Rapid enumeration of T lymphocytes by a flow-cytometric immunofluorescence method.

Abstract Combined with current advances in microprocessor-controlled flow cytometers, monoclonal antibodies provide a rapid means of phenotyping individual cell surface markers for a large number of clinical samples accurately and reproducibly, which may provide useful information in diagnosing disease and monitoring patients. We have developed a one-step flow-cytometric immunofluorescence procedure for enumerating E-rosette lymphocytes from whole blood by using the monoclonal antibody OKT11. This antibody recognizes the sheep erythrocyte receptor on the lymphocyte surface and can block sheep E-rosette formation. The flow cytometer we use, an Ortho Spectrum III, distinguishes lymphocytes from other leukocytes by measuring the narrow forward and right-angle light-scattering properties of the cells. The instrument further differentiates T lymphocytes frm non-T lymphocytes by measuring the green fluorescence signal of the OKT11-positive lymphocytes. In a typical sample, 1500--2500 lymphocytes are counted in 25 s. In a study of 158 patient samples, ranging from 1% to greater than 90% E-rosette-positive lymphocytes, the correlation coefficient between the manual E-rosette count and the flow immunofluorescence measurement is 0.943.

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Ultrarapid, Ultrasensitive One-Step Kinetic Immunoassay for C-Reactive Protein (CRP) in Whole Blood Samples: Measurement of the Entire CRP Concentration Range with a Single Sample Dilution

Clinical Chemistry ◽

10.1093/clinchem/48.2.269 ◽

2002 ◽

Vol 48 (2) ◽

pp. 269-277 ◽

Cited By ~ 12

Author(s):

Piia Tarkkinen ◽

Tom Palenius ◽

Timo Lövgren

Keyword(s):

Whole Blood ◽

Solid Phase ◽

Point Of Care ◽

High Sensitivity ◽

Clinical Samples ◽

Cardiac Complications ◽

Fluorescence Signal ◽

C Reactive Protein ◽

Reactive Protein ◽

One Step

Abstract Background: Recently, measurement of very low concentrations of C-reactive protein (CRP) has gained popularity as a potential new means for predicting the risk of future cardiac complications. In this study, we demonstrate the feasibility of a kinetic, one-step microparticle assay for quantitative determination of extremely low and high CRP concentrations in the limited timeframe typical for point-of-care testing. Methods: A noncompetitive, kinetic CRP immunoassay was developed that uses individual, porous microparticles as the solid phase. The microparticles were covalently coated with a monoclonal capture antibody, and the monoclonal detection antibody was labeled with europium. The one-step binding reaction was stopped by washing after 2 min of incubation, and the fluorescence signal of individual particles was measured. Results: The analytical detection limit (mean of zero calibrator + 3 SD) was 0.00016 mg/L CRP. Clinical samples were diluted 400-fold before assay to cover the CRP concentration range of 0.064–1200 mg/L. The assay correlated well with the Dade Behring N High Sensitivity CRP assay (for 0–10 mg/L, r = 0.969, Sy|x = 0.68, n = 54; for 0–350 mg/L, r = 0.969, Sy|x = 11.7, n = 100). The within- and between-run CVs based on calculated concentrations were, respectively, 9–16% and 14% at 0.11 mg/L, 4.5–12% and 8.2% at 4.2 mg/L, and 3.5–6.3% and 4.4% at 105 mg/L, with a CV <15% at 0.2 mg/L and above. Conclusions: Use of the kinetic microparticle approach combined with time-resolved fluorometry allows ultrasensitive quantification of CRP in whole blood in 2 min with a linear assay range spanning more than four orders of magnitude.

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Flow cytometric analysis of T lymphocytes and cytokines in aqueous humor of patients with varicella zoster virus-mediated acute retinal necrosis

BMC Ophthalmology ◽

10.1186/s12886-021-01951-1 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Hao Kang ◽

Yunbo Wei ◽

Ming Liu ◽

Di Yu ◽

Yong Tao

Keyword(s):

T Lymphocytes ◽

Varicella Zoster Virus ◽

Aqueous Humor ◽

T Lymphocyte ◽

Lymphocyte Subsets ◽

Flow Cytometric Analysis ◽

T Lymphocyte Subsets ◽

Varicella Zoster ◽

Flow Cytometric ◽

Cd8 T Lymphocytes

Abstract Background The purpose of this study is to investigate the aqueous humor (AH) T lymphocyte subsets and cytokines of acute retinal necrosis (ARN) to elucidate the immunologic inflammatory features of this disorder. Methods Three patients with ARN infected with varicella zoster virus (VZV) who underwent multiple intravitreal injections of ganciclovir were enrolled in this study. The control group consisted of four non-infectious patients with acute anterior uveitis (AAU). Flow cytometric analysis was performed on the lymphocyte subsets from the AH and peripheral blood (PB) samples during the active phase of intraocular inflammation. Five inflammatory cytokines were measured in each AH sample and various clinical characteristics were also assessed. Results VZV deoxyribonucleic acid (DNA) was detected by real-time polymerase chain reaction (PCR) in AH from all the ARN patients, who showed higher CD8+ T lymphocytes population in AH than the AAU patients (p = 0.006). CD4/CD8 ratios of T lymphocytes and the percentage of CD8 + CD25+ T lymphocytes in AH were significantly lower in ARN than in AAU (p = 0.006; p = 0.012). In the ARN patients, the percentages of CD4+ and CD8+ T lymphocytes in AH were higher than those found in PB. The percentage of CD4 + CD25+ T lymphocytes in AH was significantly higher than the proportion in PB in the AAU patients (p = 0.001). Immunoregulatory cytokine Interleukin-10 in AH was significantly elevated in the ARN patients in comparison with the case of the AAU patients (p = 0.036). In ARN, the copy number of VZV DNA in AH positively correlated with the percentage of CD8+ T lymphocytes in AH and negatively correlated with the CD4/CD8 ratio in AH during the course of disease treatment (p = 0.009, r = 0.92; p = 0.039, r = − 0.834). Conclusion The ARN patients caused by VZV had different intraocular T lymphocyte subsets and cytokines profile than those of the non-infectious patients. High percentages of CD8+ T lymphocytes and low CD4/CD8 T cell ratios may be a potential biomarker for diagnosis of viral-infectious uveitis. T lymphocytes examination at the inflammatory sites has the potential to become a useful research tool for differentiating viral and non-viral uveitis.

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Analysis and validation of a highly sensitive one-step nested quantitative real-time polymerase chain reaction assay for specific detection of severe acute respiratory syndrome coronavirus 2

Virology Journal ◽

10.1186/s12985-020-01467-y ◽

2020 ◽

Vol 17 (1) ◽

Author(s):

Yang Zhang ◽

Chunyang Dai ◽

Huiyan Wang ◽

Yong Gao ◽

Tuantuan Li ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Quantitative Polymerase Chain Reaction ◽

Clinical Samples ◽

Chain Reaction ◽

Pcr Assay ◽

Qrt Pcr ◽

Highly Sensitive ◽

One Step ◽

Polymerase Chain

Abstract Background Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is posing a serious threat to global public health. Reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. Due to technical limitations, the reported positive rates of qRT-PCR assay of throat swab samples vary from 30 to 60%. Therefore, the evaluation of alternative strategies to overcome the limitations of qRT-PCR is required. A previous study reported that one-step nested (OSN)-qRT-PCR revealed better suitability for detecting SARS-CoV-2. However, information on the analytical performance of OSN-qRT-PCR is insufficient. Method In this study, we aimed to analyze OSN-qRT-PCR by comparing it with droplet digital PCR (ddPCR) and qRT-PCR by using a dilution series of SARS-CoV-2 pseudoviral RNA and a quality assessment panel. The clinical performance of OSN-qRT-PCR was also validated and compared with ddPCR and qRT-PCR using specimens from COVID-19 patients. Result The limit of detection (copies/ml) of qRT-PCR, ddPCR, and OSN-qRT-PCR were 520.1 (95% CI: 363.23–1145.69) for ORF1ab and 528.1 (95% CI: 347.7–1248.7) for N, 401.8 (95% CI: 284.8–938.3) for ORF1ab and 336.8 (95% CI: 244.6–792.5) for N, and 194.74 (95% CI: 139.7–430.9) for ORF1ab and 189.1 (95% CI: 130.9–433.9) for N, respectively. Of the 34 clinical samples from COVID-19 patients, the positive rates of OSN-qRT-PCR, ddPCR, and qRT-PCR were 82.35% (28/34), 67.65% (23/34), and 58.82% (20/34), respectively. Conclusion In conclusion, the highly sensitive and specific OSN-qRT-PCR assay is superior to ddPCR and qRT-PCR assays, showing great potential as a technique for detection of SARS-CoV-2 in patients with low viral loads.

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Rapid electrochemical detection of coronavirus SARS-CoV-2

Nature Communications ◽

10.1038/s41467-021-21121-7 ◽

2021 ◽

Vol 12 (1) ◽

Cited By ~ 2

Author(s):

Thanyarat Chaibun ◽

Jiratchaya Puenpa ◽

Tatchanun Ngamdee ◽

Nimaradee Boonapatcharoen ◽

Pornpat Athamanolap ◽

...

Keyword(s):

Electrochemical Biosensor ◽

Rolling Circle Amplification ◽

Clinical Samples ◽

Rolling Circle ◽

Qrt Pcr ◽

Reverse Transcription Pcr ◽

Sandwich Hybridization ◽

Redox Active ◽

One Step ◽

The One

AbstractCoronavirus disease 2019 (COVID-19) is a highly contagious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Diagnosis of COVID-19 depends on quantitative reverse transcription PCR (qRT-PCR), which is time-consuming and requires expensive instrumentation. Here, we report an ultrasensitive electrochemical biosensor based on isothermal rolling circle amplification (RCA) for rapid detection of SARS-CoV-2. The assay involves the hybridization of the RCA amplicons with probes that were functionalized with redox active labels that are detectable by an electrochemical biosensor. The one-step sandwich hybridization assay could detect as low as 1 copy/μL of N and S genes, in less than 2 h. Sensor evaluation with 106 clinical samples, including 41 SARS-CoV-2 positive and 9 samples positive for other respiratory viruses, gave a 100% concordance result with qRT-PCR, with complete correlation between the biosensor current signals and quantitation cycle (Cq) values. In summary, this biosensor could be used as an on-site, real-time diagnostic test for COVID-19.

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A sensitive one-step TaqMan amplification approach for detection of rubella virus clade I and II genotypes in clinical samples

Archives of Virology ◽

10.1007/s00705-016-3131-1 ◽

2016 ◽

Vol 162 (2) ◽

pp. 477-486 ◽

Cited By ~ 5

Author(s):

C. Claus ◽

S. Bergs ◽

N. C. Emmrich ◽

J. M. Hübschen ◽

A. Mankertz ◽

...

Keyword(s):

Rubella Virus ◽

Clinical Samples ◽

One Step

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Flow cytometric determination of glutathione in clinical samples

Cytometry ◽

10.1002/cyto.990210113 ◽

1995 ◽

Vol 21 (1) ◽

pp. 68-71 ◽

Cited By ~ 12

Author(s):

Sue Chow ◽

David Hedley

Keyword(s):

Clinical Samples ◽

Flow Cytometric

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Direct freezing of whole blood enables analysis of leucocyte markers by flow cytometry: a proof-of-concept study

Future Microbiology ◽

10.2217/fmb-2021-0034 ◽

2021 ◽

Author(s):

Pénélope Bourgoin ◽

Inès Ait Belkacem ◽

Isabelle Arnoux ◽

Pierre-Emmanuel Morange ◽

Fabrice Malergue

Keyword(s):

Flow Cytometry ◽

Whole Blood ◽

Blood Cells ◽

White Blood Cells ◽

Proof Of Concept ◽

Fresh Blood ◽

Blood Samples ◽

Step Flow ◽

Freezing Method ◽

One Step

Aim: A new one-step flow cytometry procedure has been recently demonstrated for identifying subjects with infections, but only for fresh whole blood samples. The goal of this study was to assess its applicability on frozen samples, by proposing a new method to perform the sample freezing directly and easily. Methods: Fresh blood was tested, then frozen either directly or with dimethylsulfoxide and serum. Common markers of white blood cells as well as infection-related biomarkers were tested. Results: All percentages of leucocyte subsets and levels of infection-related biomarkers were significantly correlated between frozen and fresh samples. Conclusion: The direct freezing method enables an accurate assessment of common cellular sub-populations and of levels of important infectious biomarkers via flow cytometry.

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Flow Cytometric Analysis of T Lymphocytes and Cytokines in Aqueous Humor of Patients with Varicella Zoster Virus-mediated Acute Retinal Necrosis

10.21203/rs.3.rs-56692/v1 ◽

2020 ◽

Author(s):

Hao Kang ◽

Yunbo Wei ◽

Ming Liu ◽

Di Yu ◽

Yong Tao

Keyword(s):

T Lymphocytes ◽

Varicella Zoster Virus ◽

Aqueous Humor ◽

T Lymphocyte ◽

Lymphocyte Subsets ◽

Flow Cytometric Analysis ◽

T Lymphocyte Subsets ◽

Varicella Zoster ◽

Flow Cytometric ◽

Cd8 T Lymphocytes

Abstract Background: The purpose of this study is to investigate the aqueous humor (AH) T lymphocyte subsets and cytokines of acute retinal necrosis (ARN) to elucidate the immunologic inflammatory features of this disorder.Methods: Three patients with ARN infected with varicella zoster virus (VZV) who underwent multiple intravitreal injections of ganciclovir were enrolled in this study. The control group consisted of four non-viral infectious patients with acute anterior uveitis (AAU). Flow cytometric analysis was performed on the lymphocyte subsets from the AH and peripheral blood (PB) samples during the active phase of intraocular inflammation. Five inflammatory cytokines were measured in each AH sample and various clinical characteristics were also assessed.Results: VZV DNA was detected by real-time polymerase chain reaction (PCR) in AH from all the ARN patients, who showed higher CD8+ T lymphocytes population in AH than the AAU patients (p=0.006). CD4/CD8 ratios of T lymphocytes and the percentage of CD8+CD25+ T lymphocytes in AH were significantly lower in ARN than in AAU (p=0.006; p=0.012). In the ARN patients, the percentages of CD4+ and CD8+ T lymphocytes in AH were higher than those found in PB. The percentage of CD4+CD25+ T lymphocytes in AH was significantly higher than the proportion in PB in the AAU patients (p=0.001). Immunoregulatory cytokine Interleukin-10 in AH was significantly elevated in the ARN patients in comparison with the case of the AAU patients (p=0.036). In ARN, the copy number of VZV DNA in AH positively correlated with the percentage of CD8+ T lymphocytes in AH and negatively correlated with the CD4/CD8 ratio in AH during the course of disease treatment (p=0.009, r=0.92; p=0.039, r=-0.834).Conclusion: The ARN patients caused by VZV had different intraocular T lymphocyte subsets and cytokines profile than those of the non-viral infectious patients. High percentages of CD8+ T lymphocytes and low CD4/CD8 T cell ratios may be a potential biomarker for diagnosis of viral-infectious uveitis. T lymphocytes examination at the inflammatory sites has the potential to become a useful research tool for differentiating viral and non-viral uveitis.

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Two-site enzyme immunoassay of CD4 and CD8 molecules on the surface of T lymphocytes from healthy subjects and HIV-1-infected patients

Clinical Chemistry ◽

10.1093/clinchem/40.1.30 ◽

1994 ◽

Vol 40 (1) ◽

pp. 30-37 ◽

Cited By ~ 9

Author(s):

D Carriere ◽

C Fontaine ◽

A M Berthier ◽

A M Rouquette ◽

P Carayon ◽

...

Keyword(s):

Monoclonal Antibody ◽

T Lymphocytes ◽

Enzyme Immunoassay ◽

Cd8 T Cells ◽

Envelope Glycoprotein ◽

Healthy Subjects ◽

Gp 120 ◽

Highly Sensitive ◽

One Step ◽

Hiv 1

Abstract A highly sensitive two-site enzyme immunoassay (Capcellia) was developed to determine the concentration of CD4 and CD8 molecules expressed on the surface of human T lymphocytes. This assay, performed in one step (20 min), involves the specific immunocapture of T lymphocytes and reaction of the CD4 or CD8 molecules with an enzyme-labeled monoclonal antibody (mAb). The results were expressed as molar concentrations of the T-cell markers on the basis of results obtained with calibrated CD4 and CD8 standards. The assay was sensitive enough to detect 0.4 pmol/L CD4 or 0.8 pmol/L CD8, which corresponded to approximately 20 x 10(6) CD4+ or CD8+ T cells per liter of blood. Mean concentrations in healthy adults were 17.2 pmol/L for CD4 and 22.1 pmol/L for CD8. The CD4 concentration was < 8 pmol/L in 50% of HIV-1-infected patients and in 95% of AIDS patients. Given the epitopic specificity of the mAb to CD4 we used, these values correspond to the concentration of CD4 molecules free of envelope glycoprotein (gp)120.

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