BACKGROUND

Because of the life-consuming treatment and severe consequences associated with thalassemia, it is more effective to prevent than cure thalassemia. Rapid and sensitive detection is critical for controlling thalassemia. In this study, we developed a rapid and accurate test to genotype nondeletional α- and β-thalassemia mutations by an electrochemical DNA sensor.

METHODS

Screen-printed electrodes were used as electrochemical transducers for the sensor, in which the capture probe DNA was attached to the golden surface of the working electrode via an S-Au covalent bond, which is highly suitable for immobilizing the biological element. In addition, two types of ferrocene with varying redox potentials for modified signal probe DNA were adopted. The hybridization signal is detected by alternating current voltammetry when the capture probe and signal probe hybridize with the target DNA.

RESULTS

With this technique, 12 types of nondeletional α- and β-thalassemia mutations were detected, which constitute more than 90% of all the nondeletional types of thalassemia mutation determinants found in China, including the CD142 (TAA>CAA) Constand spring, CD125 (CTG>CCG) Quonsze, CD122 (CAC>CAG) Weastmead, -28 (A>G), Cap+1 (A>C), initiation codon (ATG>AGG), CD17 (AAG>TAG), CD26 (GAG>AAG), CD31(-C), CD41-42 (-CTTT), CD71-72 (+A), and IVS-II-654 (C>T) mutations. Concordance levels were 100% within the 20 blood samples of homozygous wild-type individuals and 238 blood samples of heterozygous mutant individuals.

CONCLUSIONS

The electrochemical DNA sensor developed here can be applied for rapid genotyping of thalassemia or other clinical genotyping applications and is useful for early screening of thalassemia in high-risk groups by minimizing the time and investment cost.

Bone marrow transplantation (BMT) is the only curable option for thalassemia major, β-thalassemia/HbE. However, some patients still have the risk of hypercoagulable complications. We used a whole blood flow cytometric analysis to measure the circulating microparticle (MP) levels, activated platelets, and leukocyte-platelet aggregates in 59 young β-thalassemia/HbE patients compared with 20- and 28-matched healthy and patients receiving regular blood transfusion (RT), respectively. Results from the studies showed that blood samples from BMT group contained a significantly higher numbers of circulating MPs originated from platelets (ann-V+CD41a+), leukocyte (ann-V+CD45+) and endothelial cells (ann-V+CD146+) when compared to samples from healthy subjects and RT patients. In contrast, the percentages of activated/procoagulant platelets (CD62P and CD142 expressing platelets) were decreased in BMT group. In addition, monocytes forming microaggregates were the major population among other leukocyte-platelet complexes. Different patterns of CD11b, CD62P and CD142 expression on platelet-leukocyte microaggregate surface were also found. These data suggest that circulating MPs together with leukocyte-platelet aggregates may be responsible, in part, in pathogenesis of hypercoagulable state in β-thalassemia/HbE patients who undergone BMT.

BACKGROUND

In this part of the study, where we determined the causes of preeclampsia and other obstetric complications, we focused on the role of tissue factor (TF) in the activation of these pathophysiological processes. Recent findings attribute a significant part of the activation of coagulation creation of autoantibodies. Once this mechanism is activated, the antibodies induce expression of tissue factor (TF, CD142) on monocytes and vascular endothelial cells.

METHODS

We have proposed a monitor activation model of the coagulation system in preeclampsia and other pregnancy complications using TF expression on monocytes by flow cytometry and simultaneous determination the TF-induced thrombin generation in plasma. To determine expression of tissue factor (CD142) on monocytes, we proposed a method of multicolor flow cytometry using anti CD45 PerCP, anti CD14 APC, anti CD16b FITC, and anti CD142 PE antibodies and the corresponding isotype controls.

RESULTS

We verified the model on patients with severe antiphospholipid syndrome, which is a high expression of antibodies, in particular against beta-2GPI.

CONCLUSIONS

We demonstrated complete inhibition of TF expression on monocytes and a significant reduction of thrombin generation in plasma.

Portal vein thrombosis (PVT) is a critical complication in cirrhotic patients. We explored the role of the activated factor VII-antithrombin (FVIIa-AT) complex and enhanced monocytic tissue factor (TF) expression in the development and prediction of non-neoplastic PVT in cirrhotic patients.

A total of 30 HCV-cirrhosis patients were included in our study. They were compared to 35 cirrhotic patients without PVT, 15 non-cirrhotic patients with PVT, and 15 healthy controls. The plasma level of the FVIIa-AT complexes was analyzed by ELISA. MIF CD142, CD86, and HLA-DR on monocytes (CD14) were determined by flow cytometry.

Compared with cirrhotic patients without PVT, cirrhotic patients with PVT had comparable plasma values of FVIIa, AT, and the FVIIa-AT complex. However, they had significantly lower values compared to non-cirrhotic patients with PVT and healthy controls. Cirrhotic patients with PVT had increased monocytic TF expression (MIF CD142) compared to non-PVT cirrhotic patients and healthy controls [86.5 (93.5) vs. 18 (32.0) and 11.0 (6.0), respectively; p < 0.001 for each]. However, cirrhosis PVT could not be distinguished from non-cirrhosis PVT. The area under the ROC curve of MIF CD142 was 0.759 (0.641- 0.876; p = 0.000) at an optimal cut-off value of 45, which yielded a sensitivity of 60% and a specificity of 77.1%, as well as a PPV and NPV of 69.2% for each.

Enhanced expression of monocytic TF may have a role in the development and prediction of non-neoplastic PVT in HCV-cirrhosis patients. Large multicenter studies are necessary to validate our results.

Human pancreatic exocrine cells were cultured in 3D suspension and formed pancreatospheres composed of acinar-derived and duct-like cells. We investigated, up to 6 days, the fate of human pancreatic acinar cells using fluorescein-conjugated Ulex Europaeus Agglutinin 1 lectin, a previously published acinar-specific non-genetic lineage tracing strategy. At day 4, fluorescence-activated cell sort for the intracellularly incorporated FITC-conjugated UEA1 lectin and the duct-specific CA19.9 surface marker, distinguished acinar-derived cells (UEA1⁺CA19.9^-) from duct-like cells (UEA1^-CA19.9⁺) and acinar-to-duct-like transdifferentiated cells (UEA1⁺CA19.9⁺). mRNA expression analysis of the acinar-derived (UEA1⁺CA19.9^-) and duct-like (UEA1^-CA19.9⁺) cell fractions with concomitant immunocytochemical analysis of the pancreatospheres revealed acquisition of an embryonic signature in the UEA1⁺CA19.9^- acinar-derived cells characterized by de novo expression of SOX9 and CD142, robust expression of PDX1 and surface expression of GP2. The colocalisation of CD142, a multipotent pancreatic progenitor surface marker, PDX1, SOX9 and GP2 is reminiscent of a cellular state present during human embryonic development. Addition of TGF-beta signalling inhibitor Alk5iII, induced a 28-fold increased KI67-labeling in pancreatospheres, more pronounced in the CD142⁺GP2⁺ acinar-derived cells. These findings with human cells underscore the remarkable plasticity of pancreatic exocrine acinar cells, previously described in rodents, and could find applications in the field of regenerative medicine.

This study sought to identify different subpopulations of extracellular vesicles (EVs) in plasma from female patients with established rheumatoid arthritis (RA) in relation to the activation of coagulation and fibrin formation in these patients. Forty women were included in the study, 20 patients and 20 age-matched healthy controls. The mean disease duration in patients was 13.0 (5.0-25.0) years, with medium to high disease activity despite ongoing treatment with low-dose prednisolone and methotrexate. There were no differences between the investigated groups regarding the presence of traditional cardiovascular risk factors. The concentration of phosphatidylserine-positive (PS⁺) EVs; platelet (CD42a⁺), leucocyte (CD45⁺), monocyte (CD14⁺), and endothelial (CD144⁺)-derived EVs; and EVs-expressing tissue factor (CD142⁺), P-selectin (CD62P⁺), and E-selectin (CD62E⁺) were determined by flow cytometry analysis. Overall hemostasis potential (OHP) was assessed to follow the hemostatic disturbances, including the parameters for overall coagulation potential (OCP) and overall fibrinolytic potential (OFP). Fibrin clot turbidity was measured together with clot lysis time, and scanning electron microscopy was performed. Increased concentrations of PS⁺, CD42a⁺, CD142⁺, CD45⁺, CD14⁺, and CD62P⁺ EVs were found in plasma from patients with RA compared to healthy controls, and the concentrations of PS⁺, CD42a⁺, CD14⁺, and CD62P⁺ EVs were positively correlated with the inflammatory parameters in RA patients. Positive correlations were also found between the levels of PS⁺ and CD42a⁺ EVs and OCP as well as between the levels of PS⁺, CD42a⁺, and CD62P⁺EVs and OHP. The levels of PS⁺, CD42a⁺, CD14⁺, CD62P⁺, and CD62E⁺ EVs were negatively correlated with OFP. Elevated levels of circulating EVs of different cell origins were found in patients with established RA, in relation to the inflammatory burden and coagulation activation in the disease.

Keywords: extracellular vesicles; fibrin structure; hemostasis; inflammation; rheumatoid arthritis.

Low flow rate pumping of cell suspensions finds current applications in bioreactors for short-term dynamic cell culture and adhesion assays. The aim of this study was to develop an atraumatic pump and hemodynamically adapted test circuit to allow operating periods of at least several hours. A computer-controlled mini-pump (MP) was constructed based on non-occlusive local compression of an elastic tube with commercial bi-leaflet valves directing the pulsatile flow into a compliant circuit. Cell damage and activation in the system were tested with whole blood in comparison with a set with a conventional peristaltic pump (PP). Activation of circulating THP-1 monocytes was tested by measuring the expression of CD54 (ICAM-1). Additionally, monocyte-endothelial interactions were monitored using a parallel-plate flow chamber with an artificial stenosis. The system required a priming volume of only 20 mL, delivering a peak pulsatile flow of up to 35 mL/min. After 8 h, blood hemolysis was significantly lower for MP with 11 ± 3 mg/dL compared with PP with 100 ± 16 mg/dL. CD142 (tissue factor) expression on blood monocytes was 50% lower for MP. With MP, THP-1 cells could be pumped for extended periods (17 h), with no enhanced expression of CD54 permitting the long-term co-culture of THP-1 with endothelial cells and the analysis of flow pattern effects on cell adhesion. A low-damage assay setup was developed, which allows the pulsatile flow of THP-1 cells and investigation of their interaction with other cells or surfaces for extended periods of time.

: To evaluate the plasma levels of soluble endothelial cell molecules in patients with venous thromboembolism (VTE) out of the acute phase as compared with healthy individuals. We also investigated the possible associations of the soluble endothelial cell molecules among them, as well as with other clinical and laboratory data, including the numbers of circulating endothelial cells (CEC), circulating endothelial progenitor cells (CEP), and CEC expressing activation-related [cluster of differentiation (CD)54 and CD62E] and procoagulant (CD142) markers. In total, 15 patients with VTE and 20 normal individuals were studied. The CEC and CEP were quantified and characterized by flow cytometry. The soluble molecules studied included P-selectin, E-selectin, intercellular cell adhesion molecule 1, vascular cell adhesion molecule 1 and tissue factor (ELISA), and von Willebrand factor antigen (immunoturbidimetry). VTE patients had significantly higher levels of vascular cell adhesion molecule 1 and von Willebrand factor antigen and lower levels of soluble E-selectin than controls. They also showed significantly higher numbers of CEC, as of activated/procoagulant CEC and lower numbers of CEP, compared with controls. We did not find any correlation between the levels of soluble molecules and the numbers of endothelial cell in circulation, but there was with several clinical and laboratory data in VTE patients. Our results would suggest that in VTE patients, the endothelium remains activated and in some hypercoagulable state. The levels of soluble endothelial cell molecules did not seem to be directly related to the numbers of CEC and CEP neither reflected the number of activated CEC, which may be because of the different function that surface and soluble molecules may have.

Mesenchymal stromal cells (MSCs) are under active consideration as a treatment strategy for controlling the hyper-inflammation and slow disease progression associated with coronavirus disease 2019 (COVID-19). The possible mechanism of protection through their immunoregulatory and paracrine action has been reviewed extensively. However, the importance of process control in achieving consistent cell quality, maximum safety and efficacy-for which the three key questions are which, when and how much-remains unaddressed. Any commonality, if it exists, in ongoing clinical trials has yet to be analyzed and reviewed. In this review, the authors have therefore compiled study design data from ongoing clinical trials to address the key questions of "which" with regard to tissue source, donor profile, isolation technique, culture conditions, long-term culture and cryopreservation of MSCs; "when" with regard to defining the transplantation window by identifying and staging patients based on their pro-inflammatory profile; and "how much" with regard to the number of cells in a single administration, number of doses and route of transplantation. To homogenize MSC therapy for COVID-19 on a global scale and to make it readily available in large numbers, a shared understanding and uniform agreement with respect to these fundamental issues are essential.

Keywords: CD142; MSC dosage; SARS-CoV-2; cytokine storm; hyper-coagulopathy; immunomodulation.

SARS-CoV-2 attaches to the angiotensin-converting enzyme 2 (ACE-2) receptor on human cells. The virus causes hypercytokinemia, capillary leak, pulmonary edema, acute respiratory distress syndrome, acute cardiac injury, and leads to death. Mesenchymal stem cells (MSCs) are ACE-2 negative cells; therefore, can escape from SARS-CoV-2. MSCs prevent hypercytokinemia and help the resolution of the pulmonary edema and other damages occurred during the course of COVID-19. In addition, MSCs enhance the regeneration of the lung and other tissues affected by SARS-CoV-2. The case series reported beneficial effect of MSCs in COVID-19 treatment. However, there are some concerns about the safety of MSCs, particularly referring to the increased risk of disseminated intravascular coagulation, and thromboembolism due to the expression of TF/CD142. Prospective, randomized, large scale studies are needed to reveal the optimum dose, administration way, time, efficacy, and safety of MSCs in the COVID-19 treatment.

Keywords: COVID-19; Mesenchymal stem cell; SARS-CoV-2; Transfusion; Transplantation.

Balkan endemic nephropathy (BEN) is a chronic tubulointerstitial disease frequently accompanied by urothelial carcinoma (UC). In light of the increased UC incidence and the markers observed in BEN patients with developed UC, the aim of the current case-control study is to assess survivin, p53 protein, growth factors and receptors (VEGF, VEGFR1, IGF I, IGF-1R and IGFBP5), tumor marker (TF)/CD142, circulating soluble Fas receptor and neopterin, as potentially predictive markers for UC in patients with BEN (52 patients), compared to healthy, age-matched subjects (40). A threefold increase was registered in both circulating and urinary survivin level in BEN patients. Especially noticeable was the ratio of U survivin/U Cr level five times the ratio of BEN patients associated with standard renal markers in multivariate regression models. The concentrations of VEGF, VEGFR1, (TF)/CD142, (sFas) were not significantly different in BEN patients, while urinary/plasma level demonstrated a significant decrease for VEGF. The levels of IGF I, IGFBP5 and IGF-1R were significantly reduced in the urine of BEN patients. Plasma concentration of neopterin was significantly higher, while urinary neopterin value was significantly lower in BEN patients compared to healthy controls, which reflected a significantly lower urine/plasma ratio and low local predictive value. As BEN is a slow-progressing chronic kidney disease, early detection of survivin may be proposed as potential predictor for malignant alteration and screening tool in BEN patients without the diagnosis of UC.

Keywords: Balkan endemic nephropathy (BEN); aristolochic acid nephropathy; proteomic tumor markers; survivin; upper urothelial cancer.

Recent findings have suggested that the primary factors for development of chronic venous disease (CVD), which commonly manifests as varicose veins (VV), are due to structural and biochemical modifications of the vessel wall. The aim of this exploratory study was to characterize by flow cytometry the endothelial cells (EC) mechanically extracted from the varicose saphenous veins (VSV) segments of patients submitted to VV surgery, and to compare the expression of cell surface molecules in these EC with that observed in the EC from the graft SV (GSV) of patients undergoing bypass surgery. EC were isolated from distal- (varicose trunk) and from proximal- (nearly normal) VSV segments of 30 patients submitted to VV surgery, and from proximal GSV segments of 20 patients submitted to bypass surgery (control group), using a mechanical method, and their immunophenotype was characterized by flow cytometry. EC were identified as being CD45^negCD146^brightCD31^bright, and analyzed for expression of activation-related (CD54, CD62E, CD106), procoagulant (CD142), and cell junction (CD31, CD146) molecules, and for the scavenger receptor, CD36. The EC harvested from the SV segments of CVD patients had lower expression of all the molecules evaluated, in comparison to controls; these differences were more evident for the EC isolated from the distal-VSV. The EC extracted from the proximal- and distal-VSV segments of the CVD patients also differ from each other, the first having lower levels of CD62E, CD106, CD142 and CD36. Groups did not match for gender and controls were heterogeneous concerning the underlying pathologies, which may have a confounding effect. Our study revealed that the EC isolated from varicose (distal) and nearly normal (proximal) VSV segments of the CVD patients differ phenotypically from each other, and from the EC of the control group. The VSV segments more affected by the CVD have the lowest expression of the studied markers. We hypothesize that CVD is associated with a decrease on the EC surface molecules, causing EC dysfunctionality. Further studies with a large number of gender-matched participants are needed, to confirm the results obtained in this exploratory study.

OBJECTIVE

Thalassaemia is a potentially lethal inherited anaemia, caused by reduced or absent synthesis of globin chains. Measurement of the minor adult haemoglobin Hb A2, combining α- with δ-globin, is critical for the routine diagnosis of carrier status for α- or β-thalassaemia. Here, we aim to characterize a novel δ-globin variant, Hb A2 Episkopi, in a single family of mixed Lebanese and Cypriot ancestry with mild hypochromic anaemia and otherwise normal globin genotype, which also presents with a coincidental 0.78-Mb sequence duplication on chromosome 1 (1q44) and developmental abnormalities.

METHODS

Analyses included comprehensive haematological analyses, cation-exchange high-performance liquid chromatography (CE-HPLC), cellulose acetate electrophoresis (CAE), Sanger sequencing and structure-based stability predictions for Hb A2 Episkopi.

RESULTS

The GCT>> GTT missense mutation, underlying Hb A2 Episkopi, HBD:c.428C>> T, introduces a cd142 codon change in the mature protein, resulting in reduced normal Hb A2 amounts and a novel, less abundant Hb A2 variant (HGVS: HBD:p.A143V), detectable as a delayed peak by CE-HPLC. The latter was in line with structure-based stability predictions, which indicated that the substitution of a marginal, non-helical and non-interface residue, five amino acids from the δ-globin chain carboxy-terminus, was moderately destabilizing.

CONCLUSIONS

Detection of the new variant depends on the diagnostic set-up and had failed by CAE and on an independent CE-HPLC system, which, in unfavourable circumstances, may lead to misdiagnoses of β-thalassaemia as α-thalassaemia. Given the mixed background of the affected family, the ethnic origin of the mutation is unclear, and this study thus suggests awareness for possible detection of Hb A2 Episkopi in both the Cypriot and the Lebanese populations.

The Severe Acute Respiratory Syndrome Corona Virus2 (SARS-CoV2) is responsible for Corona Virus Disease 2019 (CoViD-19), the pandemic that has afflicted close to two million people worldwide, and has taken the lives of over 120,000 patients since its first report in late December 2019. Per million people globally, the infection rate is close to 250 with a death rate of close to 14 (death rate average global death rate: 6.06%; for comparison, revised estimate of the 1918 influenza pandemic had an average global death rate of 5.4% [1]). About 400,000 SARS-CoV2-positive patients have been declared 'recovered', although it is not clear to date what exactly that entails. To be clear, the natural history of SARS-CoV2 infection and of the patho-physiology of CoViD-19 remains shrouded in relative confusion, in part due to the exceedingly virulent nature of the virus, as manifest by its elevated morbidity and mortality, and the fast accumulation of clinical observations and research evidence. Many pieces of a complex puzzle are emerging all at once and their organization into a coherent and cogent picture of the natural history of CoViD-19 is arduous and still wanting. Here, we discuss the recent findings in the context of the available evidence. We propose a putative prediction model of the natural history of CoViD-19. We highlight putative loci and modes of therapeutic intervention that may become beneficial preventive and treatment modalities for individuals at risk of SARS-CoV2 infection and CoViD-19 patients.

Keywords: Basigin CD147; Corona Virus Disease 2019 (CoViD-19); Exopeptidase CD26; Peptidase Targeted Immunoregulation (PeTIr); Severe Acute Respiratory Syndrome Corona Virus2 (SARS-CoV2); angiotensin-converting enzyme-2 (ACE2); clustered regularly interspaced short palindromic repeats (CRISPR); cytokine synthesis inhibitory factor (IL10); platelet tissue factor CD142; transferrin receptor CD71; transmembrane protease serine-2 (TMPRSS2).

BACKGROUND

Platelet Monocyte Complexes (PMCs) are commonly expressed in coronary artery disease but their pathologic significance in ST elevation myocardial infarction (STEMI) is unclear. This study evaluates the relationship between locally activated PMCs and intracoronary inflammation in stable and unstable coronary disease.

METHODS

Micro catheter aspirated blood samples of 15 STEMI and 7 stable angina patients are collected from the coronary artery (CA), aorta (AO) and right atrium (RA). Samples are labelled with monoclonal antibodies and prepared for flow cytometry. CD 14 and CD 61 double positive cells are identified as PMC. P-selectin expression is identified by additional CD62P positivity and TF expression by additional CD142 positivity. Plasma TNF-alpha and IL-6 are measured using ELISA and CRP is measured in plasma using a high sensitivity automated microparticle enhanced latex turbidimetric immunoassay.

RESULTS

No site-specific difference is seen in overall PMC expression in STEMI or stable angina. Surface P-selectin expression in STEMI [median (IQR)] is significantly higher in CA [35.01 (23.15-56.99)] compared with AO [15.99 (10.3-18.85)] or RA [14.02 (10.42-26.08)] (p = 0.003). Intracoronary PMC correlates significantly with intracoronary TNF-alpha (r = 0.87, p = 0.001) and intracoronary IL-6 (r = 0.76, p = 0.03). Bound monocytes within P-selectin positive and tissue factor positive complexes correlate positively with intracoronary TNF-alpha (r = 0.81, p = 0.008 & r = 0.80, p = 0.009 respectively) and IL-6 (r = 0.54, p = 0.16 & r = 0.71, p = 0.05 respectively). No such correlation is observed in the peripheral circulation of STEMI and stable angina patients.

CONCLUSIONS

Inflammation is not attributable to PMC formation per se. However, increased intracoronary P-selectin expression by activated platelets and tissue factor expression by activated monocytes within the complexes are determinants of local intracoronary inflammatory burden in STEMI.

Objective of this study was to detect the level of tissue factor-positive microparticles (TF(+)MP) by flow cytometry (FCM) and to analyze its clinical significance in the haemostatic disorder. TF(+) MP was detected by FCM using antibody CD142-PE in 25 cases of acute promyelocytic leukemia (APL), 20 cases of hemostatic diseases and 20 healthy adults as controls. The differences of TF(+) MP between various groups were determined. The results showed that the level of TF(+) MP in the patients with thrombotic complications was significantly higher than that in the healthy adults (P < 0.05). The TF(+) MP level was higher in the patient with APL than that in the healthy adults, especially in course before therapy (P < 0.01), but the difference was not statistically significant in the patient with APL after therapy and the healthy adults. Among these patient with APL, the level of TF(+) MP in the 18 patients who complicated with disseminated intravascular coagulation (DIC) was also higher than that in the healthy adults (P < 0.05), but the level of TF(+) MP in the other 7 patients who did not complicate with DIC was similar before and after treatment. It is concluded that the method of TF(+) MP detection by FCM is feasible and simple, it is useful for the diagnosis of thrombotic disorder, and helps evaluation for the prognosis of APL patient.

In a global context of advanced aging, geriatric diseases such as frailty syndrome face challenges in the search for biomarkers and preventive strategies. Frailty has been associated with atherothrombotic pathologies. Circulating microvesicles (cMVs), phospholipid-rich vesicles with a size of 0.1-1.0 μm, have been shown to participate in atherothrombosis onset and progression. We have hypothesized that cMVs from platelets, and vascular and immune cells, are increased in frail older adults. To verify this, a prevalent-case control study was designed with 28 frail older and 27 nonfrail older adults older than 64 years. Frailty was defined by Fried's phenotype. Total cMVs, annexin V positive (AV+)-cMVs, and annexin V negative (AV^- )-cMVs derived from blood and vascular cells were measured by flow cytometry. In the analysis of total cMVs, the frail group presented higher levels of CD14⁺ /CD142⁺ (p = .042), CD41a⁺ /CD142⁺ (p = .041), and CD56⁺ (p = .025), CD14⁺ cMVs (p = .043), and CD16⁺ /CD14⁺ (p = .019) cMVs levels. Within the phosphatidylserine-exposing cMVs (AV⁺ ), the frail group showed higher CD14⁺ /AV⁺ (p = .044), CD9⁺ /AV⁺ (p = .031), P2RY12⁺ /AV⁺ (p = .028), and CD235a⁺ /AV⁺ (p = .043) cMVs concentrations. Finally, within AV^- cMVs, the frail group showed higher CD142⁺ /CD41a⁺ /AV^- cMVs concentrations originated from platelets (p = .027), CD56⁺ /AV^- originated from natural killer cells (p = .022), and CD34⁺ /AV^- cMVs from hematopoietic stem cells (p = .037). In summary, frail older adults present higher concentrations of platelet-, leukocyte-, and hematopoietic cell-derived cMVs compared to robust age-matched older adults. These cMVs may be involved in the deregulation of the immune system, endothelial damage, and increased risk of thrombosis associated with frailty.

Keywords: biomarkers; frailty syndrome; microvesicles; older adults; thrombosis.

Objective: To detect the levels of microparticles (MP) in plasma of patients with esseutial thrombo-cythermia(ET) and analyze the relationship between the JAK2V617F mutant and MP in ET patients.

Methods: The numerical values of MPs were analysed by using flow cytometry. Venous blood of 56 ET patients and 28 healthy persons was collected in the morning and anticoagulated with sodium citrate (1∶9). The RMP, PMP, TF⁺MP and EMP were detected by FCM using phycoerythrin (PE)-conjugated monoclonal antibodies to CD235a for red blood cells, CD61 for platelets, CD142 for tissue factor (TF) and CD62E for endothelial cells, respectively. Forward scatter was set in scale using fluorescent microspheres of 0.8 μm. Standard fluorescent microbeads (0-0.8 μm) in diameter were used to set the microparticles gate. Genomic DNA was extracted from mononuclear cells by using a commercial DNA isolation kit and amplified by allele specific polymerase chain reaction (PCR). According to the size of molecular weight, the amplified products were separated by electrophoresis on a 2% agarose gel to screen 26 JAK2V617F mutations.

Results: The detection results showed that the MP levels in ET group were higher than those in normal control group: RMP (157.2±304.9/μl vs 21.3±18.4/μl), PMP (1378.9±2454/μl vs 113.8±97.1/μl), TF⁺MP (123±354.6/μl vs 21±15.7/μl) and EMP (1434.7±2601.9/μl vs 291.7±297.7/μl) (P<0.05). In 8 patients with thrombus, the levels of these four kinds of MP were higher than those of controls without thrombus: RMP (258.2±327.7/μl vs 55.6±63.8/μl), PMP (1853±1874.7/μl vs 444.7±712/μl), TF⁺MP (120.4±112.5/μl vs 60.3±128.3/μl) and EMP (1637.1±1563.5/μl vs 629.4±1000.2/μl) (P<0.05). The levels of those 4 kinds of MP in 17 patients with splenomegaly were significantly higher than those in 11 patients without splenomegaly: RMP (306.8±491.1/μl vs 59.3±51/μl), PMP (2944.8±3767.6/μl vs 334.8±420.2/μl), TF⁺MP (162.9±166.8/μl vs 31.0±28.7/μl) and EMP (3031.8±4048.8/μl vs 701.5±729.2/μl) (P<0.05). The level of other MP showed no difference between MPN patients with JAK2V617F mutation and without JAK2V617F mutation (P>0.05), except TF⁺MP (154.7±516.3/μl vs 100.5±126.6/μl) (P<0.05).

Conclusion: The numerical values of MP detected are more in ET patients than those in healthy controls. The number of MP is higher in patients with thrombus than that without thrombus, so do in patients with splenomegaly and without splenomegaly. Patients with JAK2V617F mutation show higher number of TF⁺MP than that without JAK2V617F mutation. But the other three kinds of MP show no this difference.

题目: ET患者血浆MP水平测定及其与JAK2V617F突变关系的研究.

目的: 检测原发性血小板增多症（ET）患者血浆中的微颗粒（MP）水平，并分析其与JAK2V617F突变的关系.

方法: 抽取56例ET患者及28例正常人空腹静脉血，枸橼酸钠抗凝(1∶9)，以藻红蛋白标记的特异性荧光抗体CD235a-PE、CD61-PE、CD142-PE和CD62E-PE 分别标记红细胞微颗粒（RMP）、血小板微颗粒（PMP）、组织因子微颗粒（TF⁺MP）及内皮细胞微颗粒（EMP），FCM检测4种MP绝对数。试剂盒抽提基因组DNA，PCR扩增目标DNA，琼脂糖凝胶电泳筛选26例JAK2V617F突变标本.

结果: ET组的RMP（157.2±304.9/μl vs 21.3±18.4/μl）、PMP（1378.9±2454/μl vs 113.8±97.1/μl）、TF⁺MP（123±354.6/μl vs 21±15.7/μl）、EMP（1434.7±2601.9/μl vs 291.7±297.7/μl）高于正常对照组（P<0.05）；8例血栓组的RMP（258.2±327.7/μl vs 55.6±63.8/μl）、PMP（1853±1874.7/μl vs 444.7±712/μl）、TF⁺MP（120.4±112.5/μl vs 60.3±128.3/μl）、EMP（1637.1±1563.5/μl vs 629.4±1000.2/μl）高于25例非血栓组（P<0.05）；17例脾肿大组的RMP（306.8±491.1/μl vs 59.3±51/μl）、PMP（2944.8±3767.6/μl vs 334.8±420.2/μl）、TF⁺MP（162.9±166.8/μl vs 31.0±28.7/μl）、EMP（3031.8±4048.8/μl vs 701.5±729.2/μl）高于11例非脾肿大组（P<0.05）；26例JAK2V617F突变组的TF⁺MP高于未突变组（154.7±516.3/μl vs 100.5±126.6/μl）（P<0.05），另外3种MP在这2组间无明显区别（P>0.05）.

结论: ET组、血栓组、脾肿大组4种MP水平均高于对照组；JAK2V617F突变组患者TF⁺MP数高于未突变组，另3种MP水平在突变与未突变组之间无明显区别.

CD142 promotes cell mobility, which contributes to carcinogenesis. However, the role of CD142 on colorectal cancer (CRC) mobility is unclear. This study showed that CD142 expression increased in CRC tissues, especially in those with invasion or metastasis. The positive sorting or overexpression of CD142 promoted the invasion and migration of CRC cells. Overall, CD142 may be responsible for CRC mobility.

Keywords: CD142; CRC; invasion; metastasis; mobility.