A Compound Heterozygosis of Two Novel Mutations in vWF Exacerbates vWD in a Chinese Pedigree.

BACKGROUND: von Willebrand disease (vWD), caused by mutations in the von Willebrand factor (vWF) coding gene, is a disease characterized by abnormal coagulation activity and a severe tendency for hemorrhage. Therefore, identifying mutations in vWF is important for diagnosing congenital vWD. METHODS: We studied a 23-year-old male vWD patient and his parents. Clotting methods were used to determine activated partial thromboplastin time (aPTT), prothrombin time (PT), fibrinogen (FIB) levels, FVIII activity. Chromogenic substrate method was used to determine vWF antigen and activity. The platelet count was determined. Mutations were searched using whole-exome sequencing and certified by Sanger sequencing. Clinical data, including activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), fibrinogen levels, FX activity, FX antigen levels, and the platelet count were collected. A mixing study was performed to eliminate the presence of coagulation factor inhibitors and lupus anticoagulants. Mutations were screened by using whole-exome sequencing (WES) and were verified by using Sanger sequencing. RESULTS: The proband showed severely decreased vWF antigen, vWF activity, and FVIII activity. RIPA (RISTO-CETIN-induced platelet aggregation) was 0%. Data from WES showed that the proband carried compound heterozygous variants vWF: NM_000552.5 (c.3213C>A p.Cys1071Ter) and vWF: NM_000552.5 (c.6598+2T>C). The proband's mother carried variant vWF: NM_000552.5 (c.3213C>A p.Cys1071Ter) while the proband's father carried variant vWF: NM_000552.5 (c.6598+2T>C). All laboratory test indexes of the proband's parents, including vWF antigen, vWF activity, and FVIII activity, were within the normal ranges. CONCLUSIONS: We identified a compound heterozygosis with two novel mutations in vWF (c.3213C>A, c.6598+2T >C) in a family pedigree, and our results demonstrate that the compound heterozygous mutations probably exacerbate vWD.

Thromboelastography Parameters Change Significantly in Patients with Endometrial Cancer and Uterine Fibroids.

BACKGROUND: The goal was to clarify the changes of TEG parameters in patients with uterine fibroids and endometrial cancer and the clinical diagnostic values of TEG parameters. METHODS: A total of 57 patients with uterine fibroids and 43 patients with endometrial cancer were included, and their TEG parameters were analyzed and compared with 45 healthy women. Routine coagulation indicators were also collected and compared. For significantly changed TEG indicators, the ROC curves were used to evaluate their diagnostic efficacy and determine the cutoff values. The TEG indicators of patients with endometrial cancer of stag I and II were also compared. RESULTS: APTT, and PT levels in endometrial cancer patients were significantly shorter than those in healthy controls. FIB level in endometrial cancer patients were significantly higher than those in healthy controls. Angle, MA, CI, E, G, and TPI levels were significantly upregulated in endometrial cancer patients while TMA was significantly decreased. According to ROC curve analysis, G and E had a good auxiliary diagnostic efficiency for the detection of uterine fibroids (cutoff value 6,691 d/sec and 133.8 d/sec) and TPI has good sensitivity and specificity for the diagnosis of endometrial cancer (cutoff value 51.3 dyn/cm2). The TEG index of patients with stage I and II endometrial cancer did not reach statistical difference. CONCLUSIONS: Thromboelastography parameters change significantly in patients with endometrial cancer and uterine fibroids.

Evaluation of Activated Carbon Treatment on Overcoming the Interference of Rivaroxaban on the Detection of Functional Lupus Anticoagulant.

OBJECTIVE: The aim of this study was to review the role of activated carbon (AC) in eliminating the interference of rivaroxaban in the detection of lupus anticoagulants (LAs). METHODS: Normal pooled plasma was obtained as group N1, group N2 took 1 mL plasma from N1 and added AC, group N3 was prepared by mixing normal plasma with rivaroxaban, and group N3 was treated with AC according to our procedure, as group N4. Plasma from 22 patients was collected before and 6-12 h after rivaroxaban therapy, described as group P1 and group P2, respectively, and 1 mL plasma was taken from group P2 and treated with AC, as group P3. Anti-Xa and diluted Russell's viper venom time (dRVVT)/silica clotting time (SCT) index in each group were measured and compared. RESULTS: Rivaroxaban concentrations and anti-Xa had high intercorrelations in group N3, and the levels of anti-Xa and dRVVT/SCT index had high intercorrelations. After treatment with AC, influence of rivaroxaban was removed, with LA and coagulation factor assays not influenced. Rivaroxaban administration could affect LA assay results in patients, with all LA results increased. After treatment with AC, results of anti-Xa and LA tests recovered to the level before rivaroxaban therapy. CONCLUSIONS: We proposed a reference procedure for the LA detection of patients using rivaroxaban by AC, and activated carbon was proven to be a simple product to eliminate the interference of rivaroxaban.

Upregulation of CX3CR1 expression in circulating T cells of systemic lupus erythematosus patients as a reflection of autoimmune status through characterization of cytotoxic capacity.

OBJECTIVE: This study investigated CX3CR1 expression in human peripheral blood T lymphocytes and their subsets, exploring changes in SLE patients and its diagnostic potential. METHODS: Peripheral blood samples from 31 healthy controls and 50 SLE patients were collected. RNA-Seq data from SLE patient PBMCs were used to analyze CX3CR1 expression in T cells. Flow cytometry determined CX3CR1-expressing T lymphocyte subset proportions in SLE patients and healthy controls. Subset composition and presence of GZMB, GPR56, and perforin in CX3CR1+ T lymphocytes were analyzed. T cell-clinical indicator correlations were assessed. ROC curves explored CX3CR1's diagnostic potential for SLE. RESULTS: CX3CR1+CD8+ T cells exhibited higher GPR56, perforin, and GZMB expression than other T cell subsets. The proportion of CX3CR1+ was higher in TEMRA and lower in Tn and TCM. PMA activation reduced CX3CR1+ T cell proportions. Both RNA-Seq and flow cytometry revealed elevated CX3CR1+ T cell proportions in SLE patients. Significantly lower perforin+ and GPR56+ proportions were observed in CX3CR1+CD8+ T cells in SLE patients. CX3CR1+ T cells correlated with clinical indicators. CONCLUSION: CX3CR1+ T cells display cytotoxic features, with heightened expression in CD8+ T cells, particularly in adult SLE patients. Increased CX3CR1 expression in SLE patient T cells suggests its potential as an adjunctive diagnostic marker for SLE.

Changes in the expression of T-cell factor-1 in follicular helper T cells reflect the condition of systemic lupus erythematosus patients.

OBJECTIVE: This study aimed to identify changes in T-cell factor-1 (TCF1) in circulating T-cell subsets of systemic lupus erythematosus (SLE) patients and to explore their significance in SLE. METHODS: Peripheral blood was collected from 41 SLE patients and 45 healthy controls (HCs). TCF1 in follicular helper T (TFH), follicular regulatory T (TFR) and regulatory T (Treg) cells was analyzed by flow cytometry. Interleukin-21 (IL-21), programmed death receptor 1 (PD-1) and killer cell lectin-like receptor G1 (KLRG1) were detected and compared among TFH subsets sorted according to TCF1 and CD62L expression. Correlation analyses were conducted between TCF1-related TFH cell subsets and autoantibody levels, systemic lupus erythematosus disease activity index (SLEDAI), and plasmablast levels of SLE patients. RESULTS: Absolute numbers of TCF1- TFH and TCF1+ Treg cells were increased in SLE patients. According to TCF1 and CD62L expression, circulating TFH cells and Tregs were sorted into four subsets. CD62L+TCF1+ TFH cell percentages were decreased, and CD62L-TCF1- TFH cells were increased. CD62L+TCF1+ Treg cell percentages were increased, and CD62L-TCF1- Treg cell percentages were decreased. KLRG1+ percentages in CD62L-TCF1-TFH were higher in SLE patients than in HCs. Functionally, CD62L+TCF1+ TFH cells had stronger IL-21 secretion, while CD62L-TCF1-TFH cells had weaker IL-21 secretion and lower PD-1 expression. TCF1- and CD62L-TCF1- TFH numbers were positively correlated with anti-ribosomal P, anti-dsDNA and SLEDAI. CONCLUSION: The increased TFH cells in SLE patients were mostly TCF1-negative subsets with weakened function. Changes in TCF1-related subsets can reflect the condition and abnormal humoral immunity of SLE patients.

Inflammatory Cytokine-Neutralizing Antibody Treatment Prevented Increases in Follicular Helper T Cells and Follicular Regulatory T Cells in a Mouse Model of Arthritis.

Background: Follicular T helper (TFH) and follicular regulatory T (TFR) cells play important roles in humoral immunity. Nevertheless, their significance in rheumatoid arthritis (RA) pathogenesis has not been fully elucidated. As an important treatment strategy, the effect of inflammatory factor-neutralizing antibodies on TFH and TFR in RA remains unclear. Methods: We used the collagen-induced arthritis (CIA) mouse model to illustrate the quantity and functional changes in TFH and TFR cells. The changes of plasmablast, TFH and TFR cells in the spleen and peripheral blood of CIA mice were analyzed by flow cytometry. The levels of TFH and TFR and their functional subsets in the spleen after anti-inflammatory antibody treatment were analyzed and compared. The functional changes of TFH and TFR in CIA mice before and after treatment were detected by in vitro culture experiments. Results: Plasmablast levels were increased in CIA spleen and peripheral blood and both TFH and TFR cell levels were upregulated. TFH and TFR cells were decreased significantly after the anti-inflammatory antibody treatment. TIGIT+ and TIGIT+CD226- TFH cells in CIA mouse spleen were elevated and PD-1 and ICOS expression on spleen TFH and TFR cells was increased. Both the ability of TFH cells to secrete IL-21 and aid B cells and the ability of TFR cells to secrete IL-10 and inhibit TFH cells were enhanced in the CIA mice. After antibody treatment, the cell subsets and functions were recovered. Conclusion: Germinal center TFH and TFR cells were increased and their functions were enhanced. With inflammatory factor-neutralizing antibody treatment, TFH and TFR subsets and their functions returned to normal. These findings provide important information on the dynamics of humoral immune-related cell subsets in RA and the effects of treatment on them.

Circulating CD4+ FoxP3- CXCR5- CXCR3+ PD-1hi cells are elevated in active rheumatoid arthritis and reflect the severity of the disease.

OBJECTIVE: To examine the expression and clinical significance of circulating CD4+  FoxP3-  CXCR5-  CXCR3+  PD-1hi cells in rheumatoid arthritis (RA). METHODS: CD4+  FoxP3-  CXCR5-  CXCR3+  PD-1hi cells in peripheral blood of 35 patients with active RA, 17 with RA in stable remission, and 24 healthy controls were analyzed by flow cytometry. Serum IgG and circulating plasmablast percentages were measured and correlations with CD4+  FoxP3-  CXCR5-  CXCR3+  PD-1hi cells were systematically analyzed. Disease Activity Scale 28 (DAS28) scores were also calculated and correlation analysis with CD4+  FoxP3-  CXCR5-  CXCR3+  PD-1hi cells was conducted. The levels of CD4+  FoxP3-  CXCR5-  CXCR3+  PD-1hi cells were compared before and after disease-modifying anti-rheumatic drug treatment. Cytokine levels in plasma and cytokine secretion in CD4 cells were measured and their correlations with CD4+  FoxP3-  CXCR5-  CXCR3+  PD-1hi cells were further analyzed. RESULTS: The levels of CD4+  FoxP3-  CXCR5-  CXCR3+  PD-1hi cells in the peripheral blood of patients with active RA were significantly increased compared with healthy controls. CD4+  FoxP3-  CXCR5-  CXCR3+  PD-1hi cells in patients with active RA were positively correlated with serum IgG and DAS28 scores. CD4+  FoxP3-  CXCR5-  CXCR3+  PD-1hi cells were significantly decreased in patients after treatment. Plasma interleukin-10 concentrations and interleukin-10-positive CD4 cell percentages were significantly positively correlated with CD4+  FoxP3-  CXCR5-  CXCR3+  PD-1hi cell levels. CONCLUSION: Circulating CD4+  FoxP3-  CXCR5-  CXCR3+  PD-1hi cells in patients with active RA are increased and could reflect the severity of the disease, which may play a potential role in the pathogenesis of RA.

Altered TEG Parameters Identify Hypercoagulablilty and are of Diagnosis Value for Papillary Thyroid Carcinoma Patients.

OBJECTIVE: The goal of this study was to clarify the changes and clinical significance of thromboelastography (TEG) parameters in papillary thyroid carcinomas and nodular goiters. METHODS: 62 nodular goiter (NG) patients and 53 papillary thyroid carcinoma (PTC) patients were included. Coagulation indicators, together with a series of TEG parameters were collected and analyzed, compared with results of 61 healthy controls (HC). Correlation analysis was conducted between routine coagulation indicators and TEG parameters. ROC curves were used to evaluate the potential diagnosis value of the TEG parameters that were altered in papillary thyroid carcinoma patients. RESULTS: APTT and PT in papillary thyroid carcinoma group were statistically significant higher than that in control group (p<0.05). MPV was found to be higher in PTC than NG and healthy controls.R, K and SP levels were significantly lower in PTC group than that in HC group; Angle, CI and TPI levels were significantly higher in PTC group than that in HC group. Areas under the ROC curve of CI, TPI, and angle were 0.725, 0.714, and 0.687 for distinguishing PTC from HC, 0.662, 0.668 and 0.591 for distinguishing PTC from NG. CONCLUSION: TEG parameters are altered and indicate hypercoagulablilty status of papillary thyroid carcinoma patients; CI, TPI, and angle could be potential diagnosis indicators for detecting papillary thyroid carcinoma.

ADAMTS-13 activity reduction in plasma of acute myeloid leukemia predicts poor prognosis after bone marrow transplantation.

OBJECTIVE: This research aimed to explore the significance of low activity of ADAMTS-13 in acute myeloid leukemia (AML) after bone morrow transplantation (BMT), and to evaluate the disease progress and prognosis of the patients with low or normal activity of ADAMTS-13 after BMT. METHODS: 46 AML patients were included in our research. ADAMTS-13 activity was measured before BMT. Their medical indicators were recorded one month after BMT. All the patients were followed up and their disease progression was evaluated afterwards. The medical indicators and prognosis situation were compared between Low ADAMTS13 Group (<481 ng/ml) and Normal Group (481-785 ng/ml) according to the reference range of local laboratory. ROC curves were used to evaluate the predictive value of ADAMTS13 for infection complications and survival. RESULTS: Low ADAMTS13 Group show extended APTT, PT and elevated CRP and D-Dimer levels, compared with Normal Group. Low ADAMTS13 Group suffered more BMT-related complications than Normal Group. In addition, Low ADAMTS13 Group underwent higher mortality than Normal Group in the one-year follow up after BMT and two-year follow up after onset of AML. ADAMTS13 has AUC of 0.7675, 0.7254, 0.8019 for lung infection, CMV infection and death within one year after BMT, suggesting that ADAMTS13 has predictive value for prognosis of AML patients after BMT. DISCUSSION: For patients with low ADAMTS13 activity, the prognosis was worse and the probability of serious complications and mortality was significantly higher than AML patients with normal ADAMTS13 activity, which suggest predictive role of ADAMTS13 activity for the prognosis of AML after BMT. CONCLUSION: AML patients with low activity of ADAMTS-13 had worse prognosis after BMT.

Increased Circulating Follicular Treg Cells Are Associated With Lower Levels of Autoantibodies in Patients With Rheumatoid Arthritis in Stable Remission.

OBJECTIVE: To examine the expression and changes in function of circulating CD4+CXCR5+FoxP3+ follicular Treg (Tfr) cells in patients with active rheumatoid arthritis (RA) and in patients with RA in stable remission, and to clarify the role of Tfr cells in the pathogenesis of RA. METHODS: Levels of Tfr cells and follicular helper T (Tfh) cells in the peripheral blood of 39 patients with active RA, 39 patients with RA in stable remission, and 33 healthy controls were detected by flow cytometry. The function of Tfr cells was measured by coculturing them with Tfh cells and B cells. Activated CD45RA-FoxP3high Tfr cells were also analyzed. Clinical indicators, including serum Ig and autoantibody levels, were tested, and correlations with Tfr cells were systematically analyzed. The Disease Activity Score in 28 joints (DAS28) was calculated, and correlation analysis with Tfr cells was conducted. RESULTS: The level of CD4+CXCR5+FoxP3+ Tfr cells and the Tfr cell:Tfh cell ratio in peripheral blood from patients with RA in stable remission were significantly increased compared with the same measures in patients with active RA and in healthy controls. The function of Tfr cells was enhanced, and the activated CD45RA-FoxP3high Tfr cell subset was increased in patients with RA in stable remission compared with healthy controls. Furthermore, the number of Tfr cells in RA patients was inversely correlated with IgG, rheumatoid factor, and anti-cyclic citrullinated peptide as well as with the DAS28. CONCLUSION: Circulating Tfr cells are increased as patients with RA achieve stable remission of disease, and increased Tfr cells can suppress autoimmunity in RA patients to stabilize their condition. Our results provide novel insight into RA pathogenesis.

Decreased ADAMTS-13 level is related to inflammation factors and risk stratification of acute lymphoblastic leukemia patients.

As a kind of metalloprotease of the ADAMTS family, ADAMTS-13 is crucial for maintaining the normal size of von Willebrand factor. Reduced ADAMTS-13 had been reported in patients with both localized and disseminated malignancies. However, the expression and potential role of ADAMTS-13 in hematological malignancies remain unclear. In this research, we measured and compared ADAMTS-13 levels in plasma of 35 acute lymphoblastic leukemia (ALL) patients and 30 healthy controls and found that ALL patients possessed lower level of ADAMTS-13 than controls. Correlations between ADAMTS-13 and inflammation factors were calculated and ADAMTS-13 was negatively correlated with C-reactive protein and interleukin-1ß. ALL patients with infections had lower level of ADAMTS-13 than patients without infections. In addition, high-risk ALL patients possessed lower ADAMTS-13 than patients at low risk. To conclude, ADAMTS-13 level is decreased in the plasma of ALL patients and the level of ADAMTS-13 is related to plasma inflammation factors and risk stratification of ALL patients, which could contribute to better understanding of the clinical significance of ADAMTS-13.

Reduced ADAMTS-13 level negatively correlates with inflammation factors in plasma of acute myeloid leukemia patients.

ADAMTS-13 is crucial for maintaining the normal size of vWF. Besides thrombotic thrombocytopenic purpura (TTP), decreased ADAMTS-13 had also been reported in patients with malignancy. However, the knowledge of expression and potential role of ADAMTS-13 in hematological malignancies is still limited. We measured and compared ADAMTS-13 levels in the plasma of 82 acute myeloid leukemia (AML) patients and 34 healthy controls and found that AML patients possessed lower ADAMTS-13 than controls. AML patients with infections possessed lower level of ADAMTS-13 than patients without infections and ADAMTS-13 levels were negatively correlated with C-reactive protein(CRP), IL-6, TNFα and IL-1ß. Furthermore, high risk AML patients are with lower ADAMTS-13 than patients with low risk. ADAMTS-13 negatively correlated with ISTH scores and patients accompanying DIC possessed lower ADAMTS-13.Multivariate analyses proved that low level of ADAMTS-13 is an independent risk factor for AML outcome. To conclude, ADAMTS-13 levels are decreased in plasma of AML patients and the level of ADAMTS-13 is related to inflammation and infection of AML patients. Besides, low ADAMTS-13 level is one potential risk factor for AML patients.

Relation of thromboelastography parameters to conventional coagulation tests used to evaluate the hypercoagulable state of aged fracture patients.

Fractures are common among aged people, and rapid assessment of the coagulation status is important. The thromboelastography (TEG) test can give a series of coagulation parameters and has been widely used in clinics. In this research, we looked at fracture patients over 60 and compared their TEG results with those of healthy controls. Since there is a paucity of studies comparing TEG assessments with conventional coagulation tests, we aim to clarify the relationship between TEG values and the values given by conventional coagulation tests.Forty fracture patients (27 femur and 13 humerus) over 60 years old were included in the study. The change in their coagulation status was evaluated by TEG before surgery within 4 hours after the fracture. Changes in TEG parameters were analyzed compared with controls. Conventional coagulation test results for the patients, including activated partial thromboplastin time (APTT), international normalized ratio (INR), fibrinogen, and platelets, were also acquired, and correlation analysis was done with TEG parameters, measuring similar aspects of the coagulation cascade. In addition, the sensitivity and specificity of TEG parameters for detecting raised fibrinogen levels were also analyzed.The K (time to 20 mm clot amplitude) and R (reaction time) values of aged fracture patients were lower than controls. The values for angle, maximal amplitude (MA), and coagulation index (CI) were raised compared with controls, indicating a hypercoagulable state. Correlation analysis showed that there were significant positive correlations between fibrinogen and MA/angle, between platelets and MA, and between APTT and R as well. There was significant negative correlation between fibrinogen and K. In addition, K values have better sensitivity and specificity for detecting elevated fibrinogen concentration than angle and MA values.Aged fracture patients tend to be in a hypercoagulable state, and this could be effectively reflected by a TEG test. There were correlations between TEG parameters and corresponding conventional tests. K values can better predict elevated fibrinogen levels in aged fracture patients.

Elevated D-dimer and fibrinogen levels in serum of preoperative bone fracture patients.

The changes of coagulation parameters in preoperative fracture patients reflect the coagulation status before surgery. We did retrospective assessment of preoperative fracture patients (n = 113) admitted to the hospital between September 2013 and September 2014. The control group were selected from healthy adults (n = 113) with matched age and gender. Platelet, PT INR, APTT, fibrinogen (FIB) and D-dimer values were collected and analyzed. PT INR level was 1.043 ± 0.119, APTT was 31.91 ± 7.56 s, FIB was 320.6 ± 71.8 mg/dl and D-dimer was 1283 ± 1582 ng/ml for the fracture patients. For the control group, PT INR level was 0.9976 ± 0.0602, APTT was 33.22 ± 2.55 s, FIB was 277.3 ± 44.7 mg/dl and D-dimer was 97.53 ± 63.90 ng/ml. Meanwhile, D-dimer levels of different sites of fractures were also measured: Femora 2448 ± 1961 ng/ml; Humerus 792.4 ± 691.2 ng/ml; Ulna/Radius 619.4 ± 843.7 ng/ml; Vertebra 647.7 ± 787.1 ng/ml; Tibia/Fibula 496.3 ± 268.8 ng/ml; Clavicle 260.9 ± 170.9 ng/ml; Ankle 415.4 ± 286.6 ng/ml. To conclude, D-dimer and fibrinogen levels get higher in preoperative fracture patients than controls. Besides, D-dimer levels are significantly different among different locations of fractures, and our data revealed that D-dimer levels of Femora fracture were higher than other sites.