Application of Blood Lymphocyte Immunophenotype and TCR Gene Rearrangement in the Diagnosis of CTCL.

BACKGROUND: The immunophenotype of peripheral blood lymphocytes and T-cell receptor (TCR) gene rearrangement of cutaneous T cell lymphoma (CTCL) patients were retrospectively analyzed to explore their value in the diagnosis of CTCL. METHODS: A total of fifty patients' results were enrolled from 2013 to 2021, including 29 malignant skin disorders and 21 benign skin disorders. The immunophenotype of peripheral blood lymphocytes were analyzed by flow cytometry and TCR gene rearrangement was detected by capillary electrophoresis. Lymphocyte subsets, CD4/CD8 ratio, the percentage of CD3+CD4+CD7- cells and CD45RA/CD45RO ratio was calculated between malignant and benign skin disorders. Peripheral blood lymphocyte immunophenotype and TCR gene rearrangement was compared with skin biopsy to evaluate their sensitivity and specificity. RESULTS: Lymphocyte subsets between malignant and benign groups have no significant difference in percentage of T cell (p > 0.05). The CD4/CD8 ratio is higher in patients with malignant lymphoma than the healthy range. The percentage of CD3+CD4+CD7- cells in malignant groups is higher than that in benign groups and CD45RA/ CD45RO ratio has significant difference between malignant and benign groups (p < 0.05). The sensitivity and specificity of TCR rearrangement for CTCL were 51.7% and 42.9%. The sensitivity and specificity of peripheral blood lymphocyte immunophenotype for CTCL were 44.8% and 33.3%. Combining the two methods, the sensitivity and specificity reached 69.0% and 38.1%, respectively. CONCLUSIONS: CD4/CD8 ratio of lymphocyte subsets, the proportion of CD4+CD7-T cells and CD45RA/CD45RO ratio can effectively distinguish benign and malignant dermatosis. TCR rearrangement method combined with lymphocyte immunophenotype can improve the sensitivity and specificity of CTCL diagnosis.

HLA-DR Helps to Differentiate Erythrodermic Cutaneous T-cell Lymphoma from Erythrodermic Inflammatory Dermatoses in Flow Cytometry.

Differential diagnosis of erythroderma is challenging in dermatology, especially in differentiating erythrodermic cutaneous T-cell lymphoma from erythrodermic inflammatory dermatoses. This study retrospectively reviewed the peripheral blood flow cytometric results of 73 patients diagnosed with erythroderma at Peking University First Hospital from 2014 to 2019. The flow cytometry antibody panel included white blood cell markers, T-cell markers, B-cell markers, T-cell activation markers, and T helper cell differentiation markers. Features of the cell surface antigens were compared between 34 patients with erythrodermic cutaneous T-cell lymphoma and 39 patients with erythrodermic inflammatory dermatoses. The percentage of HLA-DR+/CD4+T cells was the most pronounced marker to distinguish erythrodermic cutaneous T-cell lymphoma from erythrodermic inflammatory dermatoses, with a threshold of 20.85% (sensitivity 96.77%, specificity 70.37%, p = 0.000, area under the curve (AUC) 0.882), suggesting its potential capability in the differential diagnosis of erythrodermic cutaneous T-cell lymphoma from erythrodermic inflammatory dermatoses. Moreover, in contrast to erythrodermic inflammatory dermatoses, the percentage of Th17 cells was significantly downregulated in erythrodermic cutaneous T-cell lymphoma (p = 0.001), demonstrating a dysregulated immune environment in erythrodermic cutaneous T-cell lymphoma.

A Single-Tube 21-Color Multiparameter Flow Cytometry Improve the Sensitivity of B-ALL MRD Analysis.

BACKGROUND: Detection of minimal residual disease (MRD) by multiparameter flow cytometry (MFC) is a well-established risk stratification factor and therapeutic modification strategy in B acute lymphoblastic leukemia (B-ALL). However, current 8 color (8c)-MFC for MRD detection had the sensitivity of 0.01% with false negative or positive. Hence, a more sensitive and applicable MFC-MRD method is urgently needed. The aim of this study is to establish a single-tube 21c-MFC method to detect B-ALL MRD, evaluate its performance, and to investigate its preliminary clinical application. METHODS: We selected 21 markers to establish a single-tube 21c-MFC method. The repeatability and sensitivity of this method was validated by adding Nalm-6 cells to normal bone marrow. Samples from control group (n = 6), B-ALL group (n = 7) and complete remission (CR) group (n = 26) were detected by 21c- and 8c-MFC separately. The expression characteristics of these markers was analyzed in control and B-ALL group, and the consistency of 21c- and 8c-MFC in detecting MRD was compared. RESULTS: Repeatability of this method was 1.91% of CV and sensitivity was up to 0.005%. In control group, the expression of CD81, CD97, and CD200 gradually decreased and CD44, HLA-DR, CD73, and CD72 gradually increased with the maturation of normal B cells. In B-ALL group, CD73stro, CD81low, CD44stro, CD123stro, and CD58stro showed high-frequency expression. The consistency rate of 21c- and 8c-MFC in detecting MRD was 96%. CONCLUSIONS: A single-tube 21c-MFC method was established for MRD detection in B-ALL and had higher sensitivity than the 8c-MFC method.

Artificial intelligence of digital morphology analyzers improves the efficiency of manual leukocyte differentiation of peripheral blood.

BACKGROUND AND OBJECTIVE: Morphological identification of peripheral leukocytes is a complex and time-consuming task, having especially high requirements for personnel expertise. This study is to investigate the role of artificial intelligence (AI) in assisting the manual leukocyte differentiation of peripheral blood. METHODS: A total of 102 blood samples that triggered the review rules of hematology analyzers were enrolled. The peripheral blood smears were prepared and analyzed by Mindray MC-100i digital morphology analyzers. Two hundreds leukocytes were located and their cell images were collected. Two senior technologists labeled all cells to form standard answers. Afterward, the digital morphology analyzer unitized AI to pre-classify all cells. Ten junior and intermediate technologists were selected to review the cells with the AI pre-classification, yielding the AI-assisted classifications. Then the cell images were shuffled and re-classified without AI. The accuracy, sensitivity and specificity of the leukocyte differentiation with or without AI assistance were analyzed and compared. The time required for classification by each person was recorded. RESULTS: For junior technologists, the accuracy of normal and abnormal leukocyte differentiation increased by 4.79% and 15.16% with the assistance of AI. And for intermediate technologists, the accuracy increased by 7.40% and 14.54% for normal and abnormal leukocyte differentiation, respectively. The sensitivity and specificity also significantly increased with the help of AI. In addition, the average time for each individual to classify each blood smear was shortened by 215 s with AI. CONCLUSION: AI can assist laboratory technologists in the morphological differentiation of leukocytes. In particular, it can improve the sensitivity of abnormal leukocyte differentiation and lower the risk of missing detection of abnormal WBCs.

Patterns and levels of platelet glycosylation in patients with coronary heart disease and type 2 diabetes mellitus.

Coronary heart disease (CHD) and diabetes mellitus (DM) have high reactivity of platelets and an increased risk of thrombosis. Platelet glycosylation is closely related to platelet function and survival. However, the alteration of platelet glycosylation in CHD and T2DM still remains unknown. Platelet samples were obtained from 55 healthy controls and 102 patients, including 33 CHD, 30 T2DM and 39 CHD complicated with T2DM (CHD + T2DM). Platelet glycosylation was detected using eight-lectin based assay by flow cytometry. Platelet activation markers, such as CD62P (P-Selectin) and activated integrin GPIIb/IIIa (PAC-1), were measured on resting and stimulated conditions by flow cytometry. Platelet aggregation was measured by light transmission aggregometry. In CHD group, platelet surface weakly expressed ß-Gal and 2,6-sialic acid and strongly expressed ß-GlcNAc. In T2DM group, lectins binding to platelet of ß-Gal, 2,6-sialic acid and α-mannose were decreased, while α1,6-fucose and GlcNAc were increased. There was positive correlation between ConA (specific for α-mannose) and PAC-1 in T2DM patients, while negative correlation in healthy controls. Patterns and levels of platelet glycosylation in CHD + T2DM group are a combination of CHD group and T2DM group, in addition to the level of ECL highly elevated (specific for ß-Gal). The level of ConA was significantly correlated with glucose in T2DM group, also correlated with HbA1c in CHD + T2DM. Our findings suggested that platelets decreased in sialylation, galactosylation and mannosylation, and increased in fucosylation and GlcNAcylation in CHD and T2DM patients. The changes of platelet glycosylation may be associated with high platelet reactivity and the increased risk of thrombosis in CHD and T2DM.

Efficiency of Leukocyte Differential Using Flow Cytometry with CytoDiff in Different Workflows.

BACKGROUND: To set up the review criteria for flow cytometry with CytoDiff (FCC) and evaluate the efficiency of different workflows using combinations of FCC with a hematology analyzer and microscopy. METHODS: Leucocyte differentials of the samples from 995 clinical specimens and 278 specimens from healthy donors were analyzed using a hematology analyzer, FCC, and microscopy. RESULTS: The correlations between the hematology analyzer, FCC, and microscopy were good for neutrophils, lymphocytes, and monocytes, but not in the case of basophils (r = 0.464, 0.358, 0.33) and eosinophils (r = 0.69, 0.67, 0.621). As a reference method of WBC differential using microscopy, the threshold of blasts for FCC was defined by a ROC curve at 1% (specificity 97.9%, sensitivity 97.5%, AUC 0.989). The optimal cutoff of immature granulocytes for FCC was 1% (specificity 85.8%, sensitivity 76.5%, AUC 0.866). The optimal cutoff of lymphocytes, neutrophils, and monocytes were 50%, 85% and 12%, respectively. According to the review criteria, the workflow of CBC after FCC and then microscopy had the highest sensitivity (97.07%), lower false negative rate (2.93%), and higher accuracy (80.3%) compared with others. CONCLUSIONS: Our study integrated FCC into a WBC differential workflow in a routine laboratory and, for the first time, demonstrates the efficiency of different workflows. It can be used for reference in the selection of different hematology workflows.

The expression of Fcγ receptors in Hashimoto's thyroiditis.

The pathophysiological mechanism underlying Hashimoto's thyroiditis (HT) is still unclear. Thyroglobulin antibody (TgAb) and thyroid peroxidase antibody (TPOAb) are diagnostic hallmarks of HT. These IgG antibodies regulate the balance of immunologic tolerance and autoimmunity via Fcγ receptors (FcγRs). The aim of our study was to investigate the role of FcγRs in the pathogenesis of HT. The percentage of peripheral blood mononuclear cells (PBMCs) from HT patients bearing FcγRII was significantly lower than that seen in healthy donors, and the mean fluorescence intensity (MFI) value of FcγRII on PBMCs from HT patients was significantly higher. The percentage of PBMCs positive for FcγRIII also was significantly higher in HT patients, and the percentage of B cells bearing FcγRIIB in HT patients was significantly lower than that seen in healthy donors. Our study therefore provides evidence for FcγRs, especially FcγRIIB, being involved in the pathogenesis of HT.

[Reference intervals and its verification for leukocyte differential count in peripheral blood by CytoDiff flow cytometry].

OBJECTIVE: To establish and verify the reference intervals for leukocyte differential count in peripheral blood by CytoDiff flow cytometry. METHODS: Leukocyte differentiation count was analyzed by using hematology analyzer, CytoDiff flow cytometry and morphology differential count in 278 healthy controls, 550 flagged and 102 unflagged samples. The reference intervals of leukocyte parameters were established and verified according to the healthy controls. Then the correlations of five leukocytes were analyzed among hematology analyzer, CytoDiff flow cytometry and morphology differential count. Lymphocyte subsets were analyzed and compared between healthy controls and patients with different diseases. The CytoDiff flow cytometric blast counts were explored to analyze the clinical diagnostic efficiency compared to morphology differential count as a reference method. RESULTS: CytoDiff flow cytometry can differentiate the leukocyte into 16 parameters, including percentage and absolute count, therefore 32 parameters in total. Among these parameters, 18 parameters had significant difference between male and female (P < 0. 05), and the others had no difference (P >0. 05). Except the CD16pos monocyte, there were no difference among ages in other parameters. The correlation between hematology analyzer, CytoDiff flow cytometry and morphology differential count were good for neutrophils, lymphocytes, monocytes, basophils and eosinophils in healthy controls and clinical samples. When the cutoff value of the ratio of T + NK and B lymphocyte was set at 1. 0 by ROC, the sensitivity was 90. 9% and specificity was 99. 8% for diagnosing the chronic lymphocyte leukemia (CLL). When the cutoff value of blast count by CytoDiff flow cytometry was set at 1%, the sensitivity was 100%, specificity was 96. 1% and accuracy was 96. 2% by morphology differential blast count for the reference method. CONCLUSIONS: Establish and verify the reference interval of leukocyte differential count in peripheral blood by CytoDiff flow cytometry in the adults of Beijing region. CytoDiff flow cytometry provide more parameters than hematology analyzer and morphology differential count, and therefore have excellent clinical diagnostic efficiency, especially for CLL and blasts from acute leukemia.

[Clinical evaluation of cell population data parameters in predicting engraftment after hematopoietic stem cell transplantation].

OBJECTIVE: To explore the cell population data (CPD) parameters of DxH800 hematology analyzer in predicting engraftment after hematopoietic stem cell transplantation. METHODS: Data from 109 patients who underwent hematopoietic stem cell transplantation at Peking University First Hospital between July 2013 and September 2014 were selected, all CBC data including CPD parameters from the beginning to engraftment (absolute neutrophil count > 500/µl) were gathered and analyzed to find the early markers of myeloid engraftment and establish the cut-off value. RESULTS: The makers which could predict myeloid engraftment were mean neutrophil volume (MNV) and mean monocyte volume (MMV), the cut-off value was ≥ 180 fl and ≥ 190 fl, respectively, and engraftment was predicted earlier (2.2 ± 1.3) days and (2.3 ± 1.7) days than absolute neutrophil count. CONCLUSION: By analyzing the CPD data of DxH800 hematology analyzer, engraftment can be predicted approximately 2 days earlier than absolute neutrophil count.

B cell pathway dual inhibition for systemic lupus erythematosus: a prospective single-arm cohort study of telitacicept.

Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease associated with B-cell hyperactivity. Telitacicept is a transmembrane activator, calcium modulator, and cyclophilin ligand interactor-Fc fusion protein, which can neutralize both B-cell lymphocyte stimulator and a proliferation-inducing ligand. Patients with active SLE who received telitacicept were prospectively followed at month 1, 3, 6, 9, and 12 after telitacicept initiation. Thirty-seven participants were involved and followed for 6.00 [3.00, 6.00] months. SRI-4 rate at month 6 was 44.7%. The median dosage of prednisone was decreased by 43.8% (from 10 to 5.62 mg/d) at month 6. The anti-dsDNA level was significantly decreased, while complement levels were significantly increased at month 6 from baseline. Continuously significant reductions in serum immunoglobin (Ig)G IgA, and IgM levels were also observed. Patients experienced significant decreases in the numbers of total and naive B cells, whereas memory B cells and T cell populations did not change. The number of NK cells was significantly increased during the follow-up. At month 6, 58.3% (14 out of 24) patients experienced improved fatigue accessed by FACIT-Fatigue score exceeding the minimum clinically important difference of 4. Most adverse events were mild, but one each case of severe hypogammaglobulinemia, psychosis with suicidal behavior, and B-cell lymphoma were occurred. In our first prospective real-world study, telitacicept treatment led to a significant clinical and laboratory improvement of disease activity, as well as fatigue amelioration in patients with SLE. Safety profile was favorable overall, but more studies are greatly needed.

Performance of the digital cell morphology analyzer MC-100i in a multicenter study in tertiary hospitals in China.

BACKGROUND: This study investigated the performance of the MC-100i, a pre-commercial digital morphology analyzer utilizing a convolutional neural network algorithm, in a multicentric setting involving up to 11 tertiary hospitals in China. METHODS: Blood smears were analyzed by MC-100i, verified by morphologists, and manually differentiated. The classification performance on WBCs and RBCs was evaluated by comparing the classification results using different methods. The PLT and PLT clump counting performance was also assessed. The total assay time including hands-on time was evaluated. RESULTS: The agreements between pre- and post-classification were high for normal WBCs (κ > 0.96) and lower for overall abnormal WBCs (κ = 0.90). The post-classification results correlated well with manual differentials for both normal and abnormal WBCs (r > 0.93), except for basophils (r = 0.8480) and atypical lymphocytes (r = 0.8211). The clinical sensitivity and specificity of each RBC abnormality after verification were above 90 % using microscopy reviews as the reference. The PLTs counted by the MC-100i before and after verification correlated well with those measured by the PLT-O mode (r = 0.98). Moreover, PLT clumps were successfully classified by the analyzer in EDTA-dependent pseudothrombocytopenia blood samples. CONCLUSIONS: The MC-100i is an accurate and reliable digital cell morphology analyzer, offering another intelligent option for hematology laboratories.

IGH rod-like tracer: An AlphaFold2 structural similarity extraction-based predictive biomarker for MRD monitoring in pre-B-ALL.

Sequence variation resulting from the evolution of IGH clones and immunophenotypic drift makes it difficult to track abnormal B cells in children with precursor B cell acute lymphoblastic leukemia (pre-B-ALL) by flow cytometry, qPCR, or next-generation sequencing (NGS). The V-(D)-J regions of immunoglobulin and T cell receptor of 47 pre-B-ALL samples were sequenced using the Illumina NovaSeq platform. The IGH rod-like tracer consensus sequence was extracted based on its rod-like alpha-helices structural similarity predicted by AlphaFold2. Additional data from published 203 pre-B-ALL samples were applied for validation. NGS-IGH (+) patients with pre-B-ALL had a poor prognosis. Consistent CDR3-coded protein structures in NGS-IGH (+) samples could be extracted as a potential follow-up marker for pre-B-ALL children during treatment. IGH rod-like tracer from quantitative immune repertoire sequencing may serve as a class of biomarker with significant predictive values for the dynamic monitoring of MRD in pre-B-ALL children.

Association between urine microscopy and severe acute kidney injury in critically ill patients following non-cardiac surgery: a prospective cohort study.

BACKGROUND: Acute kidney injury (AKI) is a common and adverse complication following non-cardiac surgery. Evidence have shown urine microscopy could help early detection, differentiating the causes and predicting the progression of AKI. However, little evidence is available on AKI after non-cardiac surgery. Thus, we investigated the association between urine microscopy and severe AKI in critically ill patients after non-cardiac surgery. METHODS: This was a single-center prospective cohort study. The primary outcome was severe AKI, defined as stage 2 or 3 according to maximal KDIGO criteria within 7 days following non-cardiac surgery. Urine microscopy immediately, 6 and 12 hours after surgical intensive care unit (SICU) admission were examined and graded by a urine microscopy score (UMS) based on the observed quantification of renal tubular cells and casts in the sediment. Then, multivariate Logistic regression models were used to analyze the associations between UMS and postoperative severe AKI. RESULTS: From May 20, 2019 to November 24, 2020, 661 patients were enrolled with 147 patients (22.2%) developing postoperative severe AKI. Multivariate Logistic regression model showed elevated UMS (≥1) 6 and 12 hours after SICU admission were independently associated with postoperative severe AKI (OR 2.200, 95% CI: 1.182-4.095, P=0.013 and OR 2.949, 95% CI: 1.657-5.248, P<0.001, respectively). Furthermore, higher UMS 6 hours after SICU admission demonstrated correlation with greater risk of severe AKI with OR 3.887 (95% CI: 1.430-10.563) for UMS ≥3 and OR 2.429 (95% CI: 1.237-4.770) for UMS =1-2. The specificity and sensitivity of UMS ≥1 for severe AKI was 93.8% (95% CI: 91.7-95.9%) and 15.6% (95% CI: 9.7-21.5%), respectively. While the negative and positive predictive value was 79.5% (95% CI: 76.3-82.7%) and 41.8% (95% CI: 28.8-54.8%), respectively. In addition, patients with higher UMS (≥3, 1-2 and 0) had significantly more postoperative complications and longer SICU stay; and they also showed a trend toward other adverse postoperative outcomes. CONCLUSIONS: Early urine microscopy was independently associated with severe AKI in critically ill patients following non-cardiac surgery with higher UMS related to greater risk. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT03880110.

CD11c+ B Cells Participate in the Pathogenesis of Graves' Disease by Secreting Thyroid Autoantibodies and Cytokines.

Graves' disease (GD) is a common autoimmune disorder with an elevation in pathogenic autoantibodies, specifically anti-thyrotropin receptor antibodies (TRAbs), which are secreted by autoreactive B cells. To date, there has been little research on self-reactive B cells in GD. In the current study, we reported that a unique B-cell subset, CD11c+ B cells, was expanded in the peripheral blood (PB) of GD patients, as detected by flow cytometry. The frequency of CD11c+ B cells was positively correlated with serum TRAb levels. The flow cytometry data showed that CD11c expression was higher in a variety of B-cell subsets and that CD11c+ B cells presented a distinct immunophenotype compared to paired CD11c- B cells. Immunohistochemical and immunofluorescence staining indicated the presence of CD11c+CD19+ B cells in lymphocyte infiltration areas of the GD thyroid. Flow cytometric analysis of PB and fine-needle aspiration (FNA) samples showed that compared to PB CD11c+ B cells, CD11c+ B cells in the thyroid accumulated and further differentiated. We found that CD11c+ B cells from the PB of GD patients were induced to differentiate into autoreactive antibody-secreting cells (ASCs) capable of secreting TRAbs in vitro. Luminex liquid suspension chip detection data showed that CD11c+ B cells also secreted a variety of cytokines, including proinflammatory cytokines, anti-inflammatory cytokines, and chemokines, which might play roles in regulating the local inflammatory response and infiltration of lymphocytes in the thyroid. In addition, we performed a chemotaxis assay in a Transwell chamber to verify that CD11c+ B cells were recruited by thyroid follicular cells (TFCs) via the CXCR3-CXCL10 axis. In conclusion, our study determined that CD11c+ B cells were involved in the pathogenesis of GD in multiple ways and might represent a promising immunotherapeutic target in the future.

Clinical Evaluation of the Pre-Analytical Capabilities of Hemostasis Instrument.

Objective: Evaluate the technical performance of the pre-analytical hemolysis-icterus-lipemia (HIL) check module on the ACL-TOP-750. Methods: 8433 routine coagulation samples were evaluated for HIL, the presence of clotting and low sample volume by both visual inspection and the pre-analytical HIL check module on the ACL-TOP-750. Results: 7726 samples were in agreement with both methods and 707 were not consistent. 356 samples with low volume were identified by visual inspection and 920 by the instrument (2.7 mL threshold). Visual inspection identified 56 lipemic samples while 13 of those with moderate or high lipemia were identified by the instrument. Visual inspection identified 47 hemolyzed samples while 7 with moderate or high hemolysis were identified by the instrument. Both visual inspection and the instrument identified 36 icteric samples. For triglyceride concentration and bilirubin concentration, there was good correlation between the ACL-TOP-750 and the DXC800 biochemistry analyzer. Among 30 samples with varying amounts of clotting, 27 were discovered by visual inspection and 3 were discovered by the instrument. Conclusion: The pre-analytical check module on the ACL-TOP-750 improved the detection rate of samples below the target 2.7 mL volume, and the accuracy in detection of HIL. However, the automated method could not replace visual assessment of clotting in samples.

Establishment of the reference intervals of lymphocyte subsets for healthy Chinese Han adults and its influencing factors.

BACKGROUND: Cellular immune monitoring is becoming more critical in the clinic, but its application has not yet become sufficiently widespread. One reason may be the different reference intervals among clinical laboratories due to several factors. Percentage and number of lymphocyte subsets are standard indicators of cellular immune detection. The present study aimed to establish standardized reference intervals of lymphocyte subsets in the healthy Chinese Han adult population and examine such influencing factors as age, gender, region, and measurement instruments. METHODS: A total of 496 healthy Chinese Han people aged 18-59 years from 3 China Mainland regions (north, east, and south) were enrolled. The sample of each center was simultaneously examined by three flow cytometers (FACSCantoTMII, FACSLyricTM, and FACSCaliburTM). A single-platform flow cytometry-based absolute count technique was used to quantify the percentage and number of each lymphocyte subset. The flow cytometry results were analyzed by variance analysis and Z test to determine the influence of age, gender, and instruments on lymphocyte subsets. RESULTS: Multi-center, age-specific, and gender-specific reference intervals of healthy Chinese Han adults' lymphocyte subsets were established. There was no statistical difference in the results from the three flow cytometers. Gender affected the results of CD4+ (%) and the absolute count of CD3-CD16+CD56+, where CD4+ (%) was higher in women, and the absolute count of CD3-CD16+CD56+ was higher in men. Age mainly affected the CD4+/CD8+ ratio, which was statistically higher in groups aged over 40 years; the percentage and number of CD3-CD19+ were more elevated in age groups below 30 years; however, the difference was not statistically significant. CONCLUSIONS: This study established the reference intervals of lymphocyte subsets for healthy Chinese Han adult populations under the standardized methods. This study was the first nationwide study in China to use a flow cytometry-based single-platform method to establish the reference intervals of lymphocyte subsets of the healthy Chinese Han adult population. Gender and age were shown to influence the results of lymphocyte subsets.

Cytokines in the Immune Microenvironment Change the Glycosylation of IgG by Regulating Intracellular Glycosyltransferases.

Background: Changes in IgG glycosylation, as a novel pathological feature, are observed in various autoimmune diseases (AIDs). The glycosylation patterns of IgG play a critical role in regulating the biological function and stability of IgG involved in the pathophysiology of many AIDs. However, the intracellular regulatory mechanisms underlying the effects of disturbances in various cytokines on IgG glycosylation are poorly understood. Thus, we investigated the regulatory effects of elevated cytokines in AIDs on intracellular IgG glycosylation within B cells. Methods: First, we established a controlled primary culture system in vitro to differentiate human CD19+ B cells into antibody-secreting cells (ASCs). Then, the IgG concentrations in the supernatants were measured by enzyme-linked immunoassay (ELISA) under IFN-γ, TNF-α, IL-21, IL-17A, BAFF, or APRIL stimulation. Next, the glycosylation levels of IgG under different stimuli were compared via a lectin microarray. The fine carbohydrate structures of IgG were confirmed by matrix-assisted laser desorption/ionization-quadrupole ion trap-time of flight-mass spectrometry (MALDI-TOF-MS). Finally, the expression of glycosyltransferases and glycosidases in B cells under stimulation with several cytokines was detected by real-time PCR and western blotting. Results: We found that cytokines significantly promoted IgG production in vitro and led to considerably different IgG glycan patterns. Specifically, the results of lectin microarray showed the galactose level of IgG was increased by IFN-γ stimulation (p<0.05), and the sialylation of IgG was increased by IL-21 and IL-17A (p<0.05). The MALDI-TOF-MS data showed that the frequency of agalactosylation was decreased by IFN-γ with the increased frequency of mono-galactosylation and decreased frequency of digalactosylation, accompanied by upregulation of ß-1,4-galactosyltransferase 1. Both frequencies of mono-sialylated and disialylated N-glycans were increased by IL-21 and IL-17A with decreased frequency of asialylation, and the expression of ß-galactoside α-2,6-sialyltransferase 1 was upregulated by IL-21 and IL-17A. Conclusion: Abnormally elevated cytokines in the microenvironment regulates IgG glycan patterns by regulating intracellular glycosyltransferases in human B cells.

The platelet surface glycosylation caused by glycosidase has a strong impact on platelet function.

: Platelet surface glycosylation defects has been reported to be significantly associated with many diseases. Our previous study found that platelet surface glycosylation is altered in coronary heart disease. In this study, we further investigated whether altered glycosylation affects platelet function. Platelets were obtained from ten healthy volunteers. The platelet surface terminal sialic acid was removed by neuraminidase A, and N-linked oligosaccharides was removed by PNGase F. The function of the enzyme-treated platelet was measured. The activation and platelet adhesion to von Willebrand factor (vWF) was measured by flow cytometry. Platelet aggregation induced by ADP, arachidonic acid and collagen was detected through light transmission aggregometry, and platelet-leukocyte aggregates (PLAs) was detected by flow cytometry. Neuraminidase A treatment caused sialic acid level decrease and ß-galactose level increase significantly on platelet surface. Activation marker CD62P did not change. Platelet adhesion to vWF was increased significantly (P < 0.05). ADP-induced platelet aggregation was significantly reduced (P < 0.05). Platelet-granulocytes aggregates and platelet-monocytes aggregates increased (P < 0.05). Platelet surface sialic acid was increased after PNGase F treatment. Platelet aggregation by all agonists were significantly reduced (P < 0.05). There is no difference in the binding of vWF and PLAs for PNGase F treated platelet. We demonstrated that asialoglycosylation enhances platelet binding to vWF and forming PLAs, suggest that it may be associated with high platelet reactivity and the increased risk of thrombosis.

CD26 expression is down-regulated on CD8+ T cells in patients with Hashimoto's thyroiditis.

The immune mechanism underlying Hashimoto's thyroiditis (HT) remains unclear. CD26, also known as dipeptidyl peptidase 4 (DPP-4), is a multifunctional molecule involved in the pathophysiology of autoimmune diseases. This study aimed to investigate the role of CD26 in the pathogenesis of HT. Peripheral blood was drawn from 20 healthy controls and 31 HT patients (19 mild HT patients and 12 severe HT patients). Plasma sCD26 concentrations were measured by ELISA, and sCD26 enzymatic activity was assessed using a luciferase-based assay. The expression levels of membrane-bound CD26 were analyzed by flow cytometry. Plasma sCD26 concentrations were lower in HT patients than in healthy controls, although the difference in sCD26 concentrations between the two groups did not reach statistical significance (P=0.07). The percentages of CD8+ T cells and Tc1 cells with CD26 expression were decreased in HT patients compared with those in healthy controls, and the mean fluorescence intensity (MFI) values of CD26 on CD8+ T cells and Tc17 cells in HT patients were significantly lower than in healthy controls (P<0.05). In HT patients, the expression of CD26 on CD8+ T cells and Tc subsets was decreased in the hypothyroidism group compared with that in the euthyroid group (P<0.05). These results suggest that the sCD26 concentrations and membrane-bound CD26 levels on CD8+ T cells are aberrant in HT and that the reduced CD26 expression may be involved in the progression of HT.