scholarly journals Circulating Tumour Cell Numbers Correlate with Platelet Count and Circulating Lymphocyte Subsets in Men with Advanced Prostate Cancer: Data from the ExPeCT Clinical Trial (CTRIAL-IE 15-21)

Cancers ◽  
2021 ◽  
Vol 13 (18) ◽  
pp. 4690
Author(s):  
Brian Hayes ◽  
Lauren Brady ◽  
Gráinne Sheill ◽  
Anne-Marie Baird ◽  
Emer Guinan ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Flow Cytometry ◽  
Platelet Count ◽  
Advanced Prostate Cancer ◽  
Nk Cell ◽  
Exercise Group ◽  
Coagulation Cascade ◽  
Blood Draw ◽  
Blood Samples ◽  
Platelet Counts

Interactions between circulating tumour cells (CTCs) and platelets are thought to inhibit natural killer(NK)-cell-induced lysis. We attempted to correlate CTC numbers in men with advanced prostate cancer with platelet counts and circulating lymphocyte numbers. Sixty-one ExPeCT trial participants, divided into overweight/obese and normal weight groups on the basis of a BMI ≥ 25 or <25, were randomized to participate or not in a six-month exercise programme. Blood samples at randomization, and at three and six months, were subjected to ScreenCell filtration, circulating platelet counts were obtained, and flow cytometry was performed on a subset of samples (n = 29). CTC count positively correlated with absolute total lymphocyte count (r2 = 0.1709, p = 0.0258) and NK-cell count (r2 = 0.49, p < 0.0001). There was also a positive correlation between platelet count and CTC count (r2 = 0.094, p = 0.0001). Correlation was also demonstrated within the overweight/obese group (n = 123, p < 0.0001), the non-exercise group (n = 79, p = 0.001) and blood draw samples lacking platelet cloaking (n = 128, p < 0.0001). By flow cytometry, blood samples from the exercise group (n = 15) had a higher proportion of CD3+ T-lymphocytes (p = 0.0003) and lower proportions of B-lymphocytes (p = 0.0264) and NK-cells (p = 0.015) than the non-exercise group (n = 14). These findings suggest that CTCs engage in complex interactions with the coagulation cascade and innate immune system during intravascular transit, and they present an attractive target for directed therapy at a vulnerable stage in metastasis.

Comparative Evaluation of Hematologic Analyzers Advia® 120, Advia® 2120 and Pentra® 120DX for Platelet Counts below 40 X 109/L.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1256-1256
Author(s):  
Josep M. Jou ◽  
Fulgencio Navalon ◽  
Ester Jiménez ◽  
Maribel Diaz-Ricart ◽  
Rosa Brugues ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Linear Regression ◽  
Reference Method ◽  
Cell Counting ◽  
Average Difference ◽  
Double Labeling ◽  
Blood Samples ◽  
Platelet Counts ◽  
Flow Cytometric Method ◽  
Advia 120

Abstract More aggressive therapies used for treatment of oncohematological malignancies or control of immune responses are resulting in an increased frequency of platelet counts below the 50 x 109/L limit. The recommended reference method for platelet counts was tedious and showed low reproducibility until now. In the last 2 years, flow cytometry based techniques combined with specific monoclonal antibodies (MoAbs) have been accepted as reference method. We have evaluated the accuracy for low platelet counts of several hematologic analyzers currently used in our laboratories. The new reference method approved by ISLH, ICSH y NCCLS is based on double labeling of platelets using MoAbs directed to CD41 and CD61 followed by flow cytometry analysis. Absolute platelet counts are calculated using a ratio with red blod cell (RBC) counts provided by the hematological analyzers. In our studies, 50 blood samples with platelet counts ranging from 1.5 to 39.4 x109/L were processed in duplicate through 1 Advia 2120 (Bayer Diagnostics), 2 Advia 120 (Bayer Diagnostics) and 2 Pentra 120 DX (Horiba-ABX Diagnostics). Advia analyzers use laser-based technology while Pentra analyzers use impedance one for cell counting. All samples were also processed through the reference flow cytometric method, being platelets identified by their double labeling for CD41 and CD61. The minimal number of platelets acquired in the platelet region was established at 1000. Absolute platelet counts were calculated using RBC counts provided by the respective analyzers. All blood samples were processed within 6 hours from phlebotomy. Statistical methods applied included: coefficient of variation (%CV), coefficient of correlation (r ), linear regression, Passing-Bablock (P-B) regression and Bland-Altman test. Precision of each analyzer was obtained by processing in 10 times different blood samples with counts from 4 to 39 x 109/L. Global results were evaluated, though special attention was paid to subgroups of results below or above 20 x 109/L. Correlation between reference values and counts provided by the Advia 2120 was 0.945 with a linear regression of 0.987x+2.9. P-B correlation was good and the average difference was 2.7 x 109/L. In the subgroup of samples with counts below 20 x 109/L correlation was 0.874 with 1.00x+2.7. P-B was correct and the average difference was 2.8 x 109/L. Results with Advia 120 were always similar to those calculated with the Advia 2120, though the average difference was slightly lower with a value of 1.7 x 109/L. Precision (CV) was 16% for platelet count levels at 4 x 109/L, 12% for those at 13 x 109/L and 4% for those at 39 x 109/L. Correlation with Pentra 120 Dx was 0.937 with a linear regression of 0.894x+2.7, the P-B was acceptable with an average difference of 1.2 x 109/L. Correlation index was 0.824 with a linear regression of 0.88x+2.8 for platelet counts below 20 x 109/L, average difference was of 1.4 x 109/L and a correct P-B. Precision (CV) ranged from 26% at 4 x 109/L and 8% at 20 x 109/L platelet counts. Our data demonstrate that hematological analyzers evaluated provided very reliable results at low platelet counts. Advia and Pentra analyzers investigated tend to over calculate the number of platelets (2.5 and 1.4 x109/L respectively). Correlation scattering was slightly superior with the Pentra analyzer. Overall reproducibility for low platelet counts was excellent for both laser and impedance technologies tested.

Association Of Platelet Function Markers, Independent Of Platelet Count, With Bleeding Score In Patients With Immune Thrombocytopenia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3534-3534
Author(s):  
Andrew L. Frelinger ◽  
Anja J Gerrits ◽  
Michelle A. Berny-Lang ◽  
Travis Brown ◽  
Sabrina L. Carmichael ◽  
...  
Keyword(s):  
Platelet Count ◽  
Platelet Activation ◽  
Platelet Function ◽  
Research Funding ◽  
Immune Thrombocytopenia ◽  
Univariate Analysis ◽  
Blood Draw ◽  
Platelet Surface ◽  
Platelet Counts ◽  
Bleeding Score

Abstract Background Immune thrombocytopenia (ITP) patients with similarly low platelet counts differ in their tendency to bleed. Aim To determine if differences in platelet function in ITP patients with similarly low platelet counts partly account for the variation in bleeding tendency. Methods The relationship between bleeding scores and platelet function markers was investigated in a single center cross-sectional study of pediatric patients with ITP. Following informed consent, blood was collected from ITP patients and bleeding was graded using the Buchanan and Adix Score (J Pediatr 2002) at routine clinic visits or while admitted to the hospital. Bleeding scores were obtained by one of three hematologists blinded to platelet function results, and investigators performing platelet function tests were blinded to clinical results. Platelet function was assessed by whole blood flow cytometric measurement of unstimulated, ADP- or TRAP-stimulated platelet surface activated GPIIb-IIIa (as measured by PAC1 binding), P-selectin, and GPIb and by unstimulated, convulxin-, or ADP plus TRAP-stimulated platelet surface phosphatidylserine expression (as determined by annexin V binding). Platelet count, immature platelet fraction (IPF) and mean platelet volume (MPV) were determined by a Sysmex XE-2100, and platelet forward angle light scatter (FSC) was measured by flow cytometry. Results Platelet function and bleeding scores were evaluated in 34 consecutive consenting pediatric ITP patients (16 female, 18 male, age 9.7 ± 5.7 years [mean ± SD]). ITP was newly diagnosed (< 3 months) in 10 patients, persistent (3 -- 12 months) in 7 patients, and chronic (>12 months) in 17 patients. Platelet count at the time of the blood draw was 47 ± 55 x 109/L. The median bleeding score on day of blood draw was 1 (range 0 to 4). By univariate analysis, higher IPF, and lower platelet count were significantly associated with a higher bleeding score (odds ratio [OR] >1, p<0.05) but MPV was not. Multiple measures of platelet function were associated with bleeding scores by univariate analysis: higher levels of platelet FSC (a measure affected by multiple variables including size) surface GPIb on unstimulated, ADP- or TRAP-stimulated platelets, surface P-selectin on unstimulated platelets, and platelet FSC were associated with increased odds for higher bleeding scores (ORs each >1, p<0.05), while higher ADP- and TRAP-stimulated platelet surface activated GPIIb-IIIa and P-selectin were associated with reduced odds of higher bleeding scores (ORs each <1, p<0.05). After adjustment for platelet count, higher levels of platelet surface P-selectin on unstimulated platelets, GPIb on TRAP-stimulated platelets, and FSC remained significantly associated with increased odds for higher bleeding scores (Figure), but IPF did not. Similarly, after adjustment for platelet count, higher TRAP-stimulated percentage of P-selectin and activated GPIIb-IIIa positive platelets remained significantly associated with reduced odds of higher bleeding scores (Figure). These findings were independent of recent ITP-related treatment. Conclusions In this study of pediatric ITP patients, we identified selected platelet function markers which, independent of platelet count, are associated with increased (platelet FSC, platelet surface P-selectin on unstimulated platelets, and GPIb on TRAP-stimulated platelets) or decreased (TRAP-stimulated percent P-selectin and GPIIb-IIIa positive platelets) odds of high bleeding scores. Possible hypotheses to explain these associations are as follows: 1) Increased P-selectin on unstimulated platelets demonstrates in vivo platelet activation, possibly as a consequence of the recent bleeding. 2) Because platelet activation results in a reduction in platelet surface GPIb and increases in platelet surface activated GPIIb-IIIa and P-selectin, the ORs associated with all of these markers could be explained by reduced ability of platelets in patients with higher bleeding scores to respond to agonists. 3) While platelet FSC is partly related to size, the finding that MPV and IPF, adjusted for platelet count, were not associated with bleeding score suggests that factors other than size account for the association of platelet FSC with higher bleeding scores. Further study is required to validate these findings and determine if differences in platelet function are associated with future risk for bleeding. Disclosures: Off Label Use: Eltrombopag was given to WAS/XLT patients for treatment of thrombocytopenia. Neufeld:Shire: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Apopharma: Consultancy. Michelson:Sysmex: Honoraria.

scholarly journals Cross-Strain Platelet Immunization in Selected Mouse Strains Recapitulates Clinical and Serologic Findings Typical of Post-Transfusion Purpura (PTP)

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2349-2349
Author(s):  
Daniel W. Bougie ◽  
Jessica Sutton ◽  
Richard H. Aster
Keyword(s):  
Flow Cytometry ◽  
Platelet Count ◽  
Amino Acid ◽  
Conflicts Of Interest ◽  
Human Condition ◽  
Autoimmune Response ◽  
Single Amino Acid ◽  
Mouse Strains ◽  
Igg Antibody ◽  
Platelet Counts

Post-transfusion purpura (PTP) is an uncommon but life-threatening condition characterized by profound thrombocytopenia (TP) occurring one week after transfusion of blood products. The hallmark of PTP is a potent IgG antibody specific for a transfused human platelet antigen (HPA), usually HPA-1a located on αIIb/β3 integrin (GPIIb/IIIa). It is widely thought that, in PTP, the alloantibody somehow causes destruction of the recipient's platelets even though they lack the antigen for which the alloantibody is specific. Several reports have suggested that the underlying cause of PTP is a platelet-specific autoantibody that can be difficult to detect because it is absorbed in the process of destroying autologous platelets and is overshadowed by the accompanying, very potent alloantibody but experimental support for this concept is minimal. Platelet alloantigens comparable to HPAs have not been defined in animals. Using a public database, we identified four mouse strains (C57BL/6J (C57), 129S1/Svlmj (129), PWK/PhJ (PWK), AND SPRET/EIJ) differing from each other at amino acid residues in extracellular domains of GPIIb/IIIa that could comprise potential alloantigens. Cross-strain platelet immunizations (intraperitoneal with adjuvant) were performed weekly for 5 weeks while monitoring platelet counts and platelet associated IgG (PAIgG) and saving plasma samples for serologic studies. After 2-4 immunizations, each of 39 cross-strain but none of 9 strain-identical immunizations induced "alloantibodies" that recognized donor but not recipient platelets (flow cytometry). Thrombocytopenia (<50% of maximum platelet count) developed in 28 of 39 mice (71%) given strain-disparate platelets but not in mice given strain-identical platelets; 12 of these mice (30%) developed profound TP (<15%). The most consistent and severe declines in platelet counts occurred in PWK mice immunized with 129 platelets and vice versa (N=13) in which the mean platelet count decline was 88% (range 59-96%, median 92%). Autoantibodies recognizing syngeneic platelets were identified in all animals that developed profound TP and their potency (measured by flow cytometry) correlated closely with the severity of TP (p<0.001) (Fig 1). Alloantibodies were shown by immunoprecipitation to be mainly specific for GPIIb/IIIa (N=13) and GPIb/IX (N=1) on donor platelets. Two monoclonal antibodies (mAbs MBC417.1 and MBC425.1) specific for a single polymorphic amino acid at positions 111(Gly) and 37(Val), respectively, on GPIIb of C57 and PWK mice were generated using spleen cells of two immunized mice. To our knowledge, these are the first alloantibodies in mice that are specific for single amino acid polymorphisms in a platelet membrane glycoprotein and are thus comparable to HPA antibodies found in humans. The findings define a model (platelet immunization between PWK and 129 mice) in which a routine alloantibody response recognizing GPIIb/IIIa on donor platelets regularly transitions to an autoimmune response capable of causing profound thrombocytopenia, thus mimicking the course of PTP in human patients and supporting the hypothesis that PTP is an autoimmune disorder. Successful development of the model could be related to use of the more recently developed, wild-caught PWK strain as one of the partners for immunization. The inherent utility of a mouse model is expected to facilitate further work to define the molecular basis for a transition from allo- to auto-immunity in the human condition, post-transfusion purpura. Disclosures No relevant conflicts of interest to declare.

scholarly journals Assessment of platelet biology in equine patients with systemic inflammatory response syndrome

2020 ◽  
pp. 104063872098379
Author(s):  
Carolin Ehrmann ◽  
Julia Engel ◽  
Andreas Moritz ◽  
Katja Roscher
Keyword(s):  
Platelet Count ◽  
Platelet Aggregation ◽  
Systemic Inflammatory Response Syndrome ◽  
Inflammatory Response ◽  
Systemic Inflammatory Response ◽  
Controlled Study ◽  
Blood Samples ◽  
Platelet Counts ◽  
Impedance Aggregometry ◽  
Edta Blood

In addition to maintaining hemostasis, platelets have an important role in modulating innate and adaptive immune responses. A low platelet count has been found to be a negative prognostic factor for survival in humans and horses with critical illnesses, such as sepsis or systemic inflammatory response syndrome (SIRS). Decreased platelet aggregation, caused by in vivo activation, has been found in human patients with severe sepsis. In our prospective controlled study, we assessed platelet biology in blood samples from 20 equine SIRS cases and 120 healthy control horses. Platelet variables such as platelet count, large platelet count, clumps, plateletcrit, mean platelet volume, and mean platelet component concentration were analyzed by laser flow cytometry (Advia 2120) from K3EDTA blood and from citrate blood. Hirudin blood samples were analyzed by impedance aggregometry (Multiplate analyzer; Roche) for platelet aggregation, including spontaneous aggregation and aggregation by 4 different agonists: adenosine diphosphate (ADPtest), ADP + prostaglandin E1 (ADPtestHS), arachidonic acid (ASPItest), and collagen (COLtest). SIRS cases had significantly lower platelet counts in K3EDTA blood ( p < 0.0001) compared to control horses. There were no significant differences in aggregation values between SIRS cases and controls. Non-surviving SIRS horses did not have statistically significant lower platelet counts or lower aggregation values for COLtest, ADPtest, or ADPtestHS compared to surviving SIRS horses, although 5 non-survivors were thrombocytopenic.

scholarly journals Prostate cancer peripheral blood NK cells show enhanced CD9, CD49a, CXCR4, CXCL8, MMP-9 production, and secrete monocyte-recruiting and polarizing factors

2020 ◽  
Author(s):  
Denisa Baci ◽  
Matteo Gallazzi ◽  
Mortara Lorenzo ◽  
Annalisa Bosi ◽  
Giuseppe Buono ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Flow Cytometry ◽  
Endothelial Cells ◽  
Nk Cells ◽  
Cell Lines ◽  
Peripheral Blood ◽  
Nk Cell ◽  
Cell Subset ◽  
Multicolor Flow Cytometry ◽  
Angiogenic Cytokines

ABSTRACTBackgroundNatural killer (NK) cells are effector lymphocytes of the innate immunity. Two major NK cell subsets are mostly present in the peripheral blood (pNKs): the cytotoxic CD56dimCD16+ NK cell subset (90-95% of pNKs), and the low cytotoxic, highly cytokine-producing CD56brightCD16-/low NK cell subset (5-10% of pNKs). It has been demonstrated that NK cells in peripheral blood of patients with several tumors are altered. We have shown that in NSCLC and colon cancer, tumor associated circulating NK (pTA-NK) and tumor infiltrating NK (TI-NK) are skewed towards the CD56brightCD16-/low phenotype. We have detected the production of pro-inflammatory and pro-angiogenic cytokines and chemokines. Other groups are reporting similar observations. There is still a lack of knowledge concerning the phenotype of pNK cells in prostate cancer (PCa). Here, we phenotypically and functionally characterized peripheral blood NK (pNK) from PCa patients (PCa pTA-NKs) and investigated their production of soluble factors, with endothelial cells and macrophage stimulatory action.MethodsNK cell subset distribution was investigated in the peripheral blood of PCa patients, by multicolor flow cytometry (FC) for surface antigens expression. Protein arrays were performed to characterize the secretome on FACS-sorted pNK cells. Secreted products from FACS-sorted PCa TA-NKs were used to characterize their production of pro-inflammatory molecules. Secreted products from FACS-sorted PCa pTA-NKs were also used to stimulate endothelial cells and monocytes and macrophages, determining their ability to recruit and polarize them. Alterations of endothelial cells and monocytes, following exposure to secreted products from FACS-sorted PCa pTA-NKs, was assessed by RT-PCR. To confirm these observations, secreted products from 3 different PCa (PC-3, DU-145, LNCaP) cell lines were used to assess their effects on human NK cell polarization, by multicolor flow cytometry.ResultsCirculating NK cells from prostate cancer patients have been studied before, mostly for their impaired lytic functions. However, here we are the first to report that circulating pNK cells from PCa patients acquire a CD56brightCD9+CD49a+CXCR4+ phenotype with pro-inflammatory properties. We observed a similar polarization of heathy-donor derived pNK cells exposed to secreted products of three different PCa cell lines. Increased production of CXCL8, CXCR4, MMP-9, pro-inflammatory and reduced production of TNFα, IFNγ and Granzyme-B was detected. PCa TA-NKs released factors able to support angiogenesis in vitro and increased the expression of CXCL8, ICAM-1 and VCAM-1 mRNA in endothelial cells, confirming a pro-inflammatory signature. Secretome analysis revealed the ability of PCa pTA-NKs to release pro-angiogenic cytokines/chemokines involved in monocyte recruitment and M2-like polarization. In experimental setting, secreted products from PCa pTA-NKs can recruit THP-1 monocyte and polarize THP-1-differentiated macrophage towards CD206/Arginase1/IL-10/CXCL8-expressing M2-like/TAMs.ConclusionsOur results show that PCa pTA-NKs are effector cells able to produce pro-inflammatory angiogenesis factors able to stimulate endothelial cells, attract monocytes and polarize macrophage to an M2-like type. Our data provides a rationale for the possible use of pNK profiling in clinical studies on PCaGRAPHICAL ABSTRACTGraphical Abstract:Representative cartoon illustrating the pro-angiogenic features of PCa pTA-NKs.A) direct effects of PCa pTA-NKs in supporting angiogenesis by interacting with endothelial cells. B) Proposed model for PCa pTA-NK pro-angiogenic activities via macrophage recruitment and polarization.

A feasibility study on the effects of Triton X-100 for the in vitro inactivation of Ebola virus on haematological assays

2015 ◽  
Vol 69 (7) ◽  
pp. 637-642 ◽  
Author(s):  
Antoinette Mifsud ◽  
Daphne Peelen ◽  
Patricia Brincat ◽  
Sylvana Abela ◽  
Neville Debattista ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Blood Film ◽  
T Cell Subsets ◽  
Red Cell ◽  
Mean Cell Volume ◽  
Blood Samples ◽  
Platelet Counts ◽  
Triton X ◽  
Blood Grouping

AimsThe aim of this study was to check the effect of Triton X-100 on various, commonly used haematology test parameters.MethodsAnonymised blood samples were treated with 10 µL of 10% Triton X-100 per 1 mL of blood. Treated and untreated samples were tested in parallel for blood film morphology, complete blood counts (CBCs), flow cytometry, blood grouping and antibody screens. Samples were also taken in 3.2% citrate tubes for coagulation test analyses.ResultsStatistical differences were noted in all CBC parameters apart from the mean cell volume, eosinophil and basophil counts. Platelet counts were significantly different with an apparent rise after the addition of Triton X-100. Samples were noted to have a high red cell fragmentation index. Immunological platelet counting methods using flow cytometry and fluorescent methods showed no significant differences and gave reliable results. Neither flow cytometry for T-cell subsets nor blood grouping/antibody screens were affected by Triton X-100. However, coagulation samples were severely haemolysed prohibiting analysis.ConclusionsWe have demonstrated that the addition of Triton X-100 to haematology blood samples impacts mainly on platelet counts and coagulation studies due to haemolysis. The platelet count is spuriously raised probably due to the presence of red cell fragments. The latter can be circumvented by the use of immunological platelet counting technology.

scholarly journals Perbedaan Hitung Jumlah Trombosit Menggunakan Darah Vena dan Darah Kapiler

2016 ◽  
Vol 3 (2) ◽  
pp. 81
Author(s):  
Hieronymus Rayi Prasetya ◽  
Maria Irena Dentri ◽  
Sistiyono Sistiyono
Keyword(s):  
Platelet Count ◽  
Blood Platelets ◽  
Venous Blood ◽  
Blood Platelet ◽  
Capillary Blood ◽  
Blood Capillaries ◽  
Blood Samples ◽  
Platelet Counts ◽  
Significant Difference ◽  
Blood Platelet Count

Background: Platelets play a role in hemostasis which is the body's mechanisms to prevent and stop the bleeding. Platelets participate in the effort to close the wound, so that the body does not experience a loss of blood and protected from foreign cells. Examination of the platelet count is very important in the diagnosis of diseases, one of which is the diagnosis of dengue hemorrhagic fever (DHF). Examination of blood counts, especially platelets in clinical laboratories causes blood samples in use are not always the venous blood but could use capillary blood. Capillary blood samples are used primarily in pediatric patients, because the venous blood sampling is difficult, patient loads, and also shorten the time when taking blood. The purpose of this study was to determine whether there is a difference in counting the number of platelets using samples of blood veins and capillaries. Methods: Quantitative research with observational approach using a cross sectional study design in the 30 samples of student D3 Health Analyst STIKes To Nation Yogyakarta. Statistical methods in use are independent T test. Results: The research subjects were 30 samples of student D3 Health Analyst STIKes To Nation Yogyakarta. The results of the examination of venous blood platelet count and blood capillaries have different average values ​​are 247 530 cells / ml of blood, for blood platelets veins and 184 270 cells / ml of blood for capillary blood platelets. Spearman correlation analysis Obtained results of the examination of venous blood platelet count and blood capillaries normal distribution (p> 0.05). 0.129 venous blood platelet counts, while the number of blood platelets kapilernya 0.089. Conclusion: There is a significant difference from the results of counting the number of blood platelets using veins and capillaries, where the use of capillary blood samples showed that lower platelet counts.

scholarly journals Simultaneous Assay of Agonist-Induced Endogenous and Non-Radioactive Isotopic Serotonin Secretion from Small Numbers of Human Platelets By Mass Spectrometry

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1055-1055
Author(s):  
Xin Yi ◽  
Zhiyun Cao ◽  
Ina Sandeli ◽  
Edward KY Leung ◽  
Kiang-Teck Jerry Yeo ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Flow Cytometry ◽  
Platelet Count ◽  
Platelet Function ◽  
Limit Of Detection ◽  
Dense Granule ◽  
Ionophore A23187 ◽  
Platelet Counts ◽  
Dense Granule Secretion ◽  
Granule Secretion

Abstract Background: Light transmission aggregometry (LTA) with simultaneous assay of ATP released from platelet dense granules (lumi-aggregation) provides the ability to assess platelet function in a variety of clinical settings. This methodology, however, requires the ability to obtain adequate sample volume from the patient, if a number of platelet agonists are to be assessed. The platelet count within the patient's blood is also a limiting factor, and LTA interpretations can become difficult when platelet counts in the sample fall significantly below 150,000/µL. Analysis of platelet function by flow cytometry circumvents the limitations of both blood volume and low platelet counts, typically providing information as to glycoprotein IIb/IIIa activation state and release of platelet alpha granules in response to platelet agonists. Flow cytometry, however, is not presently capable of providing quantitative analysis of the release of platelet dense granule constituents. Given the clinical importance of platelet dense granule secretion, we sought to develop a non-radioactive method for quantitation of agonist-induced release of serotonin (5-HT) from blood sample volumes obtainable from small infants and from patients with at least moderate thrombocytopenia. Methods: In the initial studies, total platelet content and released 5-HT following stimulation by a panel of agonists were assessed using 200 µL of freshly drawn, normal citrated blood. In subsequent studies, uptake and agonist-induced secretion of non-radioactive, deuterated serotonin (d-5HT) were additionally measured in parallel with the endogenous 5-HT in these 200 µL samples. In brief, the citrated blood was initially incubated for 15' at 37° with d-5HT, then diluted 10-fold with buffer, and platelet stimulus (collagen, TRAP, ADP, epinephrine, ionophore A23187, thromboxane analogue U46619, ristocetin, or buffer blank) added. Following stimulation, samples were further diluted with buffer, prostaglandin E1 added, and then centrifuged to yield supernatants for analysis. 5-HT and d-5HT total content in unstimulated platelets was additionally assayed. N-methyl-5HT was employed as an internal standard. 5-HT, d-5HT and N-methyl-5HT were subsequently detected and quantified simultaneously by liquid chromatography/tandem mass spectrometry (MS) (Eksigent MicroLC 200/QTrap 6500 Mass Spectrometer, Sciex, Framingham, MA). Results and Conclusions: MS analysis of 5-HT was linear over the range of 50-2000 pg/mL. For whole blood platelet counts of at least 120,000/µL, this sensitivity was sufficient to permit analysis of 5-HT released in response not only to strong stimuli, but even to weaker stimuli such as low concentration (2 µg/mL) collagen. Since the goal of this project, however, was to be able to extend functional analysis to patients with more significant degrees of thrombocytopenia, further efforts were made to improve sensitivity. Assay of agonist-induced release, following platelet uptake, of d-5HT was assessed in parallel with the assay of released endogenous 5-HT. Release of 5-HT and d-5HT in response to increasing concentrations of collagen showed generally similar kinetics. However, not only was the absolute signal increased for the d-5HT, but the analytic assay itself proved significantly more sensitive as compared to that for 5-HT. Combined, these effects increased the overall sensitivity of the assay by at least an order of magnitude. For example, d-5HT released in response to 2 µg/mL collagen in a donor blood having a platelet count of 188,000/µL produced an MS signal approximately 30-fold higher than the lower limit of detection for the assay. Although it will require actual study of thrombocytopenic patients to verify, these results suggest that by use of this MS approach with d-5HT it may be possible to measure platelet dense granule secretion to a panel of agonists from 200 µL of blood in patients with thrombocytopenia potentially as severe as 10,000/µL. Disclosures No relevant conflicts of interest to declare.

scholarly journals Prostate Cancer Peripheral Blood NK Cells Show Enhanced CD9, CD49a, CXCR4, CXCL8, MMP-9 Production and Secrete Monocyte-Recruiting and Polarizing Factors

2021 ◽  
Vol 11 ◽  
Author(s):  
Matteo Gallazzi ◽  
Denisa Baci ◽  
Lorenzo Mortara ◽  
Annalisa Bosi ◽  
Giuseppe Buono ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Flow Cytometry ◽  
Endothelial Cells ◽  
Nk Cells ◽  
Cell Lines ◽  
Peripheral Blood ◽  
Nk Cell ◽  
Multicolor Flow Cytometry ◽  
Inflammatory Angiogenesis

Natural killer (NK) cells, effector lymphocytes of the innate immunity, have been shown to be altered in several cancers, both at tissue and peripheral levels. We have shown that in Non-Small Cell Lung Cancer (NSCLC) and colon cancer, tumour associated circulating NK (TA-NK) and tumour infiltrating NK (TI-NK) exhibit pro-angiogenic phenotype/functions. However, there is still a lack of knowledge concerning the phenotype of peripheral blood (PB) NK (pNK) cells in prostate cancer (PCa). Here, we phenotypically and functionally characterized pNK from PCa patients (PCa TA-NKs) and investigated their interactions with endothelial cells and monocytes/macrophages. NK cell subset distribution in PB of PCa patients was investigated, by multicolor flow cytometry, for surface antigens expression. Protein arrays were performed to characterize the secretome on FACS-sorted pNK cells. Conditioned media (CM) from FACS-sorted PCa pTA-NKs were used to determine their ability to induce pro-inflammatory/pro-angiogenic phenotype/functions in endothelial cells, monocytes, and macrophages. CM from three different PCa (PC-3, DU-145, LNCaP) cell lines, were used to assess their effects on human NK cell polarization in vitro, by multicolor flow cytometry. We found that PCa pTA-NKs acquire the CD56brightCD9+CD49a+CXCR4+ phenotype, increased the expression of markers of exhaustion (PD-1, TIM-3) and are impaired in their degranulation capabilities. Similar effects were observed on healthy donor-derived pNK cells, exposed to conditioned media of three different PCa cell lines, together with increased production of pro-inflammatory chemokines/chemokine receptors CXCR4, CXCL8, CXCL12, reduced production of TNFα, IFNγ and Granzyme-B. PCa TA-NKs released factors able to support inflammatory angiogenesis in an in vitro model and increased the expression of CXCL8, ICAM-1, and VCAM-1 mRNA in endothelial cells. Secretome analysis revealed the ability of PCa TA-NKs to release pro-inflammatory cytokines/chemokines involved in monocyte recruitment and M2-like polarization. Finally, CMs from PCa pTA-NKs recruit THP-1 and peripheral blood CD14+ monocyte and polarize THP-1 and peripheral blood CD14+ monocyte-derived macrophage towards M2-like/TAM macrophages. Our results show that PCa pTA-NKs acquire properties related to the pro-inflammatory angiogenesis in endothelial cells, recruit monocytes and polarize macrophage to an M2-like type phenotype. Our data provides a rationale for a potential use of pNK profiling in PCa patients.

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