Functional Assay Screens

2001 ◽  
Author(s):  
Maria Webb ◽  
Robert Horlick ◽  
Bassam Damaj ◽  
Kirk McMillan
Keyword(s):  
Functional Assay

The uncalibrated prothrombinase-induced clotting time test

2010 ◽  
Vol 30 (04) ◽  
pp. 212-216 ◽  
Author(s):  
R. Jovic ◽  
M. Hollenstein ◽  
P. Degiacomi ◽  
M. Gautschi ◽  
A. Ferrández ◽  
...  
Keyword(s):  
Factor Xa ◽  
Heparin Therapy ◽  
Thrombin Inhibitors ◽  
Coagulation Cascade ◽  
Diagnostic Tools ◽  
Direct Thrombin Inhibitors ◽  
Functional Assay ◽  
Clotting Time ◽  
Coagulation Time ◽  
Time Test

SummaryThe activated partial thromboplastin time test (aPTT) represents one of the most commonly used diagnostic tools in order to monitor patients undergoing heparin therapy. Expression of aPTT coagulation time in seconds represents common practice in order to evaluate the integrity of the coagulation cascade. The prolongation of the aPTT thus can indicate whether or not the heparin level is likely to be within therapeutic range. Unfortunately aPTT results are highly variable depending on patient properties, manufacturer, different reagents and instruments among others but most importantly aPTT’s dose response curve to heparin often lacks linearity. Furthermore, aPTT assays are insensitive to drugs such as, for example, low molecular weight heparin (LMWH) and direct factor Xa (FXa) inhibitors among others. On the other hand, the protrombinase-induced clotting time assay (PiCT®) has been show to be a reliable functional assay sensitive to all heparinoids as well as direct thrombin inhibitors (DTIs). So far, the commercially available PiCT assay (Pefakit®-PiCT®, DSM Nutritional Products Ltd. Branch Pentapharm, Basel, Switzerland) is designed to express results in terms of units with the help of specific calibrators, while aPTT results are most commonly expressed as coagulation time in seconds. In this report, we describe the results of a pilot study indicating that the Pefakit PiCT UC assay is superior to the aPTT for the efficient monitoring of patients undergoing UFH therapy; it is also suitable to determine and quantitate the effect of LMWH therapy. This indicates a distinct benefit when using this new approach over the use of aPPT for heparin monitoring.

Clinical evaluation of a new thyroglobulin immunoradiometric assay in the follow-up of differentiated thyroid carcinoma

2003 ◽  
Vol 42 (02) ◽  
pp. 71-77 ◽  
Author(s):  
I. Schreivogel ◽  
C. Angerstein ◽  
U. Siefker ◽  
K. Lehmann ◽  
G. Altenvoerde ◽  
...  
Keyword(s):  
Thyroid Carcinoma ◽  
Residual Disease ◽  
False Negative ◽  
Differentiated Thyroid Carcinoma ◽  
Threshold Concentration ◽  
High Rate ◽  
Functional Assay ◽  
Hypothyroid Patient ◽  
Assay Sensitivity ◽  
Significant Difference

SummaryAim: Formal and clinical comparison of a new 3rd-gene-ration-Tg-IRMA (3-G-IRMA; Dynotest®Tg-plus) with a conventional Tg-IRMA (3-G-IRMA; SELco®Tg-assay) for patients with differentiated thyroid carcinoma. In addition we evaluated, if thyroglobulin (Tg) levels above a specific threshold concentration indicate the need for further investigations for residual disease. Patients, methods: Tg concentration of 105 sera of 93 consecutive patients with a differentiated thyroid cancer was determined with both assays and compared at different cut-off values (Dynotest®Tg-plus: 0.2, 1, 2 ng/ml; SELco®Tg-assay: 0.5, 1, 2 ng/ml) with the clinical results in respect to the corresponding TSH concentration. Results: Tg concentration did not show any significant difference (SELco®Tg-assay 0.5 ng/ml, Dynotest® Tg-plus 0.2 ng/ml). The Tg-values of both assays correlated with 97%. However, correlation of recovery in both assays was small (40%). The sensitivities and specificities of both assays at different cut-offs and TSH values did not reveal significant differences. In patients with TSH concentration >30 µU/ml the functional assay sensitivity was superior to arbitrary cut-offs in the decision to start further evaluations. Conclusions: In our study neither formal nor clinical significant differences between two Tg-assays were found. In a hypothyroid patient (TSH >30 µU/ml, Tg concentration exceeding the functional assay sensitivity) further investigations for residual disease are warranted. Higher thresholds are of limited value, due to a inacceptable high rate of false negative results.

A New Functional Assay for Human Protein S Activity Using Activated Factor Vas Substrate

1989 ◽  
Vol 62 (04) ◽  
pp. 1144-1145 ◽  
Author(s):  
Martine Wolf ◽  
Catherine Boyer-Neumann ◽  
Jean-Luc Martinoli ◽  
Catherine Leroy-Matheron ◽  
Amiral Jean ◽  
...  
Keyword(s):  
Protein S ◽  
Human Protein ◽  
Functional Assay ◽  
Protein S Activity

A Novel Functional Assay of Protein C in Human Plasma and fts Comparison with Amidolytic and Anticoagulant Assays

1992 ◽  
Vol 67 (01) ◽  
pp. 046-049 ◽  
Author(s):  
H A Guglielmone ◽  
M A Vides
Keyword(s):  
Oral Anticoagulant ◽  
Protein C ◽  
Activated Protein C ◽  
Protein S ◽  
Normal Subjects ◽  
Test Sample ◽  
Functional Assay ◽  
Fast Method ◽  
Factor Va ◽  
Protein C Activity

SummaryA simple and fast method for the quantitative determination of protein C activity in plasma is here described. The first step consists in the conversion of protein C in the test sample into activated protein C by means of an activator isolated from Southern Copperhead venom. Subsequently, the degradation of factor Va, in presence of protein C-deficient plasma, is measured by the prolongation of the prothrombin time which is proportional to the amount of protein C in the sample. The dose-response curve showed a linear relationship from 6 to 150% protein C activity and the inter- and intra-assay reproducibility was 3.5% and 5.6% respectively. In normal subjects, a mean of protein C level of 98 ± 15% of normal pooled plasma was found. Comparison with the anticoagulant assay in samples of patients with oral anticoagulant, liver cirrhosis, disseminated intravascular coagulation and severe preeclampsia revealed an excellent correlation (r = 0.94, p <0.001). Also, a similar correlation (r = 0.93, p <0.001) existed between amidolytic assay and the method here proposed for all the samples studied without including the oral anticoagulant group. These results allowed us to infer that this method evaluates the ability of protein C to interact with protein S, phospholipids, calcium ions and factor Va.

Behaviour and Quantitation of Extrinsic (Tissue-Type) Plasminogen Activator in Human Blood

1983 ◽  
Vol 50 (02) ◽  
pp. 518-523 ◽  
Author(s):  
C Kluft ◽  
A F H Jie ◽  
R A Allen
Keyword(s):  
Plasminogen Activator ◽  
Venous Occlusion ◽  
Tissue Type ◽  
Functional Assay ◽  
Uterine Tissue ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Baseline Values

SummaryFunctional assay of extrinsic (tissue-type) plasminogen activator (EPA) in plasma on fibrin plates was evaluated. Using specific quenching antibodies, we demonstrated the method to be specific for EPA under all conditions tested. Contributions of urokinases and intrinsic activators were excluded. The quantity of EPA in blood samples, as compared with purified uterine tissue activator, shows 1 blood activator unit (BAU) to be comparable to 0.93 ng.The median values for EPA activity for healthy volunteers were: baseline, 1.9 BAU/ml (n = 123); diurnal, 5.5 BAU/ml (n = 12); DDAVP administration, 11.7 BAU/ml (n = 39); exhaustive exercise, 25 BAU/ml (n = 24); venous occlusion (15 min), 35 BAU/ml (n = 61). A large inter-individual variation in EPA activity was found, while individual baseline values tended to be constant for periods of weeks.In vitro in blood EPA activity shows a disappearance of 50% in about 90 min at 37° C; EPA activity in euglobulin fractions is stable for ≤2 hr at 37° C.A rapid decrease in EPA activity occurs in vivo, as noted after extracorporal circulation and exercise stimulation (t½ decay, 2-5 min).

scholarly journals Primitive Human Hematopoietic Cells Are Enriched in Cord Blood Compared With Adult Bone Marrow or Mobilized Peripheral Blood as Measured by the Quantitative In Vivo SCID-Repopulating Cell Assay

Blood ◽  
1997 ◽  
Vol 89 (11) ◽  
pp. 3919-3924 ◽  
Author(s):  
Jean C.Y. Wang ◽  
Monica Doedens ◽  
John E. Dick
Keyword(s):  
Bone Marrow ◽  
Cord Blood ◽  
Peripheral Blood ◽  
Hematopoietic Cells ◽  
Allogeneic Transplantation ◽  
Functional Assay ◽  
Hematopoietic Stem ◽  
Mobilized Peripheral Blood

Abstract We have previously reported the development of in vivo functional assays for primitive human hematopoietic cells based on their ability to repopulate the bone marrow (BM) of severe combined immunodeficient (SCID) and nonobese diabetic/SCID (NOD/SCID) mice following intravenous transplantation. Accumulated data from gene marking and cell purification experiments indicate that the engrafting cells (defined as SCID-repopulating cells or SRC) are biologically distinct from and more primitive than most cells that can be assayed in vitro. Here we demonstrate through limiting dilution analysis that the NOD/SCID xenotransplant model provides a quantitative assay for SRC. Using this assay, the frequency of SRC in cord blood (CB) was found to be 1 in 9.3 × 105 cells. This was significantly higher than the frequency of 1 SRC in 3.0 × 106 adult BM cells or 1 in 6.0 × 106 mobilized peripheral blood (PB) cells from normal donors. Mice transplanted with limiting numbers of SRC were engrafted with both lymphoid and multilineage myeloid human cells. This functional assay is currently the only available method for quantitative analysis of human hematopoietic cells with repopulating capacity. Both CB and mobilized PB are increasingly being used as alternative sources of hematopoietic stem cells in allogeneic transplantation. Thus, the findings reported here will have important clinical as well as biologic implications.

scholarly journals Blood functional assay for rapid clinical interpretation of germline TP53 variants

2020 ◽  
pp. jmedgenet-2020-107059 ◽  
Author(s):  
Sabine Raad ◽  
Marion Rolain ◽  
Sophie Coutant ◽  
Céline Derambure ◽  
Raphael Lanos ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Transcriptional Response ◽  
Functional Assay ◽  
Mrna Levels ◽  
Peripheral Blood Mononuclear ◽  
Patients With Cancer ◽  
Pathogenic Variants ◽  
Functional Variants ◽  
Multiplex Ligation Probe Amplification ◽  
P53 Mrna

BackgroundThe interpretation of germline TP53 variants is critical to ensure appropriate medical management of patients with cancer and follow-up of variant carriers. This interpretation remains complex and is becoming a growing challenge considering the exponential increase in TP53 tests. We developed a functional assay directly performed on patients’ blood.MethodsPeripheral blood mononuclear cells were cultured, activated, exposed to doxorubicin and the p53-mediated transcriptional response was quantified using reverse transcription–multiplex ligation probe amplification and RT-QMPSF assays, including 10 p53 targets selected from transcriptome analysis, and two amplicons to measure p53 mRNA levels. We applied this blood functional assay to 77 patients addressed for TP53 analysis.ResultsIn 51 wild-type TP53 individuals, the mean p53 functionality score was 12.7 (range 7.5–22.8). Among eight individuals harbouring likely pathogenic or pathogenic variants, the scores were reduced (mean 4.8, range 3.1–7.1), and p53 mRNA levels were reduced in patients harbouring truncating variants. We tested 14 rare unclassified variants (p.(Pro72His), p.(Gly105Asp), p.(Arg110His), p.(Phe134Leu), p.(Arg158Cys), p.(Pro191Arg), p.(Pro278Arg), p.(Arg283Cys), p.(Leu348Ser), p.(Asp352Tyr), p.(Gly108_Phe109delinsVal), p.(Asn131del), p.(Leu265del), c.-117G>T) and 12 yielded functionally abnormal scores. Remarkably, the assay revealed that the c.*1175A>C polymorphic variant within TP53 poly-adenylation site can impact p53 function with the same magnitude as a null variant, when present on both alleles, and may act as a modifying factor in pathogenic variant carriers.ConclusionThis blood p53 assay should therefore be a useful tool for the rapid clinical classification of germline TP53 variants and detection of non-coding functional variants.

scholarly journals A non-human primate in vitro functional assay for the early evaluation of TB vaccine candidates

npj Vaccines ◽  
2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Rachel Tanner ◽  
Andrew D. White ◽  
Charelle Boot ◽  
Claudia C. Sombroek ◽  
Matthew K. O’Shea ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
Mononuclear Cells ◽  
Functional Assay ◽  
Intermediate Precision ◽  
Peripheral Blood Mononuclear ◽  
Growth Inhibition Assay ◽  
Early Evaluation ◽  
Human Primate

AbstractWe present a non-human primate mycobacterial growth inhibition assay (MGIA) using in vitro blood or cell co-culture with the aim of refining and expediting early tuberculosis vaccine testing. We have taken steps to optimise the assay using cryopreserved peripheral blood mononuclear cells, transfer it to end-user institutes, and assess technical and biological validity. Increasing cell concentration or mycobacterial input and co-culturing in static 48-well plates compared with rotating tubes improved intra-assay repeatability and sensitivity. Standardisation and harmonisation efforts resulted in high consistency agreements, with repeatability and intermediate precision <10% coefficient of variation (CV) and inter-site reproducibility <20% CV; although some systematic differences were observed. As proof-of-concept, we demonstrated ability to detect a BCG vaccine-induced improvement in growth inhibition in macaque samples, and a correlation between MGIA outcome and measures of protection from in vivo disease development following challenge with either intradermal BCG or aerosol/endobronchial Mycobacterium tuberculosis (M.tb) at a group and individual animal level.

scholarly journals Prevalence and reclassification of BRCA1 and BRCA2 variants in a large, unselected Chinese Han breast cancer cohort

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Yun Liu ◽  
Honglian Wang ◽  
Xin Wang ◽  
Jiaqi Liu ◽  
Junjian Li ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Risk Assessment ◽  
Chinese Population ◽  
Functional Assay ◽  
Healthy Controls ◽  
Brca1 And Brca2 ◽  
Chinese Han ◽  
Pathogenic Variants ◽  
Breast Cancer Cohort ◽  
Invasive Carcinomas

AbstractAccurate interpretation of BRCA1/2 variants is critical for risk assessment and precise treatment of breast cancer (BC). Hence, the establishment of an ethnicity-based BRCA1/2 variant database of the Chinese population is of paramount importance. In this study, panel-based sequencing served to detect BRCA1/2 variants in a Chinese multicenter cohort of 21,216 BC patients and 6434 healthy controls. Overall, the percentage of subjects carrying pathogenic variants was 5.5% (1174/21,216) in BC patients and 1.1% (71/6434) in healthy controls. We identified 13 pathogenic variants as high-frequency variants that had a frequency of > 0.45‰ in BC patients (≥ 10 in 21,216 patients), none of which has been reported in Caucasians. Pathogenic BRCA1/2 variants correlated with younger onset age, higher frequencies of bilateral and triple-negative BC (TNBC), invasive carcinomas, high histological grades, and family history of BC and other cancers. Furthermore, the percentage of the subjects carrying VUS was 9.8% (2071/21,216) in BC patients and 6.9% (446/6434) in healthy controls. Based on our cohort study, we unambiguously reclassified 7 out of the 858 VUS resulting in lower VUS ratio in patients (from 9.8 to 7.9%) as well as in healthy control (from 6.9 to 5.3%). We also re-analyzed the 100 variants in 13 exons (2–5 and 15–23) of the BRCA1 genes using a functional assay (saturation genome editing; SGE). 55 of the 59 VUS had distinct status in the SGE study: 24 (43.6%) were pathogenic, and 31 (56.4%) were benign. Strong ethnicity-specific occurrences of pathogenic BRCA1/2 variants were identified in the Chinese population. Hence, the findings provide rationale and sequencing information for the implementation of BRCA1/2 variants tailored to the Chinese population into clinical risk assessment.

scholarly journals A Rose Extract Protects the Skin against Stress Mediators: A Potential Role of Olfactory Receptors

Molecules ◽  
2020 ◽  
Vol 25 (20) ◽  
pp. 4743
Author(s):  
Romain Duroux ◽  
Anne Mandeau ◽  
Gaelle Guiraudie-Capraz ◽  
Yannick Quesnel ◽  
Estelle Loing
Keyword(s):  
Physiological Role ◽  
Olfactory Receptors ◽  
Major Constituent ◽  
Epidermal Keratinocytes ◽  
Functional Assay ◽  
Skin Explants ◽  
Stress Mediators ◽  
Double Blinded ◽  
Split Face

Olfactory receptors (ORs) are expressed and active in various human tissues, including the skin. Although the sense of smell plays an important physiological role in the regulation of mood and stress, a link between olfactive compounds, ORs, and skin stress has yet to be established. This study aims to investigate the role of newly identified skin ORs and agonists in the modulation of skin stress. Screening for odorant molecules was done with cAMP functional assay to identify OR agonists. RT-qPCR and immunofluorescence microscopy were conducted to identify and quantify ORs in epidermal keratinocytes (NHEKs) and human skin explants, as well as to evaluate specific markers (G6PDH, loricrin, and γH2AX) of stress-induced skin alterations. A randomized double-blinded, split-face clinical study was performed on a panel of stressed women to measure the benefits of OR agonist treatment for skin. Three new ORs (OR10A6, OR2AG2, and OR11H4) were identified in skin. A specific Rose extract and its major constituent (phenylethyl alcohol) were found to activate these ORs. The extract composition was revealed by both GC/FID and GC/MS analyses simultaneously and showed the presence of 34 volatiles molecules. Moreover, epinephrine induces a skin stress response characterized by increased expression of G6PD, loricrin, and γH2AX biomarkers, and a decrease of OR expression. These effects were prevented in the presence of rose extract and its benefits were confirmed clinically by a decrease in the appearance of under-eye dark circles. Altogether, our findings suggest that ORs may represent a new, promising way to treat stress-associated skin disorders.

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