Simultaneous Fluorescence Detection Research Articles

Simultaneous EQCM and fluorescence detection of adsorption/desorption and oxidation for pyridoxol in aqueous KOH on a gold electrode

A simultaneous EQCM–fluorescence method has been proposed as a novel and informative tool for the study of the adsorption/desorption and oxidation for vitamin B6 (pyridoxol) at a polycrystalline Au electrode in aqueous KOH. The oxidation of pyridoxol was irreversible and accompanied by a strong electrogenerated fluorescence emission peaking at 443 nm when excited at 360 nm. The adsorption and desorption of the oxidation product were found in the double layer region and were discussed by analyzing the simultaneous fluorescence and EQCM data. In addition, the voltammetrically electrogenerated fluorescence is proposed as a new and sensitive method for assay of pyridoxol with satisfactory results.

The identification of three human metabolites of a peptide-doxorubicin conjugate using HPLC-MS-MS in positive and negative ionization modes.

A peptide-doxorubicin conjugate (I) is a drug candidate that has been evaluated for the treatment of prostate cancer. During the high-performance liquid chromatographic (HPLC)-fluorescence analysis of clinical samples for compound I and its two known metabolites [doxorubicin (II) and leucine-doxorubicin (III)], additional metabolites are observed in postdose human plasma extracts. Using neutral loss, precursor, and product ion tandem mass spectrometric (MS-MS) experiments, two of these metabolites are identified as doxorubicinol (IV) and leucine-doxorubicinol (V), the active 13-hydroxy metabolites of doxorubicin and leucine-doxorubicin, respectively. A third metabolite, 7-deoxydoxorubicinol aglycone (VI), is detected using single-ion monitoring at m/z 399 in the negative ionization mode. The product ion mass spectrum of this metabolite contains a major fragment at m/z 351, resulting from the loss of water and formaldehyde from the pseudomolecular ion. An HPLC-MS-MS method for simultaneous analysis of II, III, IV, V, and VI is developed utilizing gradient HPLC with a combination of positive/negative ionization MS in the multiple reaction monitoring mode and monitoring the appropriate MS-MS transitions. Using this methodology, rat, dog, and human plasma metabolite profiles are compared and found to be qualitatively similar. Simultaneous fluorescence and MS detection experiments confirm that the peaks observed in the HPLC-fluorescence chromatograms of plasma extracts correspond to each of the five metabolites (II-VI).

Simultaneous detection of high-resolution R-banding and fluorescence in situ hybridization signals after fluorouracil-induced cellular synchronization.

A method for simultaneous detection of fluorescence in situ hybridization of DNA probes and high resolution fluorescent R banding is described. Human lymphocytes were stimulated with phytohemagglutinin and synchronized using a fluorouracil block followed by exposure to bromodeoxyuridine and Hoechst 33258 prior to harvest. Metaphase preparations were treated with Hoechst 33258 and exposed to UV light. Thereafter they were incubated in sodium phosphate buffer and dried prior to in situ hybridization with a biotin-labelled centromere-specific alpha-satellite DNA probe for chromosome 1 (pUCl.77) and two digoxigenin-labelled probes, i.e., a PCR-generated chromosome 8-specific alphoid probe (#8) and a cosmid probe for FLT4 gene on 5q33-qter (class III receptor tyrosine kinase). Hybridization signals were detected by an indirect immunofluorescence method using fluorescein isothiocyanate. The chromosomes were counterstained with propidium iodide and 4',6-diamidino-2-phenylindole dihydrochloride. This simple method allows unambiguous chromosome band identification simultaneously with detection of the hybridized probes.

Simultaneous EQCM and fluorescence detection of adsorption/desorption and oxidation for pyridoxol in aqueous KOH on a gold electrode

A simultaneous EQCM–fluorescence method has been proposed as a novel and informative tool for the study of the adsorption/desorption and oxidation for vitamin B 6 (pyridoxol) at a polycrystalline Au electrode in aqueous KOH. The oxidation of pyridoxol was irreversible and accompanied by a strong electrogenerated fluorescence emission peaking at 443 nm when excited at 360 nm. The adsorption and desorption of the oxidation product were found in the double layer region and were discussed by analyzing the simultaneous fluorescence and EQCM data. In addition, the voltammetrically electrogenerated fluorescence is proposed as a new and sensitive method for assay of pyridoxol with satisfactory results.

A Comparative Survey of the Simultaneous Ultraviolet and Fluorescence Detection in the RP-HPLC Determination of Dansylated Biogenic Amines in Alcoholic Beverages

The simultaneous use of two detection systems in the RP-HPLC determination of biogenic amines is presented. A spectrophotometric and a fluorescence detector, operated at 254 nm and at λex/λem=320/523 nm respectively, were connected in series and biogenic amines were monitored as their dansyl derivatives after pre-column derivatization, followed by solid-phase extraction clean-up and subsequent separation by reversed-phase liquid chromatography. The dansylamides were separated on an Inertsil ODS-3 column (250 × 4 mm I.D., 5 μm), using a 35-min gradient elution with a binary system of acetonitrile-water and a flow rate of 1 mL · min−1. Both detection modes were applied simultaneously to identify and quantify eleven biogenic amines in white-, red- and retsina wines and beers of Greek origin, after treatment with polyvinylpyrrolidone. Comparison of the detection systems was performed by means of significance tests (t-tests), at the 5% level, applied to within-day measurements of standard solutions, to recovery data and to results from the analysis of wine and beer samples.

Simultaneous trichromatic fluorescence detection of proteins on Western blots using an amine-reactive dye in combination with alkaline phosphatase- and horseradish peroxidase-antibody conjugates.

Three-color fluorescence detection methods are described based upon covalently coupling the dye 2-methoxy-2,4-diphenyl-2(2H)-furanone (MDPF) to proteins immobilized on poly(vinylidene difluoride) (PVDF) membranes, followed by detection of target proteins using alkaline-phosphatase-conjugated reporter molecules in combination with the fluorogenic substrate 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) phosphate (DDAO-phosphate) as well as horseradish peroxidase-conjugated reporter molecules in combination with the new fluorogenic substrate Amplex Gold reagent. This results in all proteins in the profile being visualized as fluorescent blue signal, those detected specifically with the alkaline phosphatase conjugate appearing as fluorescent red signal and those detected specifically with the horseradish peroxidase conjugate appearing as fluorescent yellow signal. Using conventional secondary antibodies, two different targets may be identified as long as primary antibodies generated from two different species are used in the analysis. However, Zenon antibody labeling technology eliminates this restriction, permitting the simultaneous use of two different mouse monoclonal antibodies or two different rabbit polyclonal antibodies in the same electroblotting experiment. The trichromatic detection system is broadly compatible with UV epi-illuminators combined with photographic or charge-coupled device (CCD) cameras, and xenon-arc sources equipped with appropriate excitation/emission filters. Alternatively, the enzyme conjugates may be detected using a laser-based gel scanner. The trichromatic method permits detection of low nanogram amounts of protein and allows for unambiguous identification of two different target proteins relative to the entire protein profile on a single electroblot, precluding any requirement for running replicate gels that would otherwise require separate visualization of total proteins and subsequent alignment with multiple chemiluminescent or colorimetric signals generated on different electroblots.

Light emitting diode-based detectors: Absorbance, fluorescence and spectroelectrochemical measurements in a planar flow-through cell

Light emitting diodes (LEDs) were first used for chemical analysis three decades ago. They are finally making their appearance in commercial analytical systems and dedicated detectors. LEDs are the most energy-efficient means of producing monochromatic light, and provide a concentrated small cool emitter ideal for miniature analytical devices. Although they rank behind fluorescent and halogen discharge lamps in total conversion efficiency (lm/W), new efficiency records are being set every year such that by next decade broadband (white) LED sources are not only likely in analytical instrumentation, but for general illumination. This paper begins with a review of analytical use of LEDs that has been advanced in the last decade. LED-based absorbance measurement and its use in pedagogy, titrations, in providing immunity to refractive index and turbidity effects, in field and process analysis, in capillary electrophoresis (CE), in liquid–liquid extraction systems, in film and drop-based analytical systems and with liquid core waveguides (LCWs) are discussed. LED-based fluorescence and spectroelectrochemical detection follows next. Multipurpose LED-based analytical instrumentation and special analytical applications and general applications are discussed. A listing of (mostly web-based) resources for fabricating LED-based detectors is then provided. Detector circuits and available components are considered and different modes of driving LEDs are compared. The temperature dependence of LED characteristics and strategies to ameliorate this problem are discussed. The review and general resource material is followed with the construction details, operation and performance observed for a simple-to-fabricate multipurpose cell that allows simultaneous multiwavelength absorbance, fluorescence and spectroelectrochemical detection.

409 多波長蛍光の同時検出による高効率 DNA チップ検出装置の開発

409 多波長蛍光の同時検出による高効率 DNA チップ検出装置の開発

Micro sequential injection: automated insulin derivatization and separation using a lab-on-valve capillary electrophoresis system.

Automated sampling and fluorogenic derivatization of islet proteins (insulin, proinsulin, c-peptide) are separated and analyzed by a novel lab-on-valve capillary electrophoresis (LOV-CE) system. This fully integrated device is based on a micro sequential injection instrument that uses a lab-on-valve manifold to integrate capillary electrophoresis. The lab-on-valve manifold is used to perform all microfluidic tasks such as sampling, fluorogenic labeling, and CE capillary rejuvenation providing a very reliable system for reproducible CE separations. Fluorescence detection was coupled to an epiluminescence fluorescence microscope using a customized capillary positioning plate. This customized plate incorporated two fused-silica fiber optic probes that allow for simultaneous absorbance and fluorescence detection, extending the utility of this device. Derivatization conditions with respect to the sequence of addition, timing, injection position, and volumes were optimized through iterative series of experiments that are executed automatically by software control. Reproducibility in fluorogenic labeling was tested with repetitive injections of 3.45 mM insulin, yielding 1.3% RSD for peak area, 0.5% RSD for electromigration time, and 2.8% RSD for peak height. Fluorescence detection demonstrated a linear dynamic range of 3.43 to 6.87 microM for insulin (r2 = 0.99999), 0.39 to 1.96 pM for proinsulin (r2 = 0.99195) and 260 to 781 nM for c-peptide (r2 = 0.99983). By including hydrodynamic flushing immediately after the detection of the last analyte, the sampling frequency for islet protein analysis was increased. Finally, an in vitro insulin assay using rat pancreatic islet excretions was tested using this lab-on-valve capillary electrophoresis system.

Laser-induced fluorescence detection on multichannel electrophoretic microchips using microprocessor-embedded acousto-optic laser beam scanning.

An improved method for fast scanning and fluorescence detection on multimicrochannel microchips is presented using acousto-optic-deflection-driven laser-beam scanning. A microprocessor embedded subsystem used in conjunction with LabView program as the human-machine interface for control of laser-beam scanning and data preprocessing allowed faster scanning and addressing speeds to be attained and improved attenuation calibration and the data sampling speed. This system allows for flexible, high-resolution fluorescence detection for multimicrochannel electrophoresis in a manner that can be applied to a number of high-throughput analysis applications. Incorporating an F-theta focusing lens into the optical set-up allowed for a laser spot as small as 10 microm to accurately be addressed to the center of microchannels. With this spot size, it will be possible to further increase the channel density in the scanning range without encountering crosstalk. Using a six-channel microchip (four separation channels, two alignment channels), the simultaneous separation and fluorescence detection of amino acids and DNA digest samples in four channels is illustrated. User-friendly interpretation of the separation data is facilitated not only by a peak alignment/normalization routine developed within the software, but also through improved signal-to-noise ratios obtained through exploitation of signal processing.

Dual fluorescence and electrochemical detection on an electrophoresis microchip.

Simultaneous amperometric and fluorescence detection in a microfabricated electrophoresis chip is reported. Detection limits of 448 nM and 1.52, 16, and 28 microM have been achieved for dopamine, catechol, NBD-arginine, and NBD-phenylalanine, respectively. These two orthogonal detection schemes allow analysis of a wider spectrum of compounds per separation, leading to higher throughput and enabling resolution of two neutral analytes, NBD-arginine and catechol. In addition, insight into the detection and separation mechanisms is realized. Differences in migration time and peak widths between the two detectors are compared, providing a better understanding of detector alignment. A common problem encountered in electrophoresis is run-to-run migration time irreproducibility for certain samples. This novel microchip dual detection system has been utilized to reduce this irreproducibility. An unknown sample is monitored with one detector while a standard (i.e., ladder) is added to the sample and monitored simultaneously with the other detector. This dual detection method is demonstrated to normalize unknown peak mobilities in a cerebral spinal fluid sample.

Simultaneous red/green dual fluorescence detection on electroblots using BODIPY TR-X succinimidyl ester and ELF 39 phosphate.

A two-color fluorescence detection method is described based upon covalently coupling the succinimidyl ester of BODIPY TR-X dye to proteins immobilized on polyvinylidene difluoride membranes, followed by detection of target proteins using the fluorogenic, precipitating substrate ELF 39-phosphate in combination with alkaline phosphatase conjugated reporter molecules. This results in all proteins in the profile being visualized as fluorescent red signal while those detected specifically with the alkaline phosphatase conjugate appear as fluorescent green signal. The dichromatic detection system is broadly compatible with ultraviolet epi- or trans-illuminators combined with photographic or charge-coupled device cameras, and xenon-arc sources equipped with appropriate excitation/emission filters. The dichromatic method permits detection of low nanogram amounts of protein and allows for unambiguous identification of target proteins relative to the entire protein profile on a single electroblot, obviating the need to run replicate gels that would otherwise require visualization of total proteins by silver staining and subsequent alignment with chemiluminescent or colorimetric signals generated on electroblots. Combining the detection approach with an Alexa Fluor 350 dye conjugated monoclonal antibody permits simultaneous fluorescence detection of two antigens and the total protein profile on the same electroblot.

Rapid and Sensitive Detection of Apple stem pitting virus in Apple Trees Through RNA Amplification and Probing with Fluorescent Molecular Beacons

ABSTRACT Currently, detection of Apple stem pitting virus (ASPV; genus Foveavirus) in apple trees for certification purposes occurs by woody indexing. This method requires a minimum of 12 to 24 weeks in greenhouse testing to up to 2 years in field testing. In this paper, the development of a single tube AmpliDet RNA system for the rapid gel-free detection of ASPV in apple tree tissues is described. The system relies on the specific amplification of the viral RNA by nucleic acid sequence-based amplification and the simultaneous fluorescent detection of the amplification product through molecular beacons. A sensitivity of a minimum of 100 molecules of transcript RNA was obtained by the ASPV-specific AmpliDet RNA. All biologically characterized ASPV isolates from a field trial and 12 of 14 isolates from a plant virus collection were readily detected with this AmpliDet RNA system. In addition, the efficiency of this method for detecting ASPV in 'Golden Delicious' and 'Gravenstein' apple trees was compared throughout the year with mechanical inoculation onto Nicotiana occidentalis 37B, a candidate indicator for ASPV. This revealed that only AmpliDet RNA consistently detected the virus in bark tissue, irrespective of the season. Season-specific tissues such as buds, petals, and fruits, but not leaves, also were reliable sources for detection of ASPV by the AmpliDet RNA system.

Development of a simple, rapid and reproducible HPLC assay for the simultaneous determination of hypericins and stabilized hyperforin in commercial St. John's Wort preparations

A reversed-phase HPLC method was developed and validated for the simultaneous determination of hypericins and stabilized hyperforin in St. John's wort extract. The sample solution was prepared by extraction of the finely powdered extract with methanol-water (80:20, v/v) containing 5% HP-beta-cyclodextrin, and adjusted to pH 2.5 with orthophosphoric acid. Diluted extract solutions, maintained at 0 degrees C, were injected into a C18 column. The samples were eluted isocratically using a mobile phase consisting of acetonitrile and 0.3% v/v phosphoric acid (90:10, v/v) at a 1.5 ml/min flow rate with simultaneous fluorescence (315/590 nm, excitation/emission) and UV (273 nm) detection. Quantification of the marker compounds (hypericin, pseudohypericin, hyperforin) was achieved by use of standard curves generated by plotting peak heights versus concentrations. Validation studies demonstrated that this HPLC method is simple, rapid, reliable, and reproducible. The standard curves were linear over the concentration ranges, 0.5-2.5 microg/ml (hypericin), 0.35-1.6 microg/ml (pseudohypericin) and 5-50 microg/ml (hyperforin). The intra-day coefficients of variation obtained for hypericin, pseudohypericin and hyperforin were < or = 4.4%, < or = 5.4%, and < or = 2.8%, respectively; inter-day CVs were < or = 5.8%, < or = 4.9%, and < or = 2.5%, respectively. This method may be applied for the routine standardization of St. John's wort products against hyperforin and the hypericins, the putative antidepressant principles in the herbal.

Retention behavior of adenine compounds in HPLC with chloroacetaldehyde for fluorescence detection on ODS treated monodispersed—Spherical particle silicas

In this work, a simple system for the simultaneous fluorescence detection of adenine compounds in mouse liver has been developed using high-performance liquid chromatography (HPLC) with ODS-treated monodispersed spherical particle silicas (MDSi).

Simultaneous Determination of the Predominant Hyperforins and Hypericins in St. John's Wort (Hypericum perforatum L.) by Liquid Chromatography

Hypericin and hyperforin are believed to be among the active constituents in common St. John's wort (Hypericum perforatum L.). Presently, dietary supplements are generally standardized to contain specified levels of hypericin and hyperforin, and the related compounds, pseudohypericin and adhyperforin. A rapid method was developed for simultaneous determination of these 4 active constituents by liquid chromatography (LC). A 1 g portion of dried, finely ground leaf/flower sample is extracted with 20 mL methanol for 2 h. A 0.6 mL aliquot of the crude extract is combined with 5.4 mL acetonitrile-methanol (9 + 1) and passed through a mixed solid-phase cleanup column. The eluate is examined by LC for hyperforin, adhyperforin, hypericin, and pseudohypericin on a Hypersil reversed-phase column by using simultaneous ultraviolet (284 nm) and fluorescence detection (excitation, 470 nm; emission, 590 nm). The compounds are easily separated isocratically within 8 min with a mobile phase of acetonitrile-aqueous 0.1 M triethylammonium acetate (8 + 2). Average recoveries of hyperforin and adhyperforin were 101.9 and 98.4%, respectively, for 3 sample mixtures containing concentrations ranging from approximately 0.2 to 1.5% combined hyperforins per gram dry weight. Average relative standard deviation (RSD) values for hyperforin and adhyperforin for all 3 mixtures were 18.9 and 18.0%, respectively. Average recoveries of hypericin and pseudohypericin were 88.6 and 93.3% respectively, from 3 sample mixtures containing concentrations ranging from approximately 0.2 to 0.4% combined hypericins per gram dry weight. Average RSD values for hypericin and pseudohypericin for all 3 mixtures were 3.8 and 4.2%, respectively.

Simultaneous Fluorescent Detection of Two mRNA Targets by High Performance Signal Amplification

Development of drug candidates, which modulate cytokine responses or metabolic pathways by targeting gene expression, requires analytical systems that measure specific messenger RNAs rapidly and accurately and that can be adapted to high throughput screening operations. Chromagen’s High Performance Signal Amplification (HPSA), system, a DNA probe hybridization method, has been enhanced to measure two different mRNA targets simultaneously in the same micro-plate well. Multi-target testing using HPSA takes advantage of a family of low molecular weight fluorescent dyes with large quantum yields, which exhibit distinct excitation and emission wavelengths. A two-target model system using cultured human THP-1 cells examined IL-1b mRNA induction in response to bacterial lipopolysaccharide (LPS) exposure and also monitored intrinsic b-actin mRNA as a housekeeping gene transcript. An oligonucleotide DNA probe complementary to IL-1b RNA was labeled with a reporter ligand while a b-actin DNA probe was linked directly to an enzyme. Sample mRNA was captured onto the surface of micro-plate wells and hybridized to both DNA probes. After removing nonhybridized probe, a second enzyme conjugate that specifically recognizes the IL-1b DNA probe reporter ligand was allowed to bind. The two enzyme systems were distinguished by using substrates labeled with different fluorescent tags (one for IL-1b and the other for b-actin). Resolution of the two fluorescent products was carried out with Chromagen’s high-sensitivity, photoncounting fluorometer. This system was capable of detecting synthetic RNA targets for IL-1b and b-actin in the attomole range. Authentic b-actin mRNA was measured in 50,000 uninduced cells and this signal could be specifically competed away by addition of excess unlabeled b-actin probe during hybridization. A time course of IL-1b mRNA induction in THP-1 cells by LPS revealed that peak induction occurred after 2-3 hours. The bactin mRNA level showed an initial decrease, but remained relatively constant throughout the remaining time points. Results obtained with the dual detection format paralleled those generated when each target was measured separately in its single-target gene expression assay. Chromagen’s HPSA two-target system not only augments screening information, but also saves testing time and conserves reagents. The potential of this system is being explored with different gene targets of therapeutic value and other housekeeping genes. This presentation was given at the 1999 International Symposium for Laboratory Automation and Robotics held in Boston, MA, October 17-20, 1999. The full manuscript is available on CD-Rom and can be acquired by contacting Christine O’Neil, 508-497-2224; email islar@islar.com or access the ISLAR website at www.islar.com.

Simultaneous Fluorescent Detection of Two mRNA Targets by High Performance Signal Amplification

Development of drug candidates, which modulate cytokine responses or metabolic pathways by targeting gene expression, requires analytical systems that measure specific messenger RNAs rapidly and accurately and that can be adapted to high throughput screening operations. Chromagen's High Performance Signal Amplification (HPSA), system, a DNA probe hybridization method, has been enhanced to measure two different mRNA targets simultaneously in the same micro-plate well. Multi-target testing using HPSA takes advantage of a family of low molecular weight fluorescent dyes with large quantum yields, which exhibit distinct excitation and emission wavelengths. A two-target model system using cultured human THP-1 cells examined IL-1β mRNA induction in response to bacterial lipopolysaccharide (LPS) exposure and also monitored intrinsic β-actin mRNA as a housekeeping gene transcript. An oligonucleotide DNA probe complementary to IL-1β RNA was labeled with a reporter ligand while a β-actin DNA probe was linked directly to an enzyme. Sample mRNA was captured onto the surface of micro-plate wells and hybridized to both DNA probes. After removing non-hybridized probe, a second enzyme conjugate that specifically recognizes the IL-1β DNA probe reporter ligand was allowed to bind. The two enzyme systems were distinguished by using substrates labeled with different fluorescent tags (one for IL-1β and the other for β-actin). Resolution of the two fluorescent products was carried out with Chromagen's high-sensitivity, photon-counting fluorometer. This system was capable of detecting synthetic RNA targets for IL-1β and β-actin in the attomole range. Authentic β-actin mRNA was measured in 50,000 uninduced cells and this signal could be specifically competed away by addition of excess unlabeled β-actin probe during hybridization. A time course of IL-1β mRNA induction in THP-1 cells by LPS revealed that peak induction occurred after 2–3 hours. The β-actin mRNA level showed an initial decrease, but remained relatively constant throughout the remaining time points. Results obtained with the dual detection format paralleled those generated when each target was measured separately in its single-target gene expression assay. Chromagen's HPSA two-target system not only augments screening information, but also saves testing time and conserves reagents. The potential of this system is being explored with different gene targets of therapeutic value and other housekeeping genes.

Simultaneous absorbance, fluorescence and refractive index (SAFRIN) detection for Micro LC

Absorbance, fluorescence and refractive index detection are simultaneously accomplished in a Micro LC system. A unique double eccentric-bend fused silica capillary is employed with axial illumination to achieve a long path length, multisensing flow cell. Two different optical sources, one that excites fluorescence through absorption and one that is not absorbed by eluting analytes, are imaged into the bends with optical fibers, placed within the capillary lumen in separate legs of the device. Axially-propagating light exits at each bend where photodetectors simultaneously collect attenuated light, one signal based on absorption and the other signal based on refractive index. A nearby cylindrical lens, optical filter and photomultiplier tube collect fluorescence in the ‘absorption’ leg of the capillary. Thus, trifunctional detection is achieved in microscale liquid chromatography.

AgNOR of Human Interphase Cells in Relation to Acrocentric Chromosomes

Using simultaneous detection of fluorescence in situ hybridization (FISH) to acrocentric chromosome centromeres and argyrophilic nucleolar organizer regions (AgNOR), we investigated the number of AgNOR and involvement pattern of acrocentric chromosomes in the nucleoli in various types of human interphase cells. The number of AgNOR of normal gastric mucosal epithelial cells was 2.27 ± 1.18 and was higher than that of lymphocytes (1.08 ± 0.28) and lower than that of gastric cancer (7.76 ± 3.21). The number of acrocentric chromosome centromere signals of normal gastric mucosal epithelial cells was higher than that of normal leukocytes ( P < 0.000), and lower than that of gastric cancer ( P < 0.000). The acrocentric chromosome centromere signals in the lymphocytes and neutrophils were only half of that expected for diploid cells, perhaps related to acrocentric chromosome association. The proportion of acrocentric chromosomes attached to AgNOR in gastric cancer (0.88 ± 0.22) was significantly higher than that of normal gastric mucosal epithelial cells (0.72 ± 0.35, P = 0.007). In conclusion, acrocentric chromosome association appears to be present in circulating leukocytes even in interphase. The number of AgNORs and proportion of acrocentric chromosomes involved in AgNORs in human interphase cells may vary according to cell types. This could play a significant role in rDNA transcription and determination of cell phenotype, including malignant change.