Platelet and Monocyte Microvesicles as Potential Biomarkers of COVID-19 Severity: A Cross-Sectional Analysis.

Background: Coronavirus disease (COVID-19) induces inflammation, coagulopathy following platelet and monocyte activation, and fibrinolysis, resulting in elevated D-dimer levels. Activated platelets and monocytes produce microvesicles (MVs). We analyzed the differences in platelet and monocyte MV counts in mild, moderate, and severe COVID-19, as well as their correlation with D-dimer levels. Methods: In this cross-sectional study, blood specimens were collected from 90 COVID-19 patients and analyzed for D-dimers using SYSMEX CS-2500. Platelet MVs (PMVs; PMVCD42b+ and PMVCD41a+), monocyte MVs (MMVs; MMVCD14+), and phosphatidylserine-binding annexin V (PS, AnnV+) were analyzed using a BD FACSCalibur instrument. Results: PMV and MMV counts were significantly increased in COVID-19 patients. AnnV+ PMVCD42b+ and AnnV+ PMVCD41a+ cell counts were higher in patients with severe COVID-19 than in those with moderate clinical symptoms. The median (range) of AnnV+ PMVCD42b+ (MV/µL) in mild, moderate, and severe COVID-19 was 1,118.3 (328.1-1,910.5), 937.4 (311.4-2,909.5), and 1,298.8 (458.2-9,703.5), respectively (P =0.009). The median (range) for AnnV+ PMVCD41a+ (MV/µL) in mild, moderate, and severe disease was 885.5 (346.3-1,682.7), 663.5 (233.8-2,081.5), and 1,146.3 (333.3-10,296.6), respectively (P =0.007). D-dimer levels (ng/mL) weak correlated with AnnV+ PMVCD41a+ (P =0.047, r=0.258). Conclusions: PMV PMVCD42b+ and PMVCD41a+ counts were significantly increased in patients with severe clinical symptoms, and PMVCD41a+ counts correlated with D-dimer levels. Therefore, MV counts can be used as a potential biomarker of COVID-19 severity.

In vitro large scale production of megakaryocytes to functional platelets from human hematopoietic stem cells.

Megakaryocytopoiesis results in the formation of platelets, which are essential for hemostasis. Decreased production or increased destruction of platelets can cause thrombocytopenia, in which platelet transfusion is the mode of treatment. The present study is aimed in generation of megakaryocytes (MKs) and platelet from human hematopoietic stem cells (HSCs). The purity of HSCs was assessed through Flow cytometry and immunocytochemistry (ICC) studies. These pure HSCs were induced with thrombopoietin (TPO), similarly with Andrographis paniculata extract (APE) for 21 days to generate MKs. The APE is mainly composed of andrographolide which stimulates TPO from the liver, and this binds to CD110 present on the surface of HSCs and triggers the proliferation of HSCs and initiate higher MKs population subsequently, a large number of platelets. The results of the present study showed increased proliferation of HSCs grown in the presence of APE and revealed a high population of CD41a and CD42b positive MKs as enumerated by Flow cytometry compared with TPO induced MKs. These results also concurred with qRT-PCR and western blot analysis. The scanning electron microscopy (SEM) revealed the morphology of differentiated MKs and platelets were similar to human blood platelets. The differentiated MKs in APE exhibited polyploidy up to 32 N while TPO induced MKs showed polyploidy of 8 N, these results corroborated with colony forming unit assay. On thrombin stimulation, high expression of P-selectin (CD62p) and fibrinogen binding were detected in APE induced platelets. Autologous transplantation of platelets generated from APE may be a useful option in thrombocytopenia condition.

The effect of amifostine on differentiation of the human megakaryoblastic Dami cell line.

Amifostine is a cytoprotective drug that was initially used to control and treat nuclear radiation injury and is currently used to provide organ protection in cancer patients receiving chemotherapy. Clinical studies have also found that amifostine has some efficacy in the treatment of cytopenia caused by conditions such as myelodysplastic syndrome and immune thrombocytopenia, both of which involve megakaryocyte maturation defects. We hypothesized that amifostine induced the differentiation of megakaryocytes and investigated this by exposing the human Dami megakaryocyte leukemia cell line to amifostine (1 mmol/L). After 12 days of amifostine exposure, optical microscopy showed that the proportion of Dami cells with diameters >20 µm had increased to 24.63%. Transmission electron microscopy identified the development of a platelet demarcation membrane system, while flow cytometry detected increased CD41a expression and decreased CD33 expression on the Dami cell surface. Ploidy analysis found that the number of polyploid cells with >4N DNA content increased to 27.96%. We did not detect any elevation in the mRNA or protein levels of megakaryocytic differentiation-associated transcription factors GATA-binding factor 1 (GATA-1) and nuclear factor, erythroid 2 (NF-E2), but nuclear import assay revealed an increased nuclear translocation of these proteins. These findings indicate that amifostine induced the differentiation of Dami cells into mature megakaryocytes via a mechanism involving increased nuclear translocation of the transcription factors, NF-E2 and GATA-1.

Study on micromegakaryocytes of patients with myelodysplastic syndrome by flow cytometry / 重庆医学

Objective To setup a measurement of human bone marrow micromegakaryocyte which based on CD41a and PI double‐labeled flow cytometric analysis ,and study the significance in the diagnosis of MDS .Methods In 42 cases of MDS patients , their bone marrow megakaryocytes were obtained by Percoll density gradient separation medium .The megakaryocyte glycoproteinⅡb/Ⅲa(CD41a)were marked with fluorescein isothiocyanate through its corresponding monoclonal antibody ,and their DNA were marked with PI .Then the megakaryocyte ploidy was analyzed by flow cytometry(FCM ) .Results The method for micromegakaryo‐cyte identification and analysis was established .In 42 patients with MDS ,the detection rate of micromegakaryocyte was 90 .5 per‐cent by FCM analysis ,but only 54 .8 percent by Wright‐Giemsa staining test and 64 .3 percent by immunohistochemistry ,the differ‐ence among them was statistically significant(χ2 = 13 .640 ,P= 0 .001) .The 42 patients with MDS were divided into two groups (low‐risk group and high‐risk group) .The detection rates of micromegakaryocyte were 81 .8 percent in low‐risk group and 100 per‐cent in high‐risk group separately by FCM analysis ,the difference was statistically significant(χ2 =4 .019 ,P=0 .045) .Conclusion The detection rate of micromegakaryocyte by FCM with CD41a and PI double marker is higher than that by cytochemical staining . The detection rate of micromegakaryocyte in the high‐risk group is higher than that of the low‐risk group ,which shows that the de‐tection of micromegakaryocyte is of great significance for MDS prognosis assessment .

Effect of amifostine on proliferation and differentiation of human megakaryocyte Dami cells / 中国药理学与毒理学杂志

OBJECTIVE To investigate the effect of amifostine(Amf)on the differentiation of human megakaryocyte cell line-Dami. METHODS Dami cells were treated with Amf 0.01-5.0 mmol · L-1 for 12 d. Dami cells were counted every day for the growth curve:only cells with a diameter>20μm. The platelet demarcation membrane system was observed by transmission electron microscopy. The expression of CD33,CD34,CD41a and DNA ploidy was detected by flow cytometry. RESULTS Amf 0.1-1.0 mmol · L-1 promoted the differentiation of Dami cells ,but inhibited their proliferation at a concentration>1.0 mmol · L-1. When these cells were treated with Amf 1.0 mmol · L-1 for 12 d,the platelet demarcation membrane system was observed,the percentage of cells with a diameter >20 μm was increased by 24.6%(P1.0 mmol·L-1).

EDTA Inhibits the Binding of Clone 96.2C1, an Anti-CD41a Monoclonal Antibody, to the Platelets and Addition of Heparin and CaCl2 to the Antibody Neutralizes the EDTA-induced Inhibitory Effect

BACKGROUND: The binding of some monoclonal antibodies platelet glycoprotein (GP) IIb/IIIa, which is frequently used for flow cytometric immnophenotyping, is known to be inhibited by EDTA. To select the ideal antibodies to be included in the 'Acute Leukemia Panel' for immunophenotyping of acute leukemia, we compared the inhibitory effect of EDTA on the binding of 5 different clones of monoclonal antibodies to platelet GP IIb/IIIa. We also discovered a simple method to neutralize this inhibitory effect. METHODS: Flow cytometric measurement of the number of platelet GP IIb/IIIa binding sites with different anticoagulants was performed using a panel of 5 clones of monoclonal antibodies against CD41 (clone PM6/248), CD41a (clone 96.2C1 & clone HIP8), CD41b (clone HIP2) and CD61 (clone VI-PL2), and the results are expressed as the mean equivalent soluble fluorochrome (MESF) values. RESULTS: The MESF value of the EDTA platelets stained with anti-CD41a, clone 96.2C1 antibody showed a significantly lower value than the MESF of platelets anticoagulated with heparin or citrate (P<0.001). The inhibitory effect of EDTA on the binding of anti-CD41a, clone 96.2C1 antibody to the platelets was neutralized by addition of heparin and CaCl2. The mean MESF value of EDTA platelets stained with anti-CD41a, clone 96.2C1 antibody was significantly increased by the addition of heparin and CaCl2 (P=0.0001). CONCLUSION: The false-negative results of the binding of anti-CD41a, clone 96.2C1 antibody to the platelets seem to be due to the calcium chelating property of EDTA, and the addition of CaCl2 and heparin could be used as an easy compensatory measure for the inhibitory effect of EDTA on other antibodies as well.