities and the observed probabilities. A Hosmer-Lemeshow test was used to assess the goodness of fit. RESULTS: When early gestational peripheral natural killer cell levels were compared with prepregnancy peripheral natural killer cell levels, 61.5% (154) of women had a comparatively lower early-gestational peripheral natural killer cell level and 38.9% (98) of women had an increase or no change in the peripheral natural killer cell level. The live birth rate in group 1 was 89.0% (137/154), which was significantly higher than the rate of 49.0% (48/98) in group 2 (P<.001). A decrease in the peripheral natural killer cell level (odds ratio, 1.36; 95% confidence interval, 1.22-1.55; P<.001) and the anti-Muellerian hormone level (odds ratio, 1.41; 95% confidence interval, 1.14-1.81; P=.003) were important predicting factors for a higher live birth rate. Female body mass index (odds ratio, 0.97; 95% confidence interval, 0.82-1.15; P=.763) and parity (odds ratio, 1.61; 95% confidence interval, 0.71-4.12; P=.287) also were predicting factors. Furthermore, the area under the receiver operating characteristic curve of the model to diagnose of live birth was 0.853 with a sensitivity of 81.6% and a specificity of 78.0% using the training data set. And the Hosmer-Lemeshow test showed that the model was a good fit (p=6.068). CONCLUSION: We report a comparative decrease in the peripheral natural killer cell levels in early gestation when compared with prepregnancy cell levels in more than 60% of women with unexplained recurrent pregnancy loss at 4 to 6 weeks of gestation. When compared with prepregnancy peripheral natural killer cell levels, a decrease in the peripheral natural killer cell level during early pregnancy might be a useful predictor of the live birth rate among women with unexplained recurrent pregnancy loss.

Artificial Intelligence-based online platform assists blood cell morphology learning: A mixed-methods sequential explanatory designed research.

BACKGROUND: The study aimed to evaluate the effectiveness of learning blood cell morphology by learning on our Artificial intelligence (AI)-based online platform. METHODS: Our study is based on mixed-methods sequential explanatory design and crossover design. Thirty-one third-year medical students were randomly divided into two groups. The two groups had platform learning and microscopy learning in diferent sequences with pretests and posttests, respectively. Students were interviewed, and the records were coded and analyzed by NVivo 12.0. RESULTS: For both groups, test scores increased significantly after online-platform learning. Feasibility was the most mentioned advantage of the platform. The AI system could inspire the students to compare the similarities and differences between cells and help them understand the cells better. Students had positive perspectives on the online-learning platform. CONCLUSION: The AI-based online platform could assist medical students in blood cell morphology learning. The AI system could function as a more knowledgeable other (MKO) and guide the students through their zone of proximal development (ZPD) to achieve mastery. It could be an effective and beneficial complement to microscopy learning. Students had very positive perspectives on the AI-based online learning platform. It should be integrated into the course and curriculum to facilitate the students.[Box: see text].

Rational design of stable heptamethine cyanines and development of a biomarker-activatable probe for detecting acute lung/kidney injuries via NIR-II fluorescence imaging.

Developing high-quality dyes to construct activatable probes for analyte sensing via NIR-II fluorescence is critical for attaining enhanced imaging depths and resolution. Heptamethine cyanines can serve this purpose; however, they usually have poor stability and a tendency to self-aggregate. Herein, we present a design strategy involving the installation of pyridinium and tert-butyl groups onto the central cyclohexenyl core to increase steric crowding, enhance water solubility, and provide a site for the incorporation of analyte-responsive elements. The resulting NP-N dyes emit NIR-II light and can outperform benchmark heptamethine cyanines such as ICG. Using HP-N1, we developed HP-H2O2 and showed that NIR-II fluorescence signals could be enhanced when treating with H2O2. HP-H2O2 was subsequently evaluated in murine models of acute lung injury and acute kidney injury. This strategy unlocks the potential of heptamethine cyanines and is applicable to examples with extended conjugation.

A Targeted Nanosystem for Detection of Inflammatory Diseases via Fluorescent/Optoacoustic Imaging and Therapy via Modulating Nrf2/NF-κB Pathways.

Inflammatory diseases are sometimes devastating and notoriously difficult to treat. Precisely modulating inflammatory signaling pathways is a promising approach for treating inflammatory diseases. Herein, a multifunctional nanosystem is developed for active targeting, activatable imaging and on-demand therapy against inflammatory diseases through modulating inflammatory pathways. A chromophore-drug dyad (QBS-FIS) is synthesized by linking a chromophore and a Nrf2 (nuclear factor E2-related factor) activator fisetin through boronate bond which serves as fluorescence quencher and ROS (reactive oxygen species)-responsive linker. QBS-FIS molecules form nanoparticles in water and are coated with macrophage cell membrane to ensure active targeting toward inflammation site. To further improve therapeutic efficacy, a NF-kB (nuclear-factor kappa-light-chain-enhancer of activated B cells) inhibitor thalidomide is co-encapsulated to afford the nanosystem (QBS-FIS&Thd@MM). Upon administration into mice, the nanosystem migrates to inflammatory site and pathological ROS therein cleaves the boronate bonds, thereby activating the chromophore for imaging liver/kidney inflammatory diseases for disease diagnosis and recovery evaluation via fluorescence and optoacoustic imaging as well as releasing the active drugs for treating acute liver inflammation through activating Nrf2 pathway and inhibiting NF-kB pathway. The 3D multispectral optoacoustic tomography imaging is applied to precisely locate the inflammatory foci in a spatiotemporal manner.

Applying low coverage whole genome sequencing to detect malignant ovarian mass.

To evaluate whether low coverage whole genome sequencing is suitable for the detection of malignant pelvic mass and compare its diagnostic value with traditional tumor markers. We enrolled 63 patients with a pelvic mass suspicious for ovarian malignancy. Each patient underwent low coverage whole genome sequencing (LCWGS) and traditional tumor markers test. The pelvic masses were finally confirmed via pathological examination. The copy number variants (CNVs) of whole genome were detected and the Stouffers Z-scores for each CNV was extracted. The risk of malignancy (RM) of each suspicious sample was calculated based on the CNV counts and Z-scores, which was subsequently compared with ovarian cancer markers CA125 and HE4, and the risk of ovarian malignancy algorithm (ROMA). Receiver Operating Characteristic Curve (ROC) were used to access the diagnostic value of variables. As confirmed by pathological diagnosis, 44 (70%) patients with malignancy and 19 patients with benign mass were identified. Our results showed that CA125 and HE4, the CNV, the mean of Z-scores (Zmean), the max of Z-scores (Zmax), the RM and the ROMA were significantly different between patients with malignant and benign masses. The area under curve (AUC) of CA125, HE4, CNV, Zmax, and Zmean was 0.775, 0.866, 0.786, 0.685 and 0.725 respectively. ROMA and RM showed similar AUC (0.876 and 0.837), but differed in sensitivity and specificity. In the validation cohort, the AUC of RM was higher than traditional serum markers. In conclusion, we develop a LCWGS based method for the identification of pelvic mass of suspicious ovarian cancer. LCWGS shows accurate result and could be complementary with the existing diagnostic methods.

Activatable Nanocomposite Probe for Preoperative Location and Intraoperative Navigation for Orthotopic Hepatic Tumor Resection via MSOT and Aggregation-Induced Near-IR-I/II Fluorescence Imaging.

The precise location of tumor and completeness of surgical resection are critical to successful tumor surgery; thus, the method capable of preoperatively locating a tumor site and intraoperatively determining tumor margins would be highly ideal. Herein, an activatable nanocomposite probe was developed for preoperatively locating orthotopic hepatic tumor via multispectral optoacoustic tomography imaging and for intraoperative navigation via near-IR-1 (NIR-I) and NIR-II fluorescence imaging. The molecular probe comprises an electronic donor, an acceptor, and a recognition moiety and forms the nanocomposite probe with bovine serum albumin. The probe specifically responds to nitroreductase overexpressed in tumor cells, which transforms the aromatic nitro group into an electron-donating amino group and thus activates the probe. The activated probe with the aggregation-induced emission feature generates strong NIR-I/NIR-II fluorescence and optoacoustic signals for dual-mode imaging. Owing to the in situ response toward nitroreductase in tumor cells in the hepatic region, the probe is found capable of detecting early stage orthotopic liver tumors. Furthermore, with the nanocomposite probe, we can obtain the 3D MSOT images to accurately locate orthotopic liver tumors preoperatively and the NIR-I/NIR-II fluorescence images to provide intraoperative guidance for tumor resection surgery.

A Novel Diagnosis Method Based on Methylation Analysis of SHOX2 and Serum Biomarker for Early Stage Lung Cancer.

OBJECTIVES: Lung cancer (LC) is often accompanied by significant methylation abnormalities. This study aimed to develop a decision tree (DT) accompanied the stature homeobox 2 gene (SHOX2) / prostaglandin E receptor 4 (PTGER4) gene DNA methylation with traditional tumor marker in the differential diagnosis of benign and malignant lung nodule. METHODS: We performed a study with 104 patients enrolled in the LC group and 36 patients in the benign lung diseases group. All the clinical data of these patients were collected through electronic medical record. Total Methylation (TM) status of both SHOX2 and PTGER4 was defined as methylation levels of SHOX2 plus methylation levels of PTGER4. One-way analysis was used to compare the concentrations of serum samples and t-test was used to compare pairwise mean values between groups. Receiver operating curve (ROC) was used to evaluate the diagnostic value. Furthermore, the strategy was validated in 19 LC patients and 11 patients with benign lung diseases. RESULTS: There were significant differences between the concentration of neuron-specific enolase (NSE), carcinoembryonic antigen (CEA), cytokeratin 19 fragments (CYFRA21 -1) and the methylation levels of SHOX2, PTGER4 and TM in lung benign diseases and cancer group. The AUCs of NSE, CEA, CYFRA21 -1, Methylation SHOX2, Methylation PTGER4 and TM were 0.721 (95% CI: 0.627-0.816), 0.753 (95% CI: 0.673-0.833) and 0.778(95% CI: 0.700-0.856), 0.851(0.786-0.916), 0.847(0.780-0.913) and 0.861(0.800-0.922) respectively. We developed a DT model with TM and CYFRA21 -1 used in this study, and the area under the curve (AUC) of DT was 0.921 and the sensitivity up to 0.856. In the validation cohort, the AUC of SHOX2, PTGER4 and TM was also much higher than traditional serum markers. CONCLUSIONS: Our results indicated that the DT model calculated from the TM and CYFRA21 -1 can accurately classify LC and benign diseases, which showed better diagnostic performance than traditional serum parameter.

Immature platelet fraction related parameters in the differential diagnosis of thrombocytopenia.

The pathogenesis of thrombocytopenia can be divided into increased destruction (ID) of platelets in the peripheral blood and decreased production (DP) of platelets in the bone marrow. This study aimed to analyze the efficacy of immature platelet fraction (IPF) related parameters, including the IPF count (IPF#), IPF percentage (IPF%) and highly fluorescence IPF percentage (H-IPF%), measured by XN-9000, in the differential diagnosis of thrombocytopenia. One hundred and twenty healthy volunteers were enrolled in the healthy control (HC) group, and 180 thrombocytopenia patients were grouped into either the increased destruction (ID) group or the decreased production (DP) group according to their final diagnosis. IPF# was significantly lower in the DP group than in the ID and HC groups (P < .01). Among the three groups, the ID group had the highest IPF% and H-IPF%, and the HC group had the lowest IPF% and H-IPF%. The differences between the three groups were all statistically significant (P < .01). In differentiating the ID patients from the DP patients, the areas under the operating characteristics curve of IPF#, IPF% and H-IPF% were 0.859, 0.944 and 0.930, respectively. False positive rates were below 0.04 when IPF#, IPF% and H-IPF% were above 2.65, 7.55 and 2.35, respectively. IPF related parameters showed high efficacy in the differential diagnosis of thrombocytopenia. However, due to the small numerical values of the IPF related parameters in some thrombocytopenia patients, the fluctuations of IPF% and H-IPF% should also be taken into consideration. Though H-IPF% is a new parameter, its effectiveness in the differential diagnosis of thrombocytopenia is not better than IPF%'s.

Differential diagnosis of tuberculous and malignant pleural effusions: comparison of the Th1/Th2 cytokine panel, tumor marker panel and chemistry panel.

The differentiation between tuberculous plural effusion (TPE) and malignant plural effusion (MPE) remains a major clinical challenge in the diagnosis and management of pleural effusions, especially in developing countries with a high incidence of tuberculosis. We aimed to evaluate the diagnostic value of cytokines, tumor markers and biochemical markers in the differentiation of TPE and MPE. Two hundred and forty-two patients were included, of whom 134 were diagnosed with MPE and 108 were diagnosed with TPE. In total, 12 markers were tested in pleural effusion samples from all subjects: Interleukin-2 (IL-2), Tumor necrosis factor alpha (TNF-α), Interferon (IFN)-γ, interleukins-4, 6, 10 (IL-4,6,10), cytokeratin-19 fragment (CYFRA 21-1), carcinoembryonic antigen (CEA), neuron specific enolase (NSE), adenosine deaminase (ADA), lactate dehydrogenase (LDH) and high sensitivity C- reactive protein (Hs-CRP). The diagnostic value of each marker was evaluated and compared by receiver operating characteristic (ROC) curves. In the 12 markers evaluated, TNF-α, IFN-γ, IL-6, CYFRA 21-1, CEA, ADA and Hs-CRP were significantly different between the TPE and MPE groups, and the areas under the ROC curves were 0.624, 0.942, 0.619, 0.808, 0.903, 0.842 and 0.917, respectively. IFN-γ showed a better diagnostic performance than the other markers. With a cut-off value of >2.45 pg/mL, the sensitivity and specificity of IFN-γ were 91.11 and 91.94%, respectively. TNF-α, IFN-γ, IL-6, CYFRA 21-1, CEA, ADA and Hs-CRP were useful in the differentiation between the TPE and MPE groups. IFN-γ showed a better diagnostic performance than the multitude of other markers evaluated in this study, which is satisfactory for the discrimination of TPE and MPE.

Nanoaggregate Probe for Breast Cancer Metastasis through Multispectral Optoacoustic Tomography and Aggregation-Induced NIR-I/II Fluorescence Imaging.

An activatable nanoprobe for imaging breast cancer metastases through near infrared-I (NIR-I)/NIR-II fluorescence imaging and multispectral optoacoustic tomography (MSOT) imaging was designed. With a dihydroxanthene moiety serving as the electron donor, quinolinium as the electron acceptor and nitrobenzyloxydiphenylamino as the recognition element, the probe can specifically respond to nitroreductase and transform into an activated D-π-A structure with a NIR emission band extending beyond 900 nm. The activated nanoprobe exhibits NIR emission enhanced by aggregation-induced emission (AIE) and produces strong optoacoustic signal. The nanoprobe was used to detect and image metastases from the orthotopic breast tumors to lymph nodes and then to lung in two breast cancer mouse models. Moreover, the nanoprobe can monitor the treatment efficacy during chemotherapeutic course through fluorescence and MSOT imaging.

Depletion of TRDMT1 affects 5-methylcytosine modification of mRNA and inhibits HEK293 cell proliferation and migration.

Human TRDMT1 is a transfer RNA (tRNA) methyltransferase for cytosine-5 methylation and has been suggested to be involved in the regulation of numerous developmental processes. However, little is known about the molecular mechanisms or their biological significance. In this study, we investigated the effects of CRISPR-based TRDMT1 knockdown on phenotypes, mRNA m5C modifications and gene expression changes in HEK293 cells. We found that knockdown of TRDMT1 significantly inhibited cell proliferation and migration but had no effect on clonogenic potential. The inhibitory effects could be attenuated by re-expression of TRDMT1 in HEK293 cells. RNA sequencing (RNA-Seq) and RNA bisulfite sequencing (RNA-BisSeq) were performed in TRDMT1 knockdown and wild-type HEK293 cells. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses indicated that the differentially expressed genes were associated with the cell cycle, RNA transport, and RNA degradation and were enriched in cancer and Notch signaling pathways. We also found that TRDMT1 knockdown could change mRNA methylation levels. For the first time, these findings clarify the role of TRDMT1 in regulating mRNA methylation and inhibiting the proliferation and migration of HEK293 cells. These results provide new insights into a new function of TRDMT1 and elucidate the molecular mechanisms of aberrant RNA m5C during tumorigenesis.

The Role of Cytokines in the Peripheral Blood of Major Depressive Patients.

BACKGROUND: Recent technological advances offer an opportunity to further elucidate the complex cytokine network in Major Depressive Disorder (MDD). The objective of this study was to investigate the role of pro-inflammatory and anti-inflammatory cytokines in the mechanism of depressive disorders. Given the activating role of cytokines on the hypothalamic-pituitary-adrenal (HPA) axis, and the relevance of its regulation in MDD, we also analyzed the relationships between several cytokines and cortisol levels. METHODS: Twenty-five unipolar depressive patients and 20 healthy controls were recruited in this study. Flow cytometric bead array system (FCM-CBA) was used to examine the concentration of cytokines (IL-2, IL-4, IL-6, IL-10, TNF, INF-α) in peripheral blood. Plasma Adrenocorticotropic Hormone (ACTH) and serum cortisol concentrations were detected. RESULTS: Compared with the controls, depressive patients had a significant increase in concentration of IL-2, TNF, serum cortisol, and TNF/IL-4 (p < 0.05). There was a significant positive correlation between serum cortisol and IL-2, as well as ACTH and IL-2 (p < 0.05) in depressive patients. There was a significant positive correlation between IL-2 and the Hamilton depression rating scale (HAMD) total scores in depressive patients, and also with TNF (p < 0.05). There was no significant difference in concentration of IL-4, IL-6, IL-10, and INF-α between two groups (p > 0.05). CONCLUSIONS: The present results suggest that depressive patients had an increase in concentration of some pro-inflammatory cytokines. Both IL-2 and TNF play important roles in the development of depressive disorders, and their concentration in peripheral blood may be used to evaluate the severity of depressive disorders.

Cyclin-dependent kinase-mediated Sox2 phosphorylation enhances the ability of Sox2 to establish the pluripotent state.

Sox2 is a key factor in maintaining self-renewal of embryonic stem cells (ESCs) and adult stem cells as well as in reprogramming differentiated cells back into pluripotent or multipotent stem cells. Although previous studies have shown that Sox2 is phosphorylated in human ESCs, the biological significance of Sox2 phosphorylation in ESC maintenance and reprogramming has not been well understood. In this study we have identified new phosphorylation sites on Sox2 and have further demonstrated that Cdk2-mediated Sox2 phosphorylation at Ser-39 and Ser-253 is required for establishing the pluripotent state during reprogramming but is dispensable for ESC maintenance. Mass spectrometry analysis of purified Sox2 protein has identified new phosphorylation sites on two tyrosine and six serine/Threonine residues. Cdk2 physically interacts with Sox2 and phosphorylates Sox2 at Ser-39 and Ser-253 in vitro. Surprisingly, Sox2 phosphorylation at Ser-39 and Ser-253 is dispensable for ESC self-renewal and cell cycle progression. In addition, Sox2 phosphorylation enhances its ability to establish the pluripotent state during reprogramming by working with Oct4 and Klf4. Finally, Cdk2 can also modulate the ability of Oct4, Sox2, and Klf4 in reprogramming fibroblasts back into pluripotent stem cells. Therefore, this study has for the first time demonstrated that Sox2 phosphorylation by Cdk2 promotes the establishment but not the maintenance of the pluripotent state. It might also help explain why the inactivation of CDK inhibitors such as p53, p21, and Arf/Ink4 promotes the induction of pluripotent stem cells.

Study on the Clinical Significance of JAK2V617F Allele Burden in Philadelphia Chromosome-Negative Myeloproliferative Neoplasm.

BACKGROUND: It was discovered that the somatic mutation in JAK2 exon 14 (JAK2V617F) totally modified the understanding and diagnosis of Philadelphia-Negative myeloproliferative neoplasm (Ph-MPNs), including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). Real-time quantitative PCR is the most widely used method for JAK2V617F detection in clinical laboratory. In this study, we aimed to evaluate the clinical significance of JAK2V617F allele burden in Ph-MPNs detected by real-time quantitative PCR. METHODS: A total of 208 bone marrow samples were collected from patients suspected to have Ph-MPNs. Real-time quantitative PCR was performed on each sample to obtain the JAK2V617F allele burden. Clinical and laboratory data from these participants were also recorded for their first visit. RESULTS: Out of 208 participants, 118 patients were confirmed with Ph-MPNs. JAK2V617F mutations were found in 59 patients in the PV group (86.8%), 31 patients in the ET group (70.5%). PV, PMF, and ET showed a significant difference in the distribution of JAK2V617F allele burden. In JAK2V617F positive patients, JAK2V617F allele burden was closely related with WBC counts, platelet counts, and hemoglobin concentration. CONCLUSIONS: JAK2V617F allele burden is a useful marker in the diagnosis, discrimination, and evaluation of PhMPNs.

Flow cytometry immunophenotypic findings in chronic myelomonocytic leukemia and its utility in monitoring treatment response.

Chronic myelomonocytic leukemia (CMML) is a myelodysplastic/myeloproliferative neoplasm, characterized by persistent monocytosis. Due to the lack of unique surface markers expressed by neoplastic monocytes and the frequent CD34-negative blast immunophenotype, the diagnostic value of flow cytometric immunophenotyping (FCI) in CMML is rarely studied. In this study, by using a multicolor FCI assay, we assessed bone marrow (BM) immunophenotypical alterations in 118 CMML patients and follow-up BM samples in 35 of these patients. The median BM monocytes as determined by FCI were 14% (1-63%), correlated with morphologic count (P = 0.0004). FCI alterations in monocytes were observed in 96% and granulocytes in 83% of cases. The percentage of CD34(+) myeloblasts by FCI was low [median 0.6% (0.02-12.6%)], but exhibiting frequent aberrancies [median 6 (2-12)]. CD34(+) B-cell precursors were absent in 93% of cases. In 35 patients with follow-up BM samples assessed, the CD34(+) myeloblasts showed persistent FCI aberrancies in all 29 patients treated with hypomethylating agents and 3 patients on observation, but became normal in 3 patients following stem cell transplant. In conclusion, CMML exhibit numerous FCI alterations in monocytes, granulocytes, and more profound/frequent in CD34(+) myeloblasts. These findings provide solid evidence for using FCI as an ancillary test in CMML diagnosis and also, in assessment of treatment responses.

The clinical significance of negative flow cytometry immunophenotypic results in a morphologically scored positive bone marrow in patients following treatment for acute myeloid leukemia.

In a patient with acute myeloid leukemia (AML) following therapy, finding ≥5% bone marrow (BM) blasts is highly concerning for residual/relapsed disease. Over an 18-month period, we performed multicolor flow cytometry immunophenotyping (MFC) for AML minimal residual disease on >4,000 BM samples, and identified 41 patients who had ≥5% myeloblasts by morphology but negative by MFC. At the time of a negative MFC study, an abnormal cytogenetic study converted to negative in 14 patients and remained positive at a low level (2.5-9.5%) by fluorescence in situ hybridization in 3 (14%), of the latter, abnormalities subsequently disappeared in the repeated BM in 2 patients. Positive pretreatment mutations, including FLT3, NPM1, IDH1, CEBPA, became negative in all 10 patients tested. Of the seven patients with favorable cytogenetics, PML/RARA, CBFB-MYH11 or RUNX1-RUNX1T1 fusion transcripts were detected at various levels in six patients but all patients remained in complete remission. With no additional chemotherapy given, 39 patients had BM repeated (median 2 weeks, range <1-21), and all cases showed <5% BM blasts and a continuously negative MFC. In the end of follow-up (median 10 months, range 1-22), 13 patients experienced relapse, 12/13 showing clonal cytogenetic evolution/switch and 11 demonstrating major immunophenotypic shifts. We conclude that MFC is useful in identifying a regenerating BM sample with ≥5% BM blasts that would otherwise be scored as positive using standard morphologic examination. We believe this conclusion is supported by the changes in molecular cytogenetic status and the patient clinical follow-up data.

Synthesis of NaYF4 and NaLuF4 Based Upconversion Nanocrystals and Comparison of Their Properties.

In this study, four kinds of upconversion nanocrystals (UCNs) have been successfully synthesized by a facile solvothermal method. The morphology, crystalline phase, composition, grain size, upconversion luminescence and cell image of the UCNs were investigated. The properties of the NaLuF4-based UCNs were compared with the counterparts of NaYF4-based UCNs. It is found that the NaLuF4-based UCNs are apt to form hexagonal phase structures, while NaYF4-based UCNs of NaYF4:Yb, Er and NaYF4:Gd, Yb, Er are cubic and hexagonal phases respectively. The upconversion emission intensities of the NaLuF4-based UCNs are higher than that of NaYF4-based UCNs, and Gd3+ presented UCNs are higher than that of Gd3+ absented UCNs. The bioimaging application of NaLuF4:Gd, Yb, Er shows that bright upconversion luminescence can be observed when UCNs-labeled HeLa cells are excited with 980 nm light.

Synthesis of fluorescent and magnetic bi-functional NaLuF4-based upconversion nanocrystals.

Fluorescent and magnetic bi-functional NaLuF4-based upconversion nanocrystals have been developed by a facile thermal decomposition method. The influences of Mn2+ and Gd3+ doping on the upconversion luminescent and magnetic properties of the nanocrystals were investigated. The results show that Mn2+ doping can promote the transition of green to red and increase the red emission significantly. When the Mn2+ and Gd3+ are co-doped in the nanocrystals, it has a synergistic effect which can further improve the transition and red emission. Moreover, the nanocrystals co-doped with Mn2+ and Gd3+ exhibit excellent superparamagnetic properties. These results suggest that the developed nanocrystals, which have strong pure red upconversion emission and superparamagnetic properties, can serve as a potential upconversion luminescence and magnetic resonance probe for dual-modality bioimaging. This work presents a facile and effective method for modulating the transition of short-wavelength emission to red emission of upconversion nanocrystals, leading the naocrystals exhibiting excellent upconversion luminescence and magnetic properties.