scholarly journals Irreversible Alteration of Extracellular Vesicle and Cell-Free Messenger RNA Profiles in Human Plasma Associated With Ex Vivo Platelet Activation

2021 ◽  
Author(s):  
Hyun Ji Kim ◽  
Matthew Rames ◽  
Samuel Tassi Yunga ◽  
Randall Armstrong ◽  
Mayu Morita ◽  
...  
Keyword(s):  
Human Plasma ◽  
Platelet Activation ◽  
Messenger Rna ◽  
Ex Vivo ◽  
Flow Cytometric Analysis ◽  
Extracellular Vesicle ◽  
Mrna Levels ◽  
Sample Collection ◽  
Freeze Thaw ◽  
Canonical Surface

Abstract The discovery and utility of clinically relevant circulating biomarkers depend on standardized methods that minimize preanalytical errors. Here, we examined the preanalytical variations of extracellular vesicles (EVs) and cell-free mRNA (cf-mRNA) profiles in human plasma and how post-freeze/thaw processing removes and retains EVs and cf-mRNA subtypes. Using multiparametric nanoscale flow cytometric analysis, which utilized forward light scatter, side scatter, and fluorescence intensity, we characterized the effect of specific preanalytical variables on vesicle subpopulations in human plasma. Cf-mRNA levels were measured by multiplexed RT-qPCR with a panel including housekeeping, platelet, and tissue-specific genes. We found that blood processing centrifugation and temperature strongly impacted the levels of residual platelets and platelet microvesicles. Intriguingly, exosome-sized events were more resistant to processing methods. We also examined the morphology of quiescent and activated platelets and platelet-derived EVs by electron microscopy. We demonstrated that platelet activation and fragmentation generated ex vivo vesicles similar to EVs in size, morphology, and canonical surface markers. Critically, freezing plasma containing residual platelets yielded irreversible ex vivo generation of new EV subpopulations and cf-mRNA transcripts, which were not removable by additional processing after freeze-thaw. In summary, we identified distinct subpopulations of EVs and cf-mRNA in human plasma that are differentially influenced by platelet activation and fragmentation during sample collection, processing, and banking via post freeze/thaw processing.

Tissue factor messenger RNA levels in leukocytes compared with tissue factor antigens in plasma from patients in hypercoagulable state caused by various diseases

2004 ◽  
Vol 92 (07) ◽  
pp. 132-139 ◽  
Author(s):  
Tomohiro Sase ◽  
Yuko Kamikura ◽  
Toshihiro Kaneko ◽  
Yasunori Abe ◽  
Junji Nishioka ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Plasma Levels ◽  
Messenger Rna ◽  
Healthy Subjects ◽  
Flow Cytometric Analysis ◽  
Antigen Expression ◽  
Normal Subjects ◽  
Hypercoagulable State ◽  
Mrna Levels ◽  
All Diseases

SummaryWe compared the levels of tissue factor (TF) mRNA in leukocytes with plasma TF antigens of patients in hypercoagulable state caused by various diseases. Flow cytometric analysis showed absence of TF antigen expression on neutrophils and monocytes in healthy subjects but strong expression in both cell types of patients with infections. TF mRNA levels in leukocytes were low in healthy subjects but they were significantly elevated in patients with underlying diseases of disseminated intravascular coagulation (DIC), especially in acute myeloid leukaemia (AML) and infections. TF mRNA levels in leukocytes were significantly high in patients with all diseases except those with thrombosis, and plasma TF antigen levels were significantly high in all diseases. TF mRNA in leukocytes and plasma TF antigen levels were significantly high in patients with overt-DIC, and TF mRNA/antigen ratio was significantly high in patients with overt-DIC. In patients with solid cancers, TF mRNA and TF mRNA/antigen ratio were significantly higher in patients with metastases than those without. TF mRNA levels in leukocytes and plasma levels of TF antigen did not correlate in normal subjects and all patients, but they tended to be correlated in patients with AML, infections or overt-DIC. Our analysis suggests that TF expression in leukocytes plays an important role in various diseases but the expression level does not always correlate with plasma levels of TF antigen.

Axl/ Mer Inhibitor ONO-9330547 As a Novel Therapeutic Agent in a Stromal Co-Culture Model of Primary Acute Myeloid Leukaemia (AML)

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2754-2754 ◽  
Author(s):  
Marie Gilmour ◽  
Anna Scholtz ◽  
Oliver G. Ottmann ◽  
Robert K Hills ◽  
Steven Knapper ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Drug Resistance ◽  
Research Funding ◽  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Flow Cytometric Analysis ◽  
Transcript Level ◽  
High Rate ◽  
Bone Marrow Niche ◽  
Mrna Levels

Abstract The TAM kinase family are a group of receptor kinases involved in the regulation of many cellular processes including proliferation, cell survival and cytokine production. Consequently this group is frequently overexpressed or aberrantly activated in a variety of cancers including AML, where overexpression of Axl or its ligand Gas6 confers a poor prognosis particularly amongst FLT3-ITD mutated AMLs. ONO-9330547 is a highly potent Axl/Mer inhibitor which shows improved target selectivity compared to other orally available Axl inhibitors in leukaemic cells and spares normal CD34+ bone marrow cells predicting minimal toxicity. We examined a cohort of 70 primary AMLs Ex vivo for ONO-9330547 drug sensitivity and determined low micromolar EC50s in 60% of patient samples (Average EC50=3.16µM+/-3.49). Phospho-Axl was rapidly reduced in response to ONO-9330547 and downstream pro-survival targets AKT, S6 and ERK1/2 were concurrently suppressed. Dose dependant induction of apoptosis was observed in conjunction with suppression of the Axl ligand, Gas6. Synergistic interaction of ONO-9330547 with the chemotherapeutic drug Cytarabine was observed (Mean combination index =0.59), suggesting combination therapy to be a useful strategy with the development of Axl inhibitors. There was no association of ONO-9330547 sensitivity with any clinical characteristics within the patient cohort, however the samples from the commonly-occurring NPM1 mutant/ cytogenetically normal AML subgroup were significantly more sensitive to ONO-9330547 than WT or FLT3-ITD samples (p=0.004 lower EC50). mRNA levels of TAM family members Axl, Mer, Tyro3 and Gas6 were generally low in diagnostic patient material, although those with high levels of ≥1 of the TAM kinases were associated with drug resistance. This was confirmed through flow cytometric analysis of phospho-Axl, where similarly high basal activation of Axl correlated with increased EC50 values. Given the high rate of relapse and drug resistance in AML and the potential for microenvironment mediated protection of AML blasts in the bone marrow niche, we investigated the effects of ONO-9330547 in stromal co-culture models. Stimulation of basal p-Axl and Gas6 was observed through the addition of cytokines and adhesion of AML blasts to the stromal layer. In contrast to low levels of Gas6 at the transcript level, Gas6 was readily detectable in patient plasma samples and blasts co-cultured on primary AML-derived stromal layers compared to normal bone marrow stroma. Co-culture completely abrogated the efficacy of ONO-9330547 on AML blasts, significantly blocked apoptotic response and Axl/Gas 6 knockdown. In summary, constitutive or stroma- inducible levels of Gas6 ligand direct ONO-9330547 sensitivity in diagnostic AML patient samples. These data provide a pre-clinical assessment of ONO-9330547 in AML and provide rationale for further investigation of this compound in combination with both traditional and novel therapies that may disrupt microenvironment-mediated up-regulation of the Axl/Gas6 signalling pathway. Disclosures Gilmour: ONO pharmaceuticals: Research Funding. Ottmann:Fusion Pharma: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Ariad: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Hills:TEVA: Honoraria. Knapper:ONO pharmaceuticals: Research Funding; Novartis: Honoraria, Other: Travel and expenses for international conferences. Zabkiewicz:ONO pharmaceuticals: Research Funding.

scholarly journals TLR2 Deficiency Exacerbates Imiquimod-Induced Psoriasis-Like Skin Inflammation through Decrease in Regulatory T Cells and Impaired IL-10 Production

2020 ◽  
Vol 21 (22) ◽  
pp. 8560
Author(s):  
Momoko Nakao ◽  
Makoto Sugaya ◽  
Hideki Fujita ◽  
Tomomitsu Miyagaki ◽  
Sohshi Morimura ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Messenger Rna ◽  
Critical Role ◽  
Flow Cytometric Analysis ◽  
Skin Inflammation ◽  
Mrna Levels ◽  
Wild Type ◽  
Flow Cytometric ◽  
Tlr2 Signaling

Emerging evidence has demonstrated that Toll-like receptors (TLRs) are associated with autoimmune diseases. In this study, we investigated the role of TLR2 in psoriasis using imiquimod-induced psoriasis-like dermatitis. Although TLR2 signaling is known to play a critical role in the induction of proinflammatory cytokines by immune cells, such as dendritic cells (DCs), macrophages, and monocytes, TLR2 deficiency unexpectedly exacerbated psoriasiform skin inflammation. Importantly, messenger RNA (mRNA) levels of Foxp-3 and IL-10 in the lesional skin were significantly decreased in TLR2 KO mice compared with wild-type mice. Furthermore, flow cytometric analysis of the lymph nodes revealed that the frequency of regulatory T cells (Tregs) among CD4-positive cells was decreased. Notably, stimulation with Pam3CSK4 (TLR2/1 ligand) or Pam2CSK4 (TLR2/6 ligand) increased IL-10 production from Tregs and DCs and the proliferation of Tregs. Finally, adoptive transfer of Tregs from wild-type mice reduced imiquimod-induced skin inflammation in TLR2 KO mice. Taken together, our results suggest that TLR2 signaling directly enhances Treg proliferation and IL-10 production by Tregs and DCs, suppressing imiquimod-induced psoriasis-like skin inflammation. Enhancement of TLR2 signaling may be a new therapeutic strategy for psoriasis.

scholarly journals Phenotypical and Functional Characteristics of Human Regulatory T Cells during Ex Vivo Maturation from CD4+ T Lymphocytes

2021 ◽  
Vol 11 (13) ◽  
pp. 5776
Author(s):  
Varvara G. Blinova ◽  
Natalia S. Novachly ◽  
Sofya N. Gippius ◽  
Abdullah Hilal ◽  
Yulia A. Gladilina ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Ex Vivo ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Inflammatory Reactions ◽  
Effector Cells ◽  
Cycle Regulation ◽  
Further Development

Regulatory T cells (Tregs) participate in the negative regulation of inflammatory reactions by suppressing effector cells. In a number of autoimmune disorders, the suppressive function and/or the number of Tregs is compromised. The lack of active functioning Tregs can be restored with adoptive transfer of expanded ex vivo autologous Tregs. In our study, we traced the differentiation and maturation of Tregs CD4+CD25+FoxP3+CD127low over 7 days of cultivation from initial CD4+ T cells under ex vivo conditions. The resulting ex vivo expanded cell population (eTregs) demonstrated the immune profile of Tregs with an increased capacity to suppress the proliferation of target effector cells. The expression of the FoxP3 gene was upregulated within the time of expansion and was associated with gradual demethylation in the promotor region of the T cell-specific demethylation region. Real-time RT-PCR analysis revealed changes in the expression profile of genes involved in cell cycle regulation. In addition to FOXP3, the cells displayed elevated mRNA levels of Ikaros zinc finger transcription factors and the main telomerase catalytic subunit hTERT. Alternative splicing of FoxP3, hTERT and IKZF family members was demonstrated to be involved in eTreg maturation. Our data indicate that expanded ex vivo eTregs develop a Treg-specific phenotype and functional suppressive activity. We suggest that eTregs are not just expanded but transformed cells with enhanced capacities of immune suppression. Our findings may influence further development of cell immunosuppressive therapy based on regulatory T cells.

scholarly journals The Caspase-1/IL-18 Axis of the Inflammasome in Tumor Cells: A Modulator of the Th1/Tc1 Response of Tumor-Infiltrating T Lymphocytes in Colorectal Cancer

Cancers ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 189
Author(s):  
Linda Bilonda Mutala ◽  
Cécile Deleine ◽  
Matilde Karakachoff ◽  
Delphine Dansette ◽  
Kathleen Ducoin ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
T Lymphocytes ◽  
Tumor Cells ◽  
Ex Vivo ◽  
Explant Culture ◽  
T Cell Response ◽  
Mrna Levels ◽  
Innate And Adaptive Immunity ◽  
Caspase 1 ◽  
Culture Model

In colorectal cancer (CRC), a high density of T lymphocytes represents a strong prognostic marker in subtypes of CRC. Optimized immunotherapy strategies to boost this T-cell response are still needed. A good candidate is the inflammasome pathway, an emerging player in cancer immunology that bridges innate and adaptive immunity. Its effector protein caspase-1 matures IL-18 that can promote a T-helper/cytotoxic (Th1/Tc1) response. It is still unknown whether tumor cells from CRC possess a functional caspase-1/IL-18 axis that could modulate the Th1/Tc1 response. We used two independent cohorts of CRC patients to assess IL-18 and caspase-1 expression by tumor cells in relation to the density of TILs and the microsatellite status of CRC. Functional and multiparametric approaches at the protein and mRNA levels were performed on an ex vivo CRC explant culture model. We show that, in the majority of CRCs, tumor cells display an activated and functional caspase-1/IL-18 axis that contributes to drive a Th1/Tc1 response elicited by TILs expressing IL-18Rα. Furthermore, unsupervised clustering identified three clusters of CRCs according to the caspase-1/IL-18/TIL density/interferon gamma (IFNγ) axis and microsatellite status. Together, our results strongly suggest that targeting the caspase-1/IL-18 axis can improve the anti-tumor immune response in subgroups of CRC.

scholarly journals Velocity Gradient Separation Reveals a New Extracellular Vesicle Population Enriched in miR-155 and Mitochondrial DNA

Pathogens ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 526
Author(s):  
Myriam Vaillancourt ◽  
Audrey Hubert ◽  
Caroline Subra ◽  
Julien Boucher ◽  
Wilfried Wenceslas Bazié ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Velocity Gradient ◽  
Messenger Rna ◽  
Sucrose Density Gradient ◽  
Extracellular Vesicle ◽  
Therapeutic Interventions ◽  
Optimal Separation ◽  
Velocity Gradients ◽  
Commercial Kits ◽  
Hiv 1

Extracellular vesicles (EVs) and their contents (proteins, lipids, messenger RNA, microRNA, and DNA) are viewed as intercellular signals, cell-transforming agents, and shelters for viruses that allow both diagnostic and therapeutic interventions. EVs circulating in the blood of individuals infected with human immunodeficiency virus (HIV-1) may provide insights into pathogenesis, inflammation, and disease progression. However, distinguishing plasma membrane EVs from exosomes, exomeres, apoptotic bodies, virions, and contaminating proteins remains challenging. We aimed at comparing sucrose and iodixanol density and velocity gradients along with commercial kits as a means of separating EVs from HIV particles and contaminating protein like calprotectin; and thereby evaluating the suitability of current plasma EVs analysis techniques for identifying new biomarkers of HIV-1 immune activation. Multiple analysis have been performed on HIV-1 infected cell lines, plasma from HIV-1 patients, or plasma from HIV-negative individuals spiked with HIV-1. Commercial kits, the differential centrifugation and density or velocity gradients to precipitate and separate HIV, EVs, and proteins such as calprotectin, have been used. EVs, virions, and contaminating proteins were characterized using Western blot, ELISA, RT-PCR, hydrodynamic size measurement, and enzymatic assay. Conversely to iodixanol density or velocity gradient, protein and virions co-sedimented in the same fractions of the sucrose density gradient than AChE-positive EVs. Iodixanol velocity gradient provided the optimal separation of EVs from viruses and free proteins in culture supernatants and plasma samples from a person living with HIV (PLWH) or a control and revealed a new population of large EVs enriched in microRNA miR-155 and mitochondrial DNA. Although EVs and their contents provide helpful information about several key events in HIV-1 pathogenesis, their purification and extensive characterization by velocity gradient must be investigated thoroughly before further use as biomarkers. By revealing a new population of EVs enriched in miR-155 and mitochondrial DNA, this study paves a way to increase our understanding of HIV-1 pathogenesis.

scholarly journals Granulosal and thecal expression of bone morphogenetic protein- and activin-binding protein mRNA transcripts during bovine follicle development and factors modulating their expression in vitro

Reproduction ◽  
2011 ◽  
Vol 142 (4) ◽  
pp. 581-591 ◽  
Author(s):  
Claire Glister ◽  
Leanne Satchell ◽  
Phil G Knight
Keyword(s):  
Bone Morphogenetic Proteins ◽  
Transforming Growth Factor ◽  
Ex Vivo ◽  
Mrna Level ◽  
Transcript Abundance ◽  
Transforming Growth Factor Β ◽  
Follicle Development ◽  
Mrna Levels ◽  
Theca Interna

Evidence supports local roles for transforming growth factor β superfamily members including activins and bone morphogenetic proteins (BMP) in follicle development. Access of these ligands to signalling receptors is likely modulated by extracellular binding proteins (BP). In this study, we comparedex vivoexpression of four BPs (chordin, gremlin, noggin and follistatin) in granulosal (GC) and theca interna (TC) compartments of developing bovine antral follicles (1–18 mm). Effects of FSH and IGF on BMP and BP expression by cultured GC, and effects of LH and BMPs on BP expression by cultured TC were also examined. Follicular expression of all four BP transcripts was higher in GC than TC compartments (P<0.001) a finding confirmed by immunohistochemistry. Follicle category affected (P<0.01) gremlin and follistatin mRNA abundance, with a significant cell-type×follicle category interaction for chordin, follistatin and noggin. Noggin transcript abundance was lower (P<0.05) in GC of large ‘E-active’ than ‘E-inactive’ follicles while follistatin mRNA level was higher (P<0.01). FSH enhanced CYP19, FSHR, INHBA and follistatin by GC without affecting BMP or BMP–BP expression. IGF increased CYP19 and follistatin, reduced BMP4, noggin and gremlin but did not affect chordin orFSHRmRNA levels. LH increased TC androgen secretion but had no effect on BMP or BP expression. BMPs uniformly suppressed TC androgen production whilst increasing chordin, noggin and gremlin mRNA levels up to 20-fold (P<0.01). These findings support the hypothesis that extracellular BP, mostly from GC, contribute to the regulation of intrafollicular BMP/activin signalling. Enhancement of thecal BP expression by BMP implies an autoregulatory feedback role to prevent excessive signalling.

scholarly journals Differential Effects of Gonadotropin-Releasing Hormone (GnRH) Pulse Frequency on Gonadotropin Subunit and GnRH Receptor Messenger Ribonucleic Acid Levels in Vitro*

Endocrinology ◽  
1997 ◽  
Vol 138 (3) ◽  
pp. 1224-1231 ◽  
Author(s):  
Ursula B. Kaiser ◽  
Andrzej Jakubowiak ◽  
Anna Steinberger ◽  
William W. Chin
Keyword(s):  
Gene Expression ◽  
Messenger Rna ◽  
Pulse Frequency ◽  
Gnrh Receptor ◽  
Pituitary Cells ◽  
Mrna Levels ◽  
Subunit Gene ◽  
Differential Effects ◽  
Messenger Ribonucleic Acid

Abstract The hypothalamic hormone, GnRH, is released and transported to the anterior pituitary in a pulsatile manner, where it binds to specific high-affinity receptors and regulates gonadotropin biosynthesis and secretion. The frequency of GnRH pulses changes under various physiological conditions, and varying GnRH pulse frequencies have been shown to regulate differentially the secretion of LH and FSH and the expression of the gonadotropin α, LHβ, and FSHβ subunit genes in vivo. We demonstrate differential effects of varying GnRH pulse frequency in vitro in superfused primary monolayer cultures of rat pituitary cells. Cells were treated with 10 nm GnRH pulses for 24 h at a frequency of every 0.5, 1, 2, or 4 h. α, LHβ, and FSHβ messenger RNA (mRNA) levels were increased by GnRH at all pulse frequencies. α and LHβ mRNA levels and LH secretion were stimulated to the greatest extent at a GnRH pulse frequency of every 30 min, whereas FSHβ mRNA levels and FSH secretion were stimulated maximally at a lower GnRH pulse frequency, every 2 h. GnRH receptor (GnRHR) mRNA levels also were increased by GnRH at all pulse frequencies and were stimulated maximally at a GnRH pulse frequency of every 30 min. Similar results were obtained when the dose of each pulse of GnRH was adjusted to maintain a constant total cumulative dose of GnRH over 24 h. These data show that gonadotropin subunit gene expression is regulated differentially by varying GnRH pulse frequencies in vitro, suggesting that the differential effects of varying GnRH pulse frequencies on gonadotropin subunit gene expression occur directly at the level of the pituitary. The pattern of regulation of GnRHR mRNA levels correlated with that of α and LHβ but was different from that of FSHβ. This suggests that α and LHβ mRNA levels are maximally stimulated when GnRHR levels are relatively high, whereas FSHβ mRNA levels are maximally stimulated at lower levels of GnRHR expression, and that the mechanism for differential regulation of the gonadotropins by varying pulse frequencies of GnRH may involve levels of GnRHR. Furthermore, these data suggest that the mechanisms whereby varying GnRH pulse frequencies stimulate α, LHβ, and GnRHR gene expression are similar, whereas the stimulation of FSHβ mRNA levels may be different.

Abstract 803: Diesel Exhaust Inhalation Enhances Thrombus Formation In Man

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Andrew J Lucking ◽  
Magnus Lundback ◽  
Nicholas L Mills ◽  
Dana Faratian ◽  
Fleming Cassee ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Acute Myocardial Infarction ◽  
Air Pollution ◽  
Platelet Activation ◽  
Diesel Exhaust ◽  
Intermittent Exercise ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Ex Vivo Model ◽  
Double Blind

Background: Transient exposure to traffic-derived air pollution may be a trigger for acute myocardial infarction although the mechanism is unclear. The aim of this study was to investigate the effect of diesel exhaust inhalation on thrombus formation in man using an ex vivo model of thrombosis. Methods and Results: In a double-blind randomized cross-over study, 20 healthy volunteers were exposed to diluted diesel exhaust (300 μg/m3) or filtered air during intermittent exercise for 1 or 2 hours. Thrombus formation, coagulation, platelet activation and inflammatory markers were measured at 2 and 6 hours after exposure. Thrombus formation was measured using the Badimon ex vivo perfusion chamber at low (212 /s) and high (1,690 /s) shear rates with porcine aortic tunica media as the thrombogenic substrate. Specimens were fixed, stained and thrombus area measured using computerized planimetry. Compared to filtered air, diesel exhaust increased thrombus formation in the low and high shear chambers by 24.2% (p<0.001) and 19.1% (p<0.001) respectively. This increased thrombogenicity was seen at two and six hours, and using two different types of diesel exposure. Although there were no effects on coagulation variables, diesel exhaust inhalation increased platelet-neutrophil (6.5% to 9.2%; P<0.05) and platelet-monocyte (21.0% to 25.0%; P<0.05) aggregates 2 hours following exposure. Conclusions: Inhalation of diesel exhaust increases ex vivo thrombus formation and causes platelet activation in man. These findings provide a potential mechanism that links exposure to traffic-derived air pollution with acute atherothrombotic events including acute myocardial infarction.

Measurement of progesterone in sheep using a commercial ELISA kit for human plasma

2021 ◽  
pp. 104063872110435
Author(s):  
Valeria Pasciu ◽  
Maria Nieddu ◽  
Elena Baralla ◽  
Cristian Porcu ◽  
Francesca Sotgiu ◽  
...  
Keyword(s):  
Enzyme Immunoassay ◽  
Human Plasma ◽  
Method Validation ◽  
Plasma Progesterone ◽  
Freeze Thaw ◽  
Elisa Kit ◽  
Stability Data ◽  
Human Use ◽  
Species Specific

Determination of serum or plasma progesterone (P4) concentrations is important to recognize pregnant and non-pregnant ewes, and also to predict the number of carried lambs. The 2 most common methodologies for the detection of plasma P4 are radioimmunoassay (RIA) and enzyme immunoassay (EIA). RIA is very expensive, and not all laboratories are equipped to perform this test; EIA is commercially available for human use, but only a few companies produce species-specific kits, which are expensive. We verified for ovine plasma a less expensive and easily available ELISA kit (DiaMetra) designed to quantify P4 in humans. Pools of ovine and human plasma were used to compare repeatability, accuracy, sensitivity, and stability of P4 measured by the DiaMetra kit. Repeatability data were within 15%, and accuracy values were ~90% for both plasma matrices. Stability data showed a loss of <20% for freeze–thaw and <30% for 30-d storage. All parameters were acceptable under international guidelines for method validation. The human ELISA kit was used successfully to quantify plasma P4 in 26 ewes during pregnancy until delivery. P4 concentrations were also correlated with the number of carried lambs.

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