Journal articles: 'Human blood assay'

1

Fedorov, N. A., I. S. Yaneva, O. I. Skotnikova, and V. N. Pan'kov. "DNA assay in human blood plasma." Bulletin of Experimental Biology and Medicine 102, no. 3 (September 1986): 1190–92. http://dx.doi.org/10.1007/bf00842228.

Full text

APA, Harvard, Vancouver, ISO, and other styles

2

Kaur, Satbir, Sangeeta Sangeeta, Kiranjeet Kaur Galhna, and Nisha Gautam. "Assessment of Radiation Induced DNA Damage in Human Peripheral Blood Lymphocytes Using COMET Assay." International Journal of Life- Sciences Scientific Research 3, no. 4 (July 6, 2017): 1208–14. http://dx.doi.org/10.21276/ijlssr.2017.3.4.17.

Full text

APA, Harvard, Vancouver, ISO, and other styles

3

Rosen, Steffen, Stefan Tiefenbacher, Mary Robinson, Mei Huang, Jaydeep Srimani, Donnie Mackenzie, Terri Christianson, et al. "Activity of transgene-produced B-domain–deleted factor VIII in human plasma following AAV5 gene therapy." Blood 136, no. 22 (November 26, 2020): 2524–34. http://dx.doi.org/10.1182/blood.2020005683.

Full text

Abstract:

Abstract Adeno-associated virus (AAV)-based gene therapies can restore endogenous factor VIII (FVIII) expression in hemophilia A (HA). AAV vectors typically use a B-domain–deleted FVIII transgene, such as human FVIII-SQ in valoctocogene roxaparvovec (AAV5-FVIII-SQ). Surprisingly, the activity of transgene-produced FVIII-SQ was between 1.3 and 2.0 times higher in one-stage clot (OS) assays than in chromogenic-substrate (CS) assays, whereas recombinant FVIII-SQ products had lower OS than CS activity. Transgene-produced and recombinant FVIII-SQ showed comparable specific activity (international units per milligram) in the CS assay, demonstrating that the diverging activities arise in the OS assay. Higher OS activity for transgene-produced FVIII-SQ was observed across various assay kits and clinical laboratories, suggesting that intrinsic molecular features are potential root causes. Further experiments in 2 participants showed that transgene-produced FVIII-SQ accelerated early factor Xa and thrombin formation, which may explain the higher OS activity based on a kinetic bias between OS and CS assay readout times. Despite the faster onset of coagulation, global thrombin levels were unaffected. A correlation with joint bleeds suggested that both OS and CS assay remained clinically meaningful to distinguish hemophilic from nonhemophilic FVIII activity levels. During clinical development, the CS activity was chosen as a surrogate end point to conservatively assess hemostatic efficacy and enable comparison with recombinant FVIII-SQ products. Relevant trials are registered on clinicaltrials.gov as #NCT02576795 and #NCT03370913 and, respectively, on EudraCT (European Union Drug Regulating Authorities Clinical Trials Database; https://eudract.ema.europa.eu) as #2014-003880-38 and #2017-003215-19.

APA, Harvard, Vancouver, ISO, and other styles

4

Irie, Minoru, Yukitaka Miyachi, and Mari SEGAWA. "Measurement IGF-I in Human Blood by Immunoenzymometric Assay." Folia Endocrinologica Japonica 68, no. 7 (1992): 688–700. http://dx.doi.org/10.1507/endocrine1927.68.7_688.

Full text

APA, Harvard, Vancouver, ISO, and other styles

5

Fisher, T. C., J. J. F. Belch, J. C. Barbenel, and A. C. Fisher. "Human whole-blood granulocyte aggregation in vitro." Clinical Science 76, no. 2 (February 1, 1989): 183–87. http://dx.doi.org/10.1042/cs0760183.

Full text

Abstract:

1. Aggregation assays are a commonly used technique for the study of granulocyte activation. These studies are usually performed using a pure cell suspension in buffer. This necessitates a separation procedure which is time-consuming and may modify the function of the cells. Interaction between different cell types is precluded. 2. To avoid these disadvantages a method was developed which quantifies granulocyte aggregation in whole blood. Samples drawn from an incubated vessel before and after the addition of a chemotactic stimulus were fixed with formaldehyde to prevent disaggregation. Erythrocytes were then removed by chemical lysis and using an electronic cell-sizing device the number of single cells and aggregates could then be easily measured. 3. Results from a group of volunteers showed a rapid and reversible response to a chemotactic tripeptide, with a fall in single granulocyte count and the appearance of doublets and triplets. Lymphocytes were unaffected. Intra-assay reproducibility was better than ± 5%. 4. Using this assay, a significant elevation in aggregability was observed in blood from patients after acute myocardial infarction. 5. This novel technique, by avoiding the separation step, is faster, simpler and more physiological than previous methods, and as such is useful for both assays of drug action in vitro and the study of cell activation in disease states.

APA, Harvard, Vancouver, ISO, and other styles

6

Russell, Bruce, Rossarin Suwanarusk, Céline Borlon, Fabio T. M. Costa, Cindy S. Chu, Marcus J. Rijken, Kanlaya Sriprawat, et al. "A reliable ex vivo invasion assay of human reticulocytes by Plasmodium vivax." Blood 118, no. 13 (September 29, 2011): e74-e81. http://dx.doi.org/10.1182/blood-2011-04-348748.

Full text

Abstract:

AbstractCurrently, there are no reliable RBC invasion assays to guide the discovery of vaccines against Plasmodium vivax, the most prevalent malaria parasite in Asia and South America. Here we describe a protocol for an ex vivo P vivax invasion assay that can be easily deployed in laboratories located in endemic countries. The assay is based on mixing enriched cord blood reticulocytes with matured, trypsin-treated P vivax schizonts concentrated from clinical isolates. The reliability of this assay was demonstrated using a large panel of P vivax isolates freshly collected from patients in Thailand.

APA, Harvard, Vancouver, ISO, and other styles

7

Shinohara, Rikio, Yoshiji Ohta, Masamitsu Yamauchi, and Isao Ishiguro. "Improved fluorometric enzymatic sorbitol assay in human blood." Clinica Chimica Acta 273, no. 2 (May 1998): 171–84. http://dx.doi.org/10.1016/s0009-8981(98)00036-9.

Full text

APA, Harvard, Vancouver, ISO, and other styles

8

Wang, Xiaoying, Hao Li, Xiaoxi Li, Yangyang Chen, Yongmei Yin, and Genxi Li. "Electrochemical assay of melanoma biomarker in human blood." Electrochemistry Communications 39 (February 2014): 12–14. http://dx.doi.org/10.1016/j.elecom.2013.12.003.

Full text

APA, Harvard, Vancouver, ISO, and other styles

9

Modestino, Augusta, Matthew Tyndall, Johnson Yu, Roy B. Lefkowitz, Geert W. Schmid-Schönbein, and Michael J. Heller. "Thrombin generation assay in untreated whole human blood." ELECTROPHORESIS 37, no. 15-16 (August 2016): 2248–56. http://dx.doi.org/10.1002/elps.201600061.

Full text

APA, Harvard, Vancouver, ISO, and other styles

10

Kashyap, V. K. "A Simple Immunosorbent Assay for Detection of Human Blood." Journal of Immunoassay 10, no. 4 (December 1989): 315–24. http://dx.doi.org/10.1080/01971528908053244.

Full text

APA, Harvard, Vancouver, ISO, and other styles

11

Tamura, Hiroshi, Yayoi Arimoto, Shigenori Tanaka, Minoru Yoshida, Taminori Obayashi, and Tadashi Kawai. "Automated kinetic assay for endotoxin and in human blood." Clinica Chimica Acta 226, no. 1 (April 1994): 109–12. http://dx.doi.org/10.1016/0009-8981(94)90110-4.

Full text

APA, Harvard, Vancouver, ISO, and other styles

12

Ruan, Qiaoqiao, Patrick J. Macdonald, Kerry M. Swift, and Sergey Y. Tetin. "Direct single-molecule imaging for diagnostic and blood screening assays." Proceedings of the National Academy of Sciences 118, no. 14 (March 31, 2021): e2025033118. http://dx.doi.org/10.1073/pnas.2025033118.

Full text

Abstract:

Every year, over 100 million units of donated blood undergo mandatory screening for HIV, hepatitis B, hepatitis C, and syphilis worldwide. Often, donated blood is also screened for human T cell leukemia–lymphoma virus, Chagas, dengue, Babesia, cytomegalovirus, malaria, and other infections. Several billion diagnostic tests are performed annually around the world to measure more than 400 biomarkers for cardiac, cancer, infectious, and other diseases. Considering such volumes, every improvement in assay performance and/or throughput has a major impact. Here, we show that medically relevant assay sensitivities and specificities can be fundamentally improved by direct single-molecule imaging using regular epifluorescence microscopes. In current microparticle-based assays, an ensemble of bound signal-generating molecules is measured as a whole. By contrast, we acquire intensity profiles to identify and then count individual fluorescent complexes bound to targets on antibody-coated microparticles. This increases the signal-to-noise ratio and provides better discrimination over nonspecific effects. It brings the detection sensitivity down to the attomolar (10−18 M) for model assay systems and to the low femtomolar (10−16 M) for measuring analyte in human plasma. Transitioning from counting single-molecule peaks to averaging pixel intensities at higher analyte concentrations enables a continuous linear response from 10−18 to 10−5 M. Additionally, our assays are insensitive to microparticle number and volume variations during the binding reaction, eliminating the main source of uncertainties in standard assays. Altogether, these features allow for increased assay sensitivity, wide linear detection ranges, shorter incubation times, simpler assay protocols, and minimal reagent consumption.

APA, Harvard, Vancouver, ISO, and other styles

13

Mavromatis, Kreton, Diane J. Sutcliffe, Giji Joseph, R. Wayne Alexander, Edmund K. Waller, Arshed A. Quyyumi, and W. Robert Taylor. "Proangiogenic Cell Colonies Grown In Vitro from Human Peripheral Blood Mononuclear Cells." Journal of Biomolecular Screening 17, no. 9 (August 17, 2012): 1128–35. http://dx.doi.org/10.1177/1087057112457043.

Full text

Abstract:

Although multiple culture assays have been designed to identify endothelial progenitor cells (EPCs), the phenotype of cells grown in culture often remains undefined. We sought to define and characterize the proangiogenic cell population within human peripheral blood mononuclear cells. Mononuclear cells were isolated from peripheral blood and grown under angiogenic conditions for 7 days. Formed colonies (CFU-As) were identified and analyzed for proliferation, mRNA and surface antigen expression, tube-forming ability, and chromosomal content. Colonies were composed of a heterogeneous group of cells expressing the leukocyte antigens CD45, CD14, and CD3, as well as the endothelial proteins vascular endothelial (VE) cadherin, von Willebrand’s factor (vWF), CD31, and endothelial nitric oxide synthase (eNOS). Colony cells expressed increased levels of proangiogenic growth factors, and they formed tubes in Matrigel. In comparison with colonies from the CFU-Hill assay, our assay resulted in a greater number of colonies (19 ± 9 vs. 13 ± 7; p < 0.0001) with a substantial number of cells expressing an endothelial phenotype (20.2% ± 7.4% vs. 2.2% ± 1.2% expressing eNOS, p = 0.0006). Chromosomal analysis indicated the colony cells were bone marrow derived. We, therefore, describe a colony-forming unit assay that measures bone marrow–derived circulating mononuclear cells with the capacity to proliferate and mature into proangiogenic leukocytic and endothelial-like cells. This assay, therefore, reflects circulating, bone marrow–derived proangiogenic activity.

APA, Harvard, Vancouver, ISO, and other styles

14

Boivin, Guy, Julie Handfield, Emil Toma, Gilles Murray, Richard Lalonde, Vincent J. Tevere, Rita Sun, and Michel G. Bergeron. "Evaluation of the AMPLICOR Cytomegalovirus Test with Specimens from Human Immunodeficiency Virus-Infected Subjects." Journal of Clinical Microbiology 36, no. 9 (1998): 2509–13. http://dx.doi.org/10.1128/jcm.36.9.2509-2513.1998.

Full text

Abstract:

The AMPLICOR cytomegalovirus (CMV) test, a new qualitative assay for the detection of CMV DNA in plasma, was compared to conventional methods and quantitative PCR (Q-PCR) assays by using leukocytes and plasma from 179 blood samples from subjects with AIDS. For the diagnosis of CMV disease, cell-based assays such as a Q-PCR with polymorphonuclear leukocytes (Q-PCR-PMNL) and a pp65 antigenemia assay had the highest sensitivities but suffered from a lack of specificity. The best agreement between the results of the Q-PCR-PMNL assay and those of the AMPLICOR test was found when a threshold diagnostic value of 690 copies per 105 cells was selected for the Q-PCR-PMNL assay. In that context, the AMPLICOR CMV test had a sensitivity of 96.4% and a specificity of 95.3% when results were compared to results of the cell-based PCR assay. This threshold was close to the one described as associated with the best sensitivity and specificity for the diagnosis of CMV disease in a recently published study (4). Blood samples that tested positive by the Q-PCR-PMNL assay but negative by the AMPLICOR CMV test were associated with viral loads (mean, 785 copies, median, 96 copies per 105leukocytes) lower than the viral loads of blood samples that tested positive by both assays (mean, 21,452 copies; median, 9,784 copies per 105 leukocytes) (P = 0.003). The AMPLICOR CMV test gave positive results at least 48 days before the development of symptomatic CMV disease in a longitudinal analysis of a limited subset of patients (n = 6) from whom sequential specimens were available for testing. In conclusion, the AMPLICOR CMV test is a very convenient assay combining rapidity, simplicity, and the possibility of batch testing. A positive result by this test seems particularly important since this implies, in most instances, the presence or the imminence of CMV disease, although a negative test result does not rule out disease.

APA, Harvard, Vancouver, ISO, and other styles

15

Tamm, Natalia N., Karina R. Seferian, Alexander G. Semenov, Kadriya S. Mukharyamova, Ekaterina V. Koshkina, Mihail I. Krasnoselsky, Alexander B. Postnikov, et al. "Novel Immunoassay for Quantification of Brain Natriuretic Peptide and Its Precursor in Human Blood." Clinical Chemistry 54, no. 9 (September 1, 2008): 1511–18. http://dx.doi.org/10.1373/clinchem.2007.100545.

Full text

Abstract:

Abstract Background: Brain natriuretic peptide (BNP) is an unstable molecule that can rapidly lose immunologic activity in blood. Conventional sandwich BNP immunoassays use 2 antibodies specific to 2 different epitopes. Larger distances between epitopes are associated with a greater probability of proteolysis sites being located between the antibody-binding sites, and thus such assays have an increased susceptibility to underdetect BNP because of the increased likelihood of proteolytic degradation. The purpose of our study was to develop a sandwich immunoassay for the precise quantification of BNP and BNP precursor (proBNP) in human blood that is not susceptible to proteolysis. Methods: Mice were immunized with an immune complex consisting of monoclonal antibody (MAb) 24C5 (specific for BNP peptide 11–22) and the entire BNP molecule. The MAb used in our assay (Ab-BNP2) recognizes the immune complex but neither free BNP nor MAb 24C5. Results: We used MAbs 24C5 and Ab-BNP2 to develop a new type of sandwich BNP assay (the “single-epitope sandwich assay”), which requires only a short BNP fragment (fragment 11–22) for immunodetection. This assay recognizes both BNP and proBNP with the same efficiency and sensitivity and demonstrates both considerably less susceptibility to antigen degradation and greater stability of the measured antigen than conventional sandwich BNP immunoassays. Conclusions: We have developed this sensitive single-epitope sandwich assay for detecting BNP, proBNP, and their fragments in human blood. This assay appears promising for use in clinical studies to assist in triage, management, and outcomes assessment in heart failure patients.

APA, Harvard, Vancouver, ISO, and other styles

16

von Lode, Piia, Jarmo Rainaho, and Kim Pettersson. "Quantitative, Wide-Range, 5-Minute Point-of-Care Immunoassay for Total Human Chorionic Gonadotropin in Whole Blood." Clinical Chemistry 50, no. 6 (June 1, 2004): 1026–35. http://dx.doi.org/10.1373/clinchem.2004.031922.

Full text

Abstract:

Abstract Background: Human chorionic gonadotropin (hCG) is among the most common analytes available for point-of-care immunotesting, with most assays currently based on simple manual assay devices. However, as the importance of good analytical performance of rapid assays is increasingly emphasized, more sophisticated immunoassay techniques are needed to meet the future challenges of rapid yet quantitative POC testing. Methods: We developed a simple, dry-reagent, all-in-one immunoassay for the quantitative measurement of hCG in whole blood, plasma, or serum. The noncompetitive assay equally measures intact, nicked, and hyperglycosylated hCG as well as nonnicked and nicked hCG β-subunit with a rapid and simple procedure consisting of a 5-min, one-step incubation and, subsequent to washing, the measurement of time-resolved fluorescence directly from a wet well surface. Results: The assay had a detection limit (background + 3 SD) of 0.4 IU/L hCG. The within-run CV was <15% down to 2 IU/L, and the assay was linear to 6000 IU/L. The within- and between-run CVs in heparinized whole blood and plasma were ≤10% throughout the measured range (4.0–4400 IU/L). The mean (95% confidence interval) difference between whole blood and plasma was −42 (−24 to −61)% without hematocrit correction and 6.5 (−14 to 27)% with hematocrit correction (n = 106). Regression analysis with the Diagnostic Products IMMULITE® 2000 hCG method yielded the following: slope (SD), 1.02 (0.01); y-intercept (SD), −6 (10) IU/L; Sy|x = 99 IU/L (n = 124; range, 1.6–4746 IU/L; r = 0.995). Conclusions: Combined with the fully automated instrumentation, the 5-min, dry-reagent assay allows quantitative and reproducible determination of hCG in whole blood while sustaining the speed and simplicity of conventional rapid assays.

APA, Harvard, Vancouver, ISO, and other styles

17

Cheon, Seon Hee, Ho Yeon Song, Eun Hee Lee, Hee Jung Oh, In Sook Kang, Ji Yoon Cho, and Young Sun Hong. "Measuring Intracellular Mycobacterial Killing Using a Human Whole Blood Assay." Tuberculosis and Respiratory Diseases 53, no. 5 (2002): 497. http://dx.doi.org/10.4046/trd.2002.53.5.497.

Full text

APA, Harvard, Vancouver, ISO, and other styles

18

Queipo-Ortuño, M. I., P. Morata, P. Ocón, P. Manchado, and J. D. Colmenero. "Rapid diagnosis of human brucellosis by peripheral-blood PCR assay." Journal of clinical microbiology 35, no. 11 (1997): 2927–30. http://dx.doi.org/10.1128/jcm.35.11.2927-2930.1997.

Full text

APA, Harvard, Vancouver, ISO, and other styles

19

Yoshikawa, Yoshihiro, Kenji Ikebuchi, Jun-ichi Ohkawara, Fumiya Hirayama, Miki Yamaguchi, Norihiro Sato, Kazuhiro J. Mori, Masaharu Kasai, and Sadayoshi Sekiguchi. "A clonal culture assay for human cord blood lymphohematopoietic progenitors." Human Immunology 60, no. 1 (January 1999): 75–82. http://dx.doi.org/10.1016/s0198-8859(98)00094-9.

Full text

APA, Harvard, Vancouver, ISO, and other styles

20

Liu, Limin, Stefanie Hood, Yuping Wang, Robert Bezverkov, Chao Dou, Abhijit Datta, and Chong Yuan. "Direct enzymatic assay for %HbA1c in human whole blood samples." Clinical Biochemistry 41, no. 7-8 (May 2008): 576–83. http://dx.doi.org/10.1016/j.clinbiochem.2008.01.013.

Full text

APA, Harvard, Vancouver, ISO, and other styles

21

Corretge, E., and J. M. Nigretto. "Electroanalytical assay of human prekallikrein activation from whole blood dilutions." Electrochimica Acta 32, no. 4 (April 1987): 583–87. http://dx.doi.org/10.1016/0013-4686(87)87045-7.

Full text

APA, Harvard, Vancouver, ISO, and other styles

22

Daneshian, Mardas, Sonja von Aulock, and Thomas Hartung. "Assessment of pyrogenic contaminations with validated human whole-blood assay." Nature Protocols 4, no. 12 (November 5, 2009): 1709–21. http://dx.doi.org/10.1038/nprot.2009.159.

Full text

APA, Harvard, Vancouver, ISO, and other styles

23

Seano, Giorgio, Giulia Chiaverina, Paolo Armando Gagliardi, Laura di Blasio, Roberto Sessa, Federico Bussolino, and Luca Primo. "Modeling human tumor angiogenesis in a three-dimensional culture system." Blood 121, no. 21 (May 23, 2013): e129-e137. http://dx.doi.org/10.1182/blood-2012-08-452292.

Full text

Abstract:

Key Points Human arterial ring assay is an innovative system for the three-dimensional study of tumor angiogenesis. This assay can be exploited for antiangiogenic drug screening and gene function analysis on human vessels.

APA, Harvard, Vancouver, ISO, and other styles

24

Aras, Omer, Arun Shet, Ronald R. Bach, Jessica L. Hysjulien, Arne Slungaard, Robert P. Hebbel, Gines Escolar, Bernd Jilma, and Nigel S. Key. "Induction of microparticle- and cell-associated intravascular tissue factor in human endotoxemia." Blood 103, no. 12 (June 15, 2004): 4545–53. http://dx.doi.org/10.1182/blood-2003-03-0713.

Full text

Abstract:

Abstract The precise role of intravascular tissue factor (TF) remains poorly defined, due to the limited availability of assays capable of measuring circulating TF procoagulant activity (PCA). As a model of inflammation-associated intravascular thrombin generation, we studied 18 volunteers receiving an infusion of endotoxin. A novel assay that measures microparticle (MP)-associated TF PCA from a number of cellular sources (but not platelets) demonstrated an 8-fold increase in activity at 3 to 4 hours after endotoxin administration (P < .001), with a return to baseline by 8 hours. TF antigen-positive MPs isolated from plasma were visualized by electron microscopy. Interindividual MP-associated TF response to lipopolysaccharide (LPS) was highly variable. In contrast, a previously described assay that measurestotal (cell and MP-borne) whole-blood TF PCA demonstrated a more modest increase, with a peak in activity (1.3-fold over baseline; P < .000 01) at 3 to 4 hours, and persistence for more than 24 hours. This surprisingly modest increase in whole-blood TF activity is likely explained by a profound although transient LPS-induced monocytopenia. MP-associated TF PCA was highly correlated with whole-blood TF PCA and total number of circulating MPs, and whole-blood TF PCA was highly correlated with TF mRNA levels. (Blood. 2004;103:4545-4553)

APA, Harvard, Vancouver, ISO, and other styles

25

Egeler, Oliver, Albertus W. Wognum, Caren Grande, Ning Yuan, Steven Woodside, and Terry Thomas. "Automation of the Hematopoietic CFC Assay for Human Cord Blood, Bone Marrow and Mobilized Peripheral Blood Samples." Blood 118, no. 21 (November 18, 2011): 3001. http://dx.doi.org/10.1182/blood.v118.21.3001.3001.

Full text

Abstract:

Abstract Abstract 3001 The hematopoietic colony-forming-cell (CFC) assay is a valuable tool to assess the potency of cell products (umbilical cord blood, apheresis products, bone marrow) for hematopoietic stem cell transplantation and for toxicity screening in therapeutic drug development. However, the manual colony enumeration that has been required is subjective and time consuming even for experienced users. This subjectivity limits the accuracy of the assay and contributes to a high degree of inter-laboratory variability. To reduce this variability, STEMCELL Technologies has developed an imaging and analysis system (STEMvision™) for automation of CFC assay colony enumeration. We have previously shown results validating the instrument for use in cord blood (CB) cell assays (Wognum et al. 37th Annual Meeting of the European Group for Blood and Marrow Transplantation, Hamburg, Germany 2011). Here we additionally present recent results comparing automated and manual colony counting for human mobilized peripheral blood (mPB), and bone marrow (BM). Samples of CB, mPB, and BM mononuclear cells were inoculated into semi-solid culture media (Methocult™ H4034, H4434, and H4435) and plated into special meniscus-free culture plates (SmartDish™). After 14 days in culture, automated colony counts were obtained from each sample using algorithms specifically optimized for each type of product (CB, mPB, and BM). The same cultures were then manually enumerated by 2–5 operators using the standard microscope method (microscope counts) and by enumerating colonies on the STEMvision™ images (image counts). For the each of the cell products (hCB, mPB, BM), the automated total colony counts were highly correlated to the average manual total colony counts. The table below compares the total manual image counts to the automated counts. Linear regression of the data shows that in addition to being highly correlated (r2 >0.90), the two counting methods give nearly identical results on average (the line of identity has a slope of 1). The efficacy of automated classification of colonies as erythroid or myeloid+mixed was evaluated by comparing the proportion of myeloid counts (myeloid / total) in each sample for image and automated counts. The % agreement was determined as as the differential between the myeloid proportions of the manual image and automated counts. The table below shows that on average, the agreement was greater than 90%. Variability of colony counting was also significantly reduced with the STEMvision™ instrument. We found that for multiple independent measurements of a given sample, the coefficient of variance (CV) of the normalized counts was 11% for the microscope counts (2–5 different operators), 7.9% for the image counts (2–5 different operators) and 4.4% for the automated counts (2–5 different instruments). The low CV for the automated counts was not operator dependent: for 6 samples analyzed by 6 operators using a single instrument, the CV for normalized total colony counts was 4.3%. Automated colony counting with the STEMvision™ instrument has thus been shown to be highly correlated to manual scoring methods for the most common hematopoietic stem cell transplantation products(CB, mPB, and BM). In addition, automation of the assay analysis significantly reduced the variability across multiple operators relative to manual counting methods. As a result, STEMvision™ has the ability to improve standardization of the CFC assay analysis through reduced intra- and inter-lab variability. We have reported previously on the internal and independent multi-center validation of this automated system for CB products. Rigorous validation of for BM and mPB cell products is currently in progress. In addition to providing standardization, this instrument reduces the time required for the assay readout and provides a means of permanent archiving of colony assay images. In the future, the image analysis output will provide quantitative information related to colony morphology that is not easily obtained by manual analysis (eg. colony size, density, symmetry). Such information would enable automated assessment of hematopoietic toxicity. Disclosures: Egeler: Stemcell Technologies: Employment. Wognum:Stemcell Technologies: Employment. Grande:Stemcell Technologies: Employment. Yuan:Stemcell Technologies: Employment. Woodside:Stemcell Technologies: Employment. Thomas:Stemcell Technologies: Employment, Patents & Royalties.

APA, Harvard, Vancouver, ISO, and other styles

26

Sioen, Simon, Karlien Cloet, Anne Vral, and Ans Baeyens. "The Cytokinesis-Block Micronucleus Assay on Human Isolated Fresh and Cryopreserved Peripheral Blood Mononuclear Cells." Journal of Personalized Medicine 10, no. 3 (September 14, 2020): 125. http://dx.doi.org/10.3390/jpm10030125.

Full text

Abstract:

The cytokinesis-block micronucleus (CBMN) assay is a standardized method used for genotoxicity studies. Conventional whole blood cultures (WBC) are often used for this assay, although the assay can also be performed on isolated peripheral blood mononuclear cell (PBMC) cultures. However, the standardization of a protocol for the PBMC CBMN assay has not been investigated extensively. The aim of this study was to optimize a reliable CBMN assay protocol for fresh and cryopreserved peripheral blood mononuclear cells (PBMCS), and to compare micronuclei (MNi) results between WBC and PBMC cultures. The G0 CBMN assay was performed on whole blood, freshly isolated, and cryopreserved PBMCS from healthy human blood samples and five radiosensitive patient samples. Cells were exposed to 220 kV X-ray in vitro doses ranging from 0.5 to 2 Gy. The optimized PBMC CBMN assay showed adequate repeatability and small inter-individual variability. MNi values were significantly higher for WBC than for fresh PBMCS. Additionally, cryopreservation of PBMCS resulted in a significant increase of MNi values, while different cryopreservation times had no significant impact. In conclusion, our standardized CBMN assay on fresh and cryopreserved PBMCS can be used for genotoxicity studies, biological dosimetry, and radiosensitivity assessment.

APA, Harvard, Vancouver, ISO, and other styles

27

Robinson, Mary, Lindsey A. George, Benjamin J. Samelson-Jones, Valder R. Arruda, Katherine A. High, Marcus E. Carr, and Stefan Tiefenbacher. "Activity of a FIX-Padua Transgene Product in Commonly Used FIX:C One-Stage and Chromogenic Assay Systems Following PF-06838435 (SPK-9001) Gene Delivery." Blood 132, Supplement 1 (November 29, 2018): 2198. http://dx.doi.org/10.1182/blood-2018-99-119616.

Full text

Abstract:

Abstract Background PF-06838435 (SPK-9001), a gene therapy candidate containing a high specific-activity factor IX variant (R338L, FIX-Padua), is currently in phase 3 of clinical development for the treatment of Hemophilia B. Initial data with this vector is promising with significant reductions in bleeding episodes and FIX consumption (George LA et al, NEJM 2017; 377:2215-2217). To date, little is known about the activity of the expressed transgene product as measured in FIX:C one-stage and chromogenic assay systems commonly used to monitor FIX replacement therapy in patients with Hemophilia B. Aim The goal of the study was to assess the activity of the PF-06838435 expressed transgene product in plasma samples collected from participants in the Phase 1/2 trial using four commonly used FIX:C aPTT reagents. For comparison, the activity of the expressed FIX-Padua gene product was also assessed in the ROX FACTOR IX chromogenic assay. In addition, the activity of the Padua variant in congenital FIX deficient plasma spiked with increasing concentrations of purified recombinant human FIX Padua protein (rHFIXp - Samelson-Jones Lab, CHOP/UPenn), as well as recombinant human FIX (rHFIX, BeneFIX®, Pfizer Inc.), was assessed in the same FIX:C assay procedures. Methods FIX:C, in four samples collected from two different patients who received FIX gene therapy, was tested in four in vitro diagnostic (IVD) approved FIX:C one-stage assay systems, STA®-PTT Automate and STA®-C.K. Prest® on the STA-R Evolution® (Diagnostica Stago Inc.), Dade Actin® FSL on the BCS®XP (Siemens Healthcare), and HemosIL® SynthASil® on the ACL TOP® (Instrumentation Laboratory). In addition, samples were also tested in the ROX FACTOR IX (Rossix AB) chromogenic assay on the BCS®XP (Siemens Healthcare). The aPTT reagents selected for this study correspond to 90% of the FIX:C assay reagents currently used in CAP accredited laboratories in the US(2014 CAP Proficiency Survey) and represent the three main types of activator commonly used in the FIX one-stage clot assay: silica (STA®-PTT Automate, HemosIL® SynthASil®), ellagic acid (Dade Actin® FSL) and kaolin (STA®-C.K. Prest®). For comparison, FIX:C in each of the five FIX:C assay systems was also determined in samples spiked with purified rHFIXp (provided by Dr. Samelson-Jones) or rHFIX (provided by Spark Therapeutics Inc.) in 20X buffer solutions. On the day of testing rHFIXp, and rHFIX 20X buffer solutions were diluted 1:20 in congenital FIX deficient plasma to achieve approximate final FIX:C concentrations of 40, 30 and 20%, extrapolated from an estimated 8-fold specific activity to antigen ratio. Results A consistent pattern in the measured FIX:C for the PF-06838435 transgene product was observed in the five FIX:C assay systems (Fig. 1). For the aPTT based FIX:C assays, Actin FSL, gave the lowest FIX:C values, whereas PTT Automate measured the highest FIX:C levels. In all cases, the chromogenic FIX:C assay gave the lowest activity values for the transgene product. A similar FIX:C assay dependent pattern was observed for the purified rHFIXp spiked at 40, 30 and 20% FIX:C (Fig. 2). In all FIX:C assays tested, rHFIXp was under-recovered to a varying degree from target, with recoveries for the 20% FIX:C samples ranging between -26.5% (STA®-PTT Automate) and -73.5% (Dade Actin® FSL) in the aPTT based FIX:C assays and -85% in the ROX FIX chromogenic assay. In contrast, rHFIX (BeneFIX®) was recovered within ±25% of expected values in all aPTT based FIX:C assays and, consistent with previously reported data, modestly under-recovered in the ROX FIX chromogenic assay (Fig. 3). Conclusion This study found differences in the FIX:C results obtained for a Padua FIX variant transgene product and recombinant human FIX-Padua when tested in commonly used IVD approved FIX:C assays in North America. These results suggest that FIX:C assay selection is important for measuring FIX-Padua activity, which will be particularly relevant in hemophilia B gene therapy following FIX-Padua gene transfer. Disclosures George: University of Pennsylvania: Equity Ownership; Pfizer: Consultancy. High:Spark Therapeutics: Employment, Equity Ownership, Patents & Royalties. Carr:Sparks Therapeutics Inc.: Consultancy. Tiefenbacher:Laboratory Corporation of America: Employment, Equity Ownership; Siemens Healthcare: Consultancy.

APA, Harvard, Vancouver, ISO, and other styles

28

Rapier, J. M., Y. Villamarzo, G. Schochetman, C. Y. Ou, C. L. Brakel, J. Donegan, W. Maltzman, S. Lee, D. Kirtikar, and D. Gatica. "Nonradioactive, colorimetric microplate hybridization assay for detecting amplified human immunodeficiency virus DNA." Clinical Chemistry 39, no. 2 (February 1, 1993): 244–47. http://dx.doi.org/10.1093/clinchem/39.2.244.

Full text

Abstract:

Abstract A nonradioactive, colorimetric microplate hybridization procedure was used to assay human immunodeficiency virus (HIV) DNA, amplified by the polymerase chain reaction (PCR). Under the PCR conditions used, four proviral copies per 150,000 cells were detected by amplifying a series of DNA mixtures that contained various copy numbers of HIV. Assays of PCR-amplified DNA from peripheral blood mononuclear cells of seronegative individuals yielded negative results (104 of 104), whereas samples from seropositive individuals yielded > 99% positive results (141 of 142). Similar results were obtained in a chemiluminescent assay with an acridinium ester-labeled probe and in a solution hybridization assay in which a 32P-labeled probe was used.

APA, Harvard, Vancouver, ISO, and other styles

29

Akdeniz, Ali, Mehmet Gokhan Caglayan, Irina Polivina, and Pavel Anzenbacher. "Detection and quantification of ATP in human blood serum." Organic & Biomolecular Chemistry 14, no. 31 (2016): 7459–62. http://dx.doi.org/10.1039/c6ob01378c.

Full text

APA, Harvard, Vancouver, ISO, and other styles

30

Hoch, Denise, Waltraud Brandl, Jasmin Strutz, Harald C. Köfeler, Mireille N. M. van Poppel, Lars Bode, Ursula Hiden, Gernot Desoye, and Evelyn Jantscher-Krenn. "Human Milk Oligosaccharides in Cord Blood Are Altered in Gestational Diabetes and Stimulate Feto-Placental Angiogenesis In Vitro." Nutrients 13, no. 12 (November 26, 2021): 4257. http://dx.doi.org/10.3390/nu13124257.

Full text

Abstract:

(1) Background: Human milk oligosaccharides (HMOs) are present in maternal serum during pregnancy and their composition is altered in gestational diabetes (GDM). HMOs are also in fetal cord blood and in contact with the feto-placental endothelium, potentially affecting its functions, such as angiogenesis. We hypothesized that cord blood HMOs are changed in GDM and contribute to increased feto-placental angiogenesis, hallmark of GDM. (2) Methods: Using HPLC, we quantified HMOs in cord blood of women with normal glucose tolerance (NGT, n = 25) or GDM (n = 26). We investigated in vitro angiogenesis using primary feto-placental endothelial cells (fpECs) from term placentas after healthy pregnancy (n = 10), in presence or absence of HMOs (100 µg/mL) isolated from human milk, 3′-sialyllactose (3′SL, 30 µg/mL) and lactose (glycan control) and determined network formation (Matrigel assay), proliferation (MTT assays), actin organization (F-actin staining), tube formation (fibrin tube formation assay) and sprouting (spheroid sprouting assay). (3) Results: 3′SL was higher in GDM cord blood. HMOs increased network formation, HMOs and 3’SL increased proliferation and F-actin staining. In fibrin assays, HMOs and 3’SL increased total tube length by 24% and 25% (p < 0.05), in spheroid assays, by 32% (p < 0.05) and 21% (p = 0.056), respectively. Lactose had no effect. (4) Conclusions: Our study suggests a novel role of HMOs in feto-placental angiogenesis and indicates a contribution of HMO composition to altered feto-placental vascularization in GDM.

APA, Harvard, Vancouver, ISO, and other styles

31

Picard, Morgane, Calaiselvy Soundaramourty, Ricardo Silvestre, Jérôme Estaquier, and Sónia André. "Leishmania infantum Infection of Primary Human Myeloid Cells." Microorganisms 10, no. 6 (June 17, 2022): 1243. http://dx.doi.org/10.3390/microorganisms10061243.

Full text

Abstract:

Circulating phagocytic cells often serve as cellular targets for a large number of pathogens such as Leishmania parasites. Studying primary human cells in an infectious context requires lengthy procedures for cell isolation that may affect the analysis performed. Using whole blood and a no-lyse and no-wash flow cytometric assay (NoNo assay), we monitored the Leishmania infantum infection of primary human cells. We demonstrated, using fluorescent parasites, that among monocyte cell populations, L. infantum preferentially infects classical (CD14+CD16−) and intermediate (CD14+CD16+) primary human monocytes in whole blood. Because classical monocytes are the preponderant population, they represent the larger L. infantum reservoir. Moreover, we also found that, concomitantly to monocyte infection, a subset of PMNs is infected early in whole blood. Of interest, in whole blood, PMNs are less infected compared to classical monocytes. Overall, by using this NoNo assay, we provided a novel avenue in our understanding of host–leishmania interactions.

APA, Harvard, Vancouver, ISO, and other styles

32

Lee, Ryang Hwa, Shu Ching Hsu, James Munoz, Jin Sup Jung, Na Rea Lee, Radhika Pochampally, and Darwin J. Prockop. "A subset of human rapidly self-renewing marrow stromal cells preferentially engraft in mice." Blood 107, no. 5 (March 1, 2006): 2153–61. http://dx.doi.org/10.1182/blood-2005-07-2701.

Full text

Abstract:

Controversies have arisen as to whether adult stem cells or progenitor cells from bone marrow can engraft into nonhematopoietic tissues in vivo. To resolve some of the controversies, we developed a highly sensitive polymerase chain reaction-based single nucleotide polymorphism (PCR-SNP) assay for competitive engraftment of mixtures of stem/progenitor cells. We used the assay to follow engraftment in immunodeficient mice of subpopulations of the stem/progenitor cells from human bone marrow referred to as either mesenchymal stem cells or marrow stromal cells (MSCs). The engraftment into adult mice without induced tissue injury was low and variable, but there was preferential engraftment of a subpopulation of rapidly self-renewing MSCs (RS-MSCs) compared with a subpopulation of slowly renewing MSCs (SR-MSCs). After intravenous infusion, there was a tendency for the cells to engraft into the hippocampal region that was previously designated a “vascular niche.” Migration assays suggested that preferential engraftment of RS-MSCs was in part explained by their expression of CXCR4 and CX3R1, the receptors for SDF-1 and fractalkine.

APA, Harvard, Vancouver, ISO, and other styles

33

Lai, Jian-Ping, Steven D. Douglas, Farida Shaheen, David E. Pleasure, and Wen-Zhe Ho. "Quantification of Substance P mRNA in Human Immune Cells by Real-Time Reverse Transcriptase PCR Assay." Clinical and Vaccine Immunology 9, no. 1 (January 2002): 138–43. http://dx.doi.org/10.1128/cdli.9.1.138-143.2002.

Full text

Abstract:

ABSTRACT We have applied a newly developed real-time reverse transcriptase (RT) PCR (RT-PCR) assay for quantification of substance P (SP) mRNA expression (the SP real-time RT-PCR assay) in human blood monocyte-derived macrophages, peripheral blood lymphocytes, and microglia isolated from fetal brain. The SP real-time RT-PCR assay had a sensitivity of 60 mRNA copies, with a dynamic range of detection between 60 and 600,000 copies of the SP gene transcript per reaction mixture. The coefficient of variation of the threshold cycle number between the SP real-time RT-PCR assays was less than 1.16%. This assay with an SP-specific primer pair efficiently recognizes all four isoforms of preprotachykinin A (the SP precursor) gene transcripts. In order to use this assay to measure the levels of SP mRNA in the human immune cells quantitatively, we designed a specific probe (molecular beacon) derived from exon 3 of the SP gene. We demonstrated that the real-time RT-PCR quantitatively detected SP mRNA in the human immune cells, among which the microglia isolated from fetal brain had the highest levels of SP mRNA. The SP real-time PCR assay yielded reproducible data, as the intra-assay variation was less than 1%. Thus, it is feasible to apply the real-time RT-PCR assay for quantification of SP mRNA levels in human immune cells, as well as in other nonneuronal cells. Since SP is a major modulator of neuroimmunoregulation, this assay has the potential for widespread application for basic and clinical investigations.

APA, Harvard, Vancouver, ISO, and other styles

34

Canavan, James B., Behdad Afzali, Cristiano Scottà, Henrieta Fazekasova, Francis C. Edozie, Thomas T. Macdonald, Maria P. Hernandez-Fuentes, Giovanna Lombardi, and Graham M. Lord. "A rapid diagnostic test for human regulatory T-cell function to enable regulatory T-cell therapy." Blood 119, no. 8 (February 23, 2012): e57-e66. http://dx.doi.org/10.1182/blood-2011-09-380048.

Full text

Abstract:

Abstract Regulatory T cells (CD4+CD25hiCD127loFOXP3+ T cells [Tregs]) are a population of lymphocytes involved in the maintenance of self-tolerance. Abnormalities in function or number of Tregs are a feature of autoimmune diseases in humans. The ability to expand functional Tregs ex vivo makes them ideal candidates for autologous cell therapy to treat human autoimmune diseases and to induce tolerance to transplants. Current tests of Treg function typically take up to 120 hours, a kinetic disadvantage as clinical trials of Tregs will be critically dependent on the availability of rapid diagnostic tests before infusion into humans. Here we evaluate a 7-hour flow cytometric assay for assessing Treg function, using suppression of the activation markers CD69 and CD154 on responder T cells (CD4+CD25− [Tresp]), compared with traditional assays involving inhibition of CFSE dilution and cytokine production. In both freshly isolated and ex vivo expanded Tregs, we describe excellent correlation with gold standard suppressor cell assays. We propose that the kinetic advantage of the new assay may place it as the preferred rapid diagnostic test for the evaluation of Treg function in forthcoming clinical trials of cell therapy, enabling the translation of the large body of preclinical data into potentially useful treatments for human diseases.

APA, Harvard, Vancouver, ISO, and other styles

35

Millen, Scott H., David I. Bernstein, Beverly Connelly, Joel I. Ward, Swei-Ju Chang, and Alison A. Weiss. "Antibody-Mediated Neutralization of Pertussis Toxin-Induced Mitogenicity of Human Peripheral Blood Mononuclear Cells." Infection and Immunity 72, no. 1 (January 2004): 615–20. http://dx.doi.org/10.1128/iai.72.1.615-620.2004.

Full text

Abstract:

ABSTRACT Antibody-mediated neutralization of pertussis toxin-induced proliferation of human peripheral blood mononuclear cells (PBMC) was assessed using alamarBlue and compared with results from the Chinese hamster ovary (CHO) cell assay using sera from vaccinated adults and convalescent children. Neutralization values for the CHO assay were similar for vaccinated and convalescent subjects; however. the convalescent group had higher titers in the PBMC assay. Results for pertussis toxin neutralization with the CHO assay appear to be distinct from those with the PBMC assay.

APA, Harvard, Vancouver, ISO, and other styles

36

Gruber, A., and JH Griffin. "Direct detection of activated protein C in blood from human subjects." Blood 79, no. 9 (May 1, 1992): 2340–48. http://dx.doi.org/10.1182/blood.v79.9.2340.2340.

Full text

Abstract:

Abstract The antithrombotic enzyme, activated protein C (APC) was measured in blood using an enzyme capture assay (ECA). The ECA involved (1) collection of blood into anticoagulant containing a reversible inhibitor of the enzyme, (2) specific affinity capture of the enzyme by an immobilized antibody that does not inhibit the enzyme, (3) removal of the reversible inhibitor by washing, and (4) direct assay of the captured enzyme's amidolytic activity. The ECA for APC used benzamidine for inhibition, anti-PC light-chain murine monoclonal antibody for capture, and the oligopeptide substrate S-2366 for enzyme assay. The sensitivity of this assay was 5 pmol/L (0.3 ng/mL) APC. The APC activity in normal pooled plasma corresponded to the amidolytic activity of 38 pmol/L (2.26 +/- 0.2 ng/mL) purified human plasma- derived APC in the ECA. APC levels in 41 normal donors ranged from 64% to 143%, averaging 104.9% +/- 19.6% (SD). Thus, APC is a measurable and normal component of circulating human blood, and this ECA may be useful for identifying APC deficiency. Moreover, similar ECAs for other enzymes in the circulation may be useful.

APA, Harvard, Vancouver, ISO, and other styles

37

Gruber, A., and JH Griffin. "Direct detection of activated protein C in blood from human subjects." Blood 79, no. 9 (May 1, 1992): 2340–48. http://dx.doi.org/10.1182/blood.v79.9.2340.bloodjournal7992340.

Full text

Abstract:

The antithrombotic enzyme, activated protein C (APC) was measured in blood using an enzyme capture assay (ECA). The ECA involved (1) collection of blood into anticoagulant containing a reversible inhibitor of the enzyme, (2) specific affinity capture of the enzyme by an immobilized antibody that does not inhibit the enzyme, (3) removal of the reversible inhibitor by washing, and (4) direct assay of the captured enzyme's amidolytic activity. The ECA for APC used benzamidine for inhibition, anti-PC light-chain murine monoclonal antibody for capture, and the oligopeptide substrate S-2366 for enzyme assay. The sensitivity of this assay was 5 pmol/L (0.3 ng/mL) APC. The APC activity in normal pooled plasma corresponded to the amidolytic activity of 38 pmol/L (2.26 +/- 0.2 ng/mL) purified human plasma- derived APC in the ECA. APC levels in 41 normal donors ranged from 64% to 143%, averaging 104.9% +/- 19.6% (SD). Thus, APC is a measurable and normal component of circulating human blood, and this ECA may be useful for identifying APC deficiency. Moreover, similar ECAs for other enzymes in the circulation may be useful.

APA, Harvard, Vancouver, ISO, and other styles

38

Pu, Xinzhu, Zemin Wang, and James E. Klaunig. "Cryopreservation of human blood for alkaline and Fpg-modified comet assay." Toxicology Mechanisms and Methods 26, no. 3 (March 16, 2016): 196–201. http://dx.doi.org/10.3109/15376516.2016.1144126.

Full text

APA, Harvard, Vancouver, ISO, and other styles

39

Tappenden, K. A., G. Evans, M. J. Gallimore, I. J. Mackie, H. P. Wendel, M. Winter, and D. W. Jones. "HUMAN VASCULAR ENDOTHELIAL CELLS INFLUENCE THE WHOLE BLOOD THROMBIN GENERATION ASSAY." Journal of Thrombosis and Haemostasis 5 (July 2007): P—M—111—P—M—111. http://dx.doi.org/10.1111/j.1538-7836.2007.tb01033.x.

Full text

APA, Harvard, Vancouver, ISO, and other styles

40

van Dieijen, G., M. P. van Dieijen-Visser, J. Franssen, and H. C. Hemker. "Spectrophotometric Method for the Assay of Human Blood Coagulation Factor VIII." Pathophysiology of Haemostasis and Thrombosis 17, no. 1-2 (1987): 14–24. http://dx.doi.org/10.1159/000215554.

Full text

APA, Harvard, Vancouver, ISO, and other styles

41

Sellar, Kathryn J., Huub H. van Rossum, Fred P. H. T. M. Romijn, Nico P. M. Smit, Johan W. de Fijter, and Johannes van Pelt. "Spectrophotometric assay for calcineurin activity in leukocytes isolated from human blood." Analytical Biochemistry 358, no. 1 (November 2006): 104–10. http://dx.doi.org/10.1016/j.ab.2006.08.013.

Full text

APA, Harvard, Vancouver, ISO, and other styles

42

Schwartz, Reinhard, Anette Walk, Heikki Toomes, and Volker Schirrmacher. "Assay for the determination of human carcinoma cells in circulating blood." Journal of Cancer Research and Clinical Oncology 109, no. 2 (March 1985): 122–29. http://dx.doi.org/10.1007/bf00391886.

Full text

APA, Harvard, Vancouver, ISO, and other styles

43

Anderson, Diana, Tian-Wei Yu, Malgorzata M. Dobrzy?ska, Gloria Ribas, and Ricardo Marcos. "Effects in the comet assay of storage conditions on human blood." Teratogenesis, Carcinogenesis, and Mutagenesis 17, no. 3 (1997): 115–25. http://dx.doi.org/10.1002/(sici)1520-6866(1997)17:3<115::aid-tcm3>3.0.co;2-k.

Full text

APA, Harvard, Vancouver, ISO, and other styles

44

Zaidi, M., S. D. Brain, J. R. Tippins, V. Di Marzo, B. S. Moonga, T. J. Chambers, H. R. Morris, and I. MacIntyre. "Structure-activity relationship of human calcitonin-gene-related peptide." Biochemical Journal 269, no. 3 (August 1, 1990): 775–80. http://dx.doi.org/10.1042/bj2690775.

Full text

Abstract:

The calcitonin-calcitonin-gene-related peptide (CGRP) gene complex encodes a small family of peptides: calcitonin, CGRP and katacalcin. Calcitonin is a circulating hormone that prevents skeletal breakdown by inhibiting the resorption of bone by osteoclasts. CGRP, a potent vasodilator, is involved in normal regulation of blood flow. The calcitonins structurally resemble the CGRP peptides, and both are known to cross-react at each others' receptors. The present study was undertaken to examine the structural prerequisites for biological activity of the intact CGRP molecule. We therefore prepared eight chymotryptic and tryptic fragments of CGRP and synthesized its acetylated and S-carboxyamidomethylcysteinyl analogues. The analogues were purified by h.p.l.c. and their structures were confirmed by fast-atom bombardment mass spectrometry. We have examined the effects of structurally modified analogues and fragments of human CGRP in a calcitonin-receptor-mediated assay, the osteoclast bone resorption assay, and in one or two CGRP-receptor-mediated assays, the rabbit skin blood flow assay and the oedema formation assay. The results showed that (1) in the osteoclast bone resorption assay, both CGRP peptides, alpha and beta, were equipotent, and were both at least 1000-fold were both approx. 1000-fold more potent than salmon calcitonin; human calcitonin had no effect; (3) the bis- and N-acetylated CGRP analogues retained reduced levels of biological activity in all assays, whereas S-carboxyamidomethylcysteinyl-human CGRP was without activity; and (4) all tryptic and chymotryptic fragments of CGRP were without biological activity, with the exception of hCGRP-(Ala1-Lys35): this fragment had much reduced activity compared with the intact peptide in inhibiting osteoclastic bone resorption and increasing blood flow in the rabbit skin. The results suggest that: (1) calcitonin and CGRP act at distinct receptors to mediate different physiological effects; (2) minor amino acid substitutions, as between the alpha and beta forms of CGRP (these two forms have 94% structural similarity) do not result in differences in biological activity; (3) the intact peptide is required for full biological activity of the CGRP molecule, and even the loss of two amino acids at the C-terminus of the molecule results in a marked decrease in activity; (4) the disulphide bridge appears to play an important role in the interaction of the intact CGRP molecule with its receptor; and (5) the C-terminal region is probably necessary for the peptide to assume the right conformation in the interaction with the receptor.

APA, Harvard, Vancouver, ISO, and other styles

45

Cardenas, Juan, Jayaprakash Kotha, Genmin Lu, Pamela B. Conley, Matthieu Bourdin, Tristan Herve, and Lisa K. Jennings. "Validation of a Modified Anti-FXa Assay on STA-Compact Analyzer for Measuring FXa Inhibitor Levels in the Presence of Andexanet Alfa." Blood 136, Supplement 1 (November 5, 2020): 37–38. http://dx.doi.org/10.1182/blood-2020-140988.

Full text

Abstract:

Introduction: The anticoagulants rivaroxaban and apixaban inhibit Factor Xa (FXa) activity and are effective therapeutic agents for the prevention and treatment of thromboembolism. Andexanet alfa (andexanet) is the only specific reversal agent approved for anticoagulation reversal in patients presenting with life-threatening bleeding treated with rivaroxaban or apixaban. Anti-FXa assays are functional assays used for measuring direct and indirect FXa inhibitor levels in plasma. Current commercially available anti-FXa assays are not suitable for accurately measuring anti-FXa activity in patient samples in the presence of andexanet due to the high sample dilutions (e.g.,1:44 with STA-Liquid Anti-Xa), which causes dissociation of the inhibitor from andexanet (due to reversible binding). This results in substantial underestimation of the reversal activity of andexanet, and in some cases with erroneously elevated anti-FXa levels in patient samples following andexanet treatment. Therefore, we modified the current anti-FXa assays to measure apixaban and rivaroxaban anti-FXa levels in the presence of andexanet. Methods: The modified anti-FXa assays were performed on STA®-Compact analyzer using reagents from STA-Liquid Anti-Xa assay kits (Stago). Andexanet was provided as a 10 mg/mL frozen stock. Plasma samples with FXa inhibitor in the presence or absence of andexanet were prepared using Stago Calibrator 1 and pooled normal human plasma (Precision Biologic). Previous anti-FXa assays (calibrator range: 0-500 ng/mL) employed 1:4 dilution of test plasma in Owren-Koller buffer followed by addition of FXa substrate and bovine FXa with an overall 1:44 sample dilution. In the modified assays, calibration curves for the anti-FXa assay were generated using apixaban or rivaroxaban calibrators in the range of 0 to 80 ng/mL; test samples were analyzed neat with an overall 1:2.6 sample dilution. Rivaroxaban and apixaban concentrations in the test samples were interpolated from the respective assay specific calibration curves. Validation of the modified anti-FXa assays was performed by assessing a) linearity and precision of the calibration curve and controls, b) lower limit of quantitation (LLOQ), c) inter-assay precision and d) sample stability. Results: The calibration curves for both apixaban and rivaroxaban assays demonstrated good correlation with a mean "r" value of 0.997 and 0.998, respectively (n=6). The percent recovery and precision of the modified anti-FXa assay are summarized in Table 1. The LLOQ for apixaban and rivaroxaban using the modified assays were <18.8 and <20.6 ng/mL, respectively, based on the preliminary results. Both apixaban and rivaroxaban assays demonstrated potent reversal of anti-FXa activity in presence of andexanet. Samples containing 232 ng/mL apixaban (0.5 µM) treated with equimolar andexanet (0.5 µM) resulted in 77.18 ng/mL of quantifiable apixaban (~66.7% reversal). Similarly, samples containing 242 ng/mL rivaroxaban (0.5 µM) treated with equimolar andexanet (0.5 µM) resulted in 29.44 ng/mL quantifiable rivaroxaban (~87.8% reversal). The short-term stability study with the modified anti-FXa assay included storage of plasma samples at 2-8oC up to 24 hours and under frozen conditions at -20oC up to 2 weeks. Conclusions: The modified anti-FXa assays intended for measuring FXa inhibitor levels in the presence of andexanet produced acceptable analytical performance characteristics. Application of the modified anti-FXa assays in plasma samples from andexanet-treated human subjects has yet to be studied. Table 1 Disclosures Cardenas: CirQuest Labs/MLM Medical Labs: Current Employment. Kotha:CirQuest Labs/MLM Medical Labs: Current Employment. Lu:Portola Pharmaceuticals, Inc.: Current Employment. Conley:Portola Pharmaceuticals, Inc.: Current Employment. Bourdin:Diagnostica Stago: Current Employment. Herve:Diagnostica Stago: Current Employment. Jennings:CirQuest Labs/MLM Medical Labs: Current Employment, Current equity holder in private company.

APA, Harvard, Vancouver, ISO, and other styles

46

Shepard, Robin N., Jody Schock, Kevin Robertson, Diane C. Shugars, John Dyer, Pietro Vernazza, Colin Hall, Myron S. Cohen, and Susan A. Fiscus. "Quantitation of Human Immunodeficiency Virus Type 1 RNA in Different Biological Compartments." Journal of Clinical Microbiology 38, no. 4 (2000): 1414–18. http://dx.doi.org/10.1128/jcm.38.4.1414-1418.2000.

Full text

Abstract:

Little information is available describing viral loads in body fluids other than blood. In addition, the suitability of commercially available assays for human immunodeficiency virus type 1 (HIV-1) RNA quantitation has not been evaluated in most nonblood fluids. We compared Organon Teknika's nucleic acid sequence-based amplification method (NASBA) and Roche's Amplicor HIV-1 Monitor (reverse transcriptase PCR [RT-PCR]) for quantitating HIV-1 RNA in cerebrospinal fluid (CSF), saliva, breast milk, seminal plasma, and cervical-vaginal lavage fluid (CVL). Saliva and breast milk frequently demonstrated some inhibition in the RT-PCR assay, similar to the inhibition previously described in seminal plasma. Inhibition of the RT-PCR assay was not observed with CSF or CVL, nor in any of the NASBA assays. When fluids from HIV-infected individuals were tested by RT-PCR and NASBA, 73 and 27% of CSF samples and 60 and 40% of breast milk specimens had detectable RNA, respectively. These differences were not statistically significant. In cross-sectional studies using RT-PCR to measure viral RNA in paired blood plasma and CSF samples, 71% of blood plasma samples and 42% of CSF samples were positive. A similar analysis using NASBA with paired blood plasma and CVL, saliva, or seminal plasma samples revealed 91% were blood plasma positive and 55% were CVL positive, 76% were blood plasma positive and 46% were saliva positive, and 83% were blood plasma positive and 63% were seminal plasma positive. NASBA worked fairly well to quantitate HIV-1 RNA from all fluids without apparent inhibition. RT-PCR performed well on CVL and CSF, frequently with greater sensitivity, although its use in other fluids appears limited due to the presence of inhibitors. These studies demonstrate that viral loads in nonblood fluids were generally lower than in blood.

APA, Harvard, Vancouver, ISO, and other styles

47

Vysyaraju, Kranthi, Felix Rivera-Mariani, Jesse Negherbon, Helena Hogberg, and Thomas Hartung. "Whole Blood Assay detects endotoxin and non-endotoxin pyrogens (P3098)." Journal of Immunology 190, no. 1_Supplement (May 1, 2013): 125.22. http://dx.doi.org/10.4049/jimmunol.190.supp.125.22.

Full text

Abstract:

Abstract Pyrogenic contaminations may precipitate adverse effects that range from fever to death. There is limited surveillance on non-endotoxin pyrogens and a sensitive technique to detect them is essential. Cells of the immune system come in contact with pyrogens; this causes the release of mediators that induce fever. The Whole Blood Assay (WBA) exploits this human physiological response; human whole blood is incubated with test samples, and interleukin-1 beta (IL-1β) induced in the presence of pyrogenic contamination is measured by ELISA. In this study, we demonstrate the ability of WBA to detect non-endotoxin pyrogens. Human whole blood was incubated with different doses of the following non-endotoxin pyrogenic stimuli: lipoteichoic acid (gram positive bacteria cell wall), Pam2CSK4, FSL-1 (bacterial diacylated lipoproteins), zymosan (fungal [1, 3] - beta glucan) and fungal spores. Levels of induced IL-1β were compared with endotoxin stimulated blood and unstimulated blood as positive and negative controls respectively. All tested stimuli showed pro-inflammatory response compared to the negative control (p < 0.05) confirming that WBA effectively detects non-endotoxin pyrogens. In conclusion, WBA is a sensitive and valuable assay relevant to the human response to a broad range of pyrogenic stimuli. It can also be easily adapted for application in quality assessment of biotherapeutics and immunotoxicology.

APA, Harvard, Vancouver, ISO, and other styles

48

Stoddard, Mark B., Valerian Pinto, Paul B. Keiser, and Wendell Zollinger. "Evaluation of a Whole-Blood Cytokine Release Assay for Use in Measuring Endotoxin Activity of Group B Neisseria meningitidis Vaccines Made from Lipid A Acylation Mutants." Clinical and Vaccine Immunology 17, no. 1 (November 18, 2009): 98–107. http://dx.doi.org/10.1128/cvi.00342-09.

Full text

Abstract:

ABSTRACT Bacterial endotoxin interacts with the human immune system via complex immunological pathways. The evaluation of endotoxicity is important in the development of safe vaccines and immunomodulatory therapeutics. The Limulus amebocyte lysate (LAL) assay is generally accepted by the FDA for use for the quantification of lipopolysaccharide (LPS), while the rabbit pyrogen test (RPT) is used to estimate pyrogenicity during early development and production. Other in vitro assays, such as cytokine release assays with human whole blood (WB) or peripheral blood mononuclear cells (PBMCs), have also been used and may better estimate the human immunological response to products containing novel LPS molecules. In this study, WB and PBMC interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) release assays were used to estimate the endotoxic activities of purified LPS and native outer membrane vesicle (NOMV) vaccines derived from wild-type (hexa-acylated lipid A) and genetically detoxified (penta- and tetra-acylated lipid A) group B Neisseria meningitidis. A method for quantification of the differences in endotoxicity observed in the WB and PBMC assays is elucidated. The LAL assay was shown to be relatively insensitive to lipid A variations, and the RPT was less sensitive than the cytokine release assay with WB. The IL-6 and TNF-α assays with WB but not the assays with PBMCs distinguished between vaccines containing LPS from penta- and tetra-acylated strains. The high degree of sensitivity of the WB system to LPS variations and the presumed relevance of the use of human tissues to predict toxicity in humans suggest that this assay may be particularly well suited for the safety evaluation of vaccines and therapeutics containing acylation variants of LPS.

APA, Harvard, Vancouver, ISO, and other styles

49

Chen, Yu-Ping, Yuan-Yuan Qiao, Xiao-Hang Zhao, Hong-Song Chen, Yan Wang, and Zhuozhi Wang. "Rapid Detection of Hepatitis B Virus Surface Antigen by an Agglutination Assay Mediated by a Bispecific Diabody against Both Human Erythrocytes and Hepatitis B Virus Surface Antigen." Clinical and Vaccine Immunology 14, no. 6 (April 18, 2007): 720–25. http://dx.doi.org/10.1128/cvi.00310-06.

Full text

Abstract:

ABSTRACT Bispecific antibodies have immense potential for use in clinical applications. In the present study, a bispecific diabody against human red blood cells (RBCs) and hepatitis B virus surface antigen (HBsAg) was used to detect HBsAg in blood specimens. The bispecific diabody was constructed by crossing over the variable region of the heavy chains and the light chains of anti-RBC and anti-HBsAg antibodies with a short linker, SRGGGS. In enzyme-linked immunosorbent assays, this bispecific diabody showed specific binding to both RBCs and HBsAg. When this bispecific diabody was mixed with human blood specimens in the presence of HBsAg, the dual binding sites of the diabody caused agglutination of human RBCs. This diabody-mediated agglutination assay was then used to test 712 clinical blood specimens and showed 97.7% sensitivity and 100% specificity when the results were compared with those of the conventional immunoassay, which was used as a reference. This autologous RBC agglutination assay provides a simple approach for rapid screening for HBsAg in blood specimens.

APA, Harvard, Vancouver, ISO, and other styles

50

Banada, Padmapriya P., Srinidhi Deshpande, Soumitesh Chakravorty, Riccardo Russo, James Occi, Gabriel Meister, Kelly J. Jones, et al. "Sensitive Detection of Francisella tularensis Directly from Whole Blood by Use of the GeneXpert System." Journal of Clinical Microbiology 55, no. 1 (November 9, 2016): 291–301. http://dx.doi.org/10.1128/jcm.01126-16.

Full text

Abstract:

ABSTRACTFrancisella tularensisis a potential bioterrorism agent that is highly infectious at very low doses. Diagnosis of tularemia by blood culture and nucleic acid-based diagnostic tests is insufficiently sensitive. Here, we demonstrate a highly sensitiveF. tularensisassay that incorporates sample processing and detection into a single cartridge suitable for point-of-care detection. The assay limit of detection (LOD) and dynamic range were determined in a filter-based cartridge run on the GeneXpert system.F. tularensisDNA in buffer or CFU ofF. tularensiswas spiked into human or macaque blood. To simulate detection in human disease, the assay was tested on blood drawn from macaques infected withF. tularensisSchu S4 at daily intervals. Assay detection was compared to that with a conventional quantitative PCR (qPCR) assay and blood culture. The assay LOD was 0.1 genome equivalents (GE) per reaction and 10 CFU/mlF. tularensisin both human and macaque blood. In infected macaques, the assay detectedF. tularensison days 1 to 4 postinfection in 21%, 17%, 60%, and 83% of macaques, respectively, compared to conventional qPCR positivity rates of 0%, 0%, 30%, and 100% and CFU detection of blood culture at 0%, 0%, 0%, and 10% positive, respectively. Assay specificity was 100%. The new cartridge-based assay can rapidly detectF. tularensisin bloodstream infections directly in whole blood at the early stages of infection with a sensitivity that is superior to that of other methods. The simplicity of the automated testing procedures may make this test suitable for rapid point-of-care detection.

APA, Harvard, Vancouver, ISO, and other styles

Journal articles on the topic 'Human blood assay'

Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles