Early molecular markers for retrospective biodosimetry and prediction of acute health effects.

Radiation-induced biological changes occurring within hours and days after irradiation can be potentially used for either exposure reconstruction (retrospective dosimetry) or the prediction of consecutively occurring acute or chronic health effects. The advantage of molecular protein or gene expression (GE) (mRNA) marker lies in their capability for early (1-3 days after irradiation), high-throughput and point-of-care diagnosis, required for the prediction of the acute radiation syndrome (ARS) in radiological or nuclear scenarios. These molecular marker in most cases respond differently regarding exposure characteristics such as e.g. radiation quality, dose, dose rate and most importantly over time. Changes over time are in particular challenging and demand certain strategies to deal with. With this review, we provide an overview and will focus on already identified and used mRNA GE and protein markers of the peripheral blood related to the ARS. These molecules are examined in light of 'ideal' characteristics of a biomarkers (e.g. easy accessible, early response, signal persistency) and the validation degree. Finally, we present strategies on the use of these markers considering challenges as their variation over time and future developments regarding e.g. origin of samples, point of care and high-throughput diagnosis.

Using Clinical Signs and Symptoms for Medical Management of Radiation Casualties - 2015 NATO Exercise.

The utility of early-phase (≤5 days) radiation-induced clinical signs and symptoms (e.g., vomiting, diarrhea, erythema and changes in blood cell counts) was examined for the prediction of later occurring acute radiation syndrome (ARS) severity and the development of medical management strategies. Medical treatment protocols for radiation accident victims (METREPOL) was used to grade ARS severities, which were assigned response categories (RCs). Data on individuals (n = 191) with mild (RC1, n = 45), moderate (RC2, n = 19), severe (RC3, n = 20) and fatal (RC4, n = 18) ARS, as well as nonexposed individuals (RC0, n = 89) were generated using either METREPOL (n = 167) or the system for evaluation and archiving of radiation accidents based on case histories (SEARCH) database (n = 24), the latter comprised of real-case descriptions. These data were converted into tables reflecting clinical signs and symptoms, and submitted to eight teams representing five participating countries. The teams were comprised of medical doctors, biologists and pharmacists with subject matter expertise. The tables comprised cumulated clinical data from day 1-3 and day 1-5 postirradiation. While it would have reflected a more realistic scenario to provide the data to the teams over the course of a 3- or 5-day period, the logistics of doing so proved too challenging. In addition, the team members participating in this exercise chose to receive the cumulated reports of day 1-3 and 1-5. The teams were tasked with predicting ARS incidence, ARS severity and the requirement for hospitalization for multiple cases, as well as providing the certainty of their diagnosis. Five of the teams also performed dose estimates. The teams did not employ harmonized methodologies, and the expertise among the members varied, as did the tools used and the means of analyzing the clinical data. The earliest report time was 3 h after the tables were sent to the team members. The majority of cases developing ARS (89.6% ± 3.3 SD) and requiring hospitalization (88.8% ± 4.6 SD) were correctly identified by all teams. Determination of ARS severity was particularly challenging for RC2-3, which was systematically overestimated. However, RC4 was correctly predicted at 94-100% by all teams. RC0 and RC1 ARS severities were more difficult to discriminate. When reported RCs (0-1 and 3-4) were merged, on average 89.6% (±3.3 SD) of all cases could be correctly classified. Comparisons on frequency distributions revealed no statistically significant differences among the following: 1. reported ARS from different teams (P > 0.2); 2. cases generated based on METREPOL or SEARCH (P > 0.5); or 3. results reported at day 3 and 5 postirradiation (P > 0.1). Dose estimates of all teams increased significantly along with ARS severity (P < 0.0001) as well as with dose estimates generated from dicentric chromosomal-aberration measurements available for SEARCH cases (P < 0.0001). In summary, early-phase radiation-induced clinical signs and symptoms proved to be useful for rapid and accurate assessment, with minor limitations, toward predicting life-threatening ARS severity and developing treatment management strategies.

Further biodosimetry investigations using murine partial-body irradiation model.

This study evaluates both the effects of physical restraint and use of candidate biomarkers in a CD2F1 male mouse partial-body irradiation model for biological dosimetry diagnostic assays. Mice were irradiated (6-Gy, 250-kVp X ray) to 3/3rd (total body), 2/3rd (gut and torso), 1/3rd (gut only) and 0/3rd (sham) of total body. Blood was sampled for haematology and blood plasma proteomic biomarkers at 1 and 2 d after exposure. Increases in the body fraction exposed showed progressive decreases in lymphocyte counts and increases in the neutrophil-to-lymphocyte ratios with no significant differences in the neutrophil and platelet counts. The radioresponse for plasma biomarker Flt3L showed proportional increases; however, G-CSF and SAA levels exhibited dramatic and non-proportional increases in levels. Physical restraint at 1 d post-exposure increased lymphocyte counts and SAA, decreased neutrophil-to-lymphocyte ratio and Flt3L and showed no effects on neutrophil and platelet counts or G-CSF.

Sample Tracking in an Automated Cytogenetic Biodosimetry Laboratory for Radiation Mass Casualties.

Chromosome aberration-based dicentric assay is expected to be used after mass casualty life-threatening radiation exposures to assess radiation dose to individuals. This will require processing of a large number of samples for individual dose assessment and clinical triage to aid treatment decisions. We have established an automated, high-throughput, cytogenetic biodosimetry laboratory to process a large number of samples for conducting the dicentric assay using peripheral blood from exposed individuals according to internationally accepted laboratory protocols (i.e., within days following radiation exposures). The components of an automated cytogenetic biodosimetry laboratory include blood collection kits for sample shipment, a cell viability analyzer, a robotic liquid handler, an automated metaphase harvester, a metaphase spreader, high-throughput slide stainer and coverslipper, a high-throughput metaphase finder, multiple satellite chromosome-aberration analysis systems, and a computerized sample tracking system. Laboratory automation using commercially available, off-the-shelf technologies, customized technology integration, and implementation of a laboratory information management system (LIMS) for cytogenetic analysis will significantly increase throughput.This paper focuses on our efforts to eliminate data transcription errors, increase efficiency, and maintain samples' positive chain-of-custody by sample tracking during sample processing and data analysis. This sample tracking system represents a "beta" version, which can be modeled elsewhere in a cytogenetic biodosimetry laboratory, and includes a customized LIMS with a central server, personal computer workstations, barcode printers, fixed station and wireless hand-held devices to scan barcodes at various critical steps, and data transmission over a private intra-laboratory computer network. Our studies will improve diagnostic biodosimetry response, aid confirmation of clinical triage, and medical management of radiation exposed individuals.

Radiation biodosimetry: applications for spaceflight.

The multiparametric dosimetry system that we are developing for medical radiological defense applications could be adapted for spaceflight environments. The system complements the internationally accepted personnel dosimeters and cytogenetic analysis of chromosome aberrations, considered the best means of documenting radiation doses for health records. Our system consists of a portable hematology analyzer, molecular biodosimetry using nucleic acid and antigen-based diagnostic equipment, and a dose assessment management software application. A dry-capillary tube reagent-based centrifuge blood cell counter (QBC Autoread Plus, Becton [correction of Beckon] Dickinson Bioscience) measures peripheral blood lymphocytes and monocytes, which could determine radiation dose based on the kinetics of blood cell depletion. Molecular biomarkers for ionizing radiation exposure (gene expression changes, blood proteins) can be measured in real time using such diagnostic detection technologies as miniaturized nucleic acid sequences and antigen-based biosensors, but they require validation of dose-dependent targets and development of optimized protocols and analysis systems. The Biodosimetry Assessment Tool, a software application, calculates radiation dose based on a patient's physical signs and symptoms and blood cell count analysis. It also annotates location of personnel dosimeters, displays a summary of a patient's dosimetric information to healthcare professionals, and archives the data for further use. These radiation assessment diagnostic technologies can have dual-use applications supporting general medical-related care.

Real-time quantitative RT-PCR assay of GADD45 gene expression changes as a biomarker for radiation biodosimetry.

PURPOSE: To assess the efficacy of fluorescent-based quantitative reverse transcription-polymerase chain reaction (QRT-PCR) technology to measure gene expression changes (GEC) for rapid, point-of-care radiation dose assessment. MATERIALS AND METHODS: A real-time QRT-PCR assay based on 5'-fluorogenic nuclease TaqMan(TM) methodology was developed, which employs both relative and absolute quantification of a candidate mRNA biomarker. Growth arrest and DNA damage gene 45 (GADD45), a cell-cycle regulation and DNA repair gene, served as the paradigm because of the reported linear dose-response relationship for mRNA induction in the human myeloid tumor cell line (ML-1) over the range of 2-50 cGy. Using an ex vivo whole-blood model, GEC was measured from total blood RNA at 24h and 48 h after (60)Co gamma-ray exposures (0-3 Gy; 0.1 Gy/min). RESULTS: A linear and reproducible up-regulation representing a twofold to fourfold change in GADD45 relative and absolute GEC was confirmed in both intra- and inter-assay analyses. CONCLUSIONS: Primer and probes to detect GADD45 targets using real-time PCR were developed. This is the first report using realtime QRT-PCR to measure radiation-induced GEC dose response. Real-time QRT-PCR using GEC as biomarkers offers rapidity, sensitivity, and reproducibility as a potential efficient biological dosimetry tool applicable in radiation therapy applications and early-response accident biodosimetry.

Proto-oncogene expression: a predictive assay for radiation biodosimetry applications.

Using a model system of in vitro human peripheral blood lymphocytes, the effect of low-dose (0.25 to 1.50 Gy) 250-kVp X ray radiation (1 Gy.min-1) on the expression of several proto-oncogenes was examined (c-Haras, c-src, c-met, c-jun, c-fos, and c-myc) and beta-actin from 0.25 to 17 h post-radiation. RNA was extracted from cells harvested at various times after exposure and examined for levels of particular mRNAs by northern blot hybridisation. A progressive time- and dose-dependent increase in mRNA levels was observed for c-Haras mRNA, while the other proto-oncogenes (c-src, c-met, c-fos, c-jun and c-myc) examined were variable during the same time period. beta-actin levels were initially decreased but at 17 h post-radiation had returned to control levels. A comparison of the rate of c-Haras transcription at 5 and 17 h post-irradiation revealed that c-Haras transcription was higher at 5 h than at 17 h. These findings suggest that the level of specific proto-oncogene expression, particularly c-Haras, may be useful early diagnostic molecular biomarkers for biodosimetry applications. The use of real-time PCR technologies to quantify gene expression changes will also be discussed.

Quantitative plasmid mixture analysis using the fluorogenic 5'-nuclease polymerase chain reaction assay.

The fluorogenic 5'-nuclease polymerase chain reaction (PCR) assay has been shown to be useful for quantifying a given DNA target in a sample. Here we show how an existing PCR protocol can be amended for quantification by incorporating distinctive dual-labeled, sequence-specific oligonucleotide probes and resulting in a two- to threefold broader and more reliable dynamic range than that of conventional end-point analysis of PCR products. Moreover, we show a multiplex situation in which two targets, one normal and one mutated, can be amplified and quantified simultaneously and in the same reaction tube. Use of this novel approach for quantitative PCR applications eliminates the need for post-PCR processing and has clinical- and research-based diagnostic applications, particularly for measuring levels of mutations in a mixture.

Biodosimety Assessment Tool: a post-exposure software application for management of radiation accidents.

The Biodosimetry Assessment Tool software application under development will equip health care providers with diagnostic information (clinical signs and symptoms, physical dosimetry, etc.) germane to the management of human radiation casualties. Designed primarily for prompt use after a radiation incident, the user-friendly program facilitates collection, integration, and archiving of data obtained from exposed persons. Data collected in templates are compared with established radiation dose responses obtained from the literature to provide multiparameter dose assessments. The program archives clinical information (e.g., extent of contamination, wounds, infection, etc.) useful for casualty management, displays relevant diagnostic information in a concise format, and can be used to manage both military and civilian radiation accidents. In addition, monitoring of diagnostic information of individuals using this program could potentially minimize the severity of psychological casualties by making a marked impact on the way that both radiation casualties and the worried well view their exposure, dose, and future risk for the development of disease.

Radiation exposure assessment using cytological and molecular biomarkers.

Chromosome aberration analysis is the conventional means of assessing radiation exposure. The Armed Forces Radiobiology Research Institute recently established an alternative method to measure radiation-induced chromosome aberrations in interphase cells. The method uses commercially available chemical agents to induce premature chromosome condensation in resting' G0 human peripheral blood lymphocytes. Then specific whole-chromosome DNA probes are used with fluorescence in situ hybridisation to detect aberrant cells rapidly over a broad dose range. In new research, the real-time fluorogenic 5'-nuclease, or TaqMan, polymerase chain reaction assay is being used to identify radiation-responsive molecular biomarkers, including gene expression targets and DNA mutations. The goal is to establish rapid, precise, high-throughput assay systems that are practical in a variety of radiation exposure scenarios. The new methodologies that have a number of other applications, together with diagnostic software now in development, could improve the United States military's emergency response capability and medical readiness.

In situ detection of a PCR-synthesized human pancentromeric DNA hybridization probe by color pigment immunostaining: application for dicentric assay automation.

We report a low cost and efficient method for synthesizing a human pancentromeric DNA probe by the polymerase chain reaction (PRC) and an optimized protocol for in situ detection using color pigment immunostaining. The DNA template used in the PCR was a 2.4 kb insert containing human alphoid repeated sequences of pancentromeric DNA subcloned into pUC9 (Miller et al. 1988) and the primers hybridized to internal sequences of the 172 bp consensus tandem repeat associated with human centromeres. PCR was performed in the presence of biotin-11-dUTP, and the product was used for in situ hybridization to detect the pancentromeric region of human chromosomes in metaphase spreads. Detection of pancentromeric probe was achieved by immunoenzymatic color pigment painting to yield a permanent image detected at high resolution by bright field microscopy. The ability to synthesize the centromeric probe rapidly and to detect it with color pigment immunostaining will lead to enhanced identification and eventually to automation of various chromosome aberration assays.

Induction of premature chromosome condensation by a phosphatase inhibitor and a protein kinase in unstimulated human peripheral blood lymphocytes: a simple and rapid technique to study chromosome aberrations using specific whole-chromosome DNA hybridization probes for biological dosimetry.

We developed a simple and rapid method to study chromosome aberrations involving specific chromosomes using unstimulated human peripheral blood lymphocytes (HPBL). Premature chromosome condensation (PCC) was induced by incubating unstimulated HPBL in the presence of okadaic acid (OA, a phosphatase inhibitor), adenosine triphosphate (ATP), and p34(cdc2)/cyclin B kinase [an essential component of mitosis-promoting factor (MPF)], which eliminated the need for fusion with mitotic cells. OA concentration and duration of incubation for PCC induction was optimized using mitogen-stimulated HPBL; a final concentration of 0.75 microM incubated for 3 h was optimum, resulting in approximately 20% PCC yield. In unstimulated HPBL, PCC was induced by the addition of p34(cdc2)/cyclin B kinase at concentrations as low as 5 units/ml to a cell culture medium containing OA. Increases in the concentration of p34(cdc2)/cyclin B kinase from 5 to 50 units/ml resulted in a concentration-dependent increase in PCC yield (30% to 42%). We demonstrate that this technique of inducing PCC in unstimulated HPBL is suitable for studying radiation-induced aberrations involving a specific chromosome (chromosome 1) after 24 h repair using a whole-chromosome in situ hybridization probe and chromosome painting. Cells with aberrant chromosome number 1 are characterized with more than two chromosome spots. The frequency of cells with aberrant chromosome 1 increased with 60Co gamma-radiation doses in the region 0-7.5 Gy. The observed dose-effect relationship for the percentage of cells with aberrant chromosome 1 (Y) was explained by using both a linear [Y=(2.77+/-0.230)D+0.90+/-0.431, r(2)=0.966] and a nonlinear power [Y=(5.70+/-0.46)D((0.61+/-0.05)), r(2)=0.9901) model. This technique can be applied to biological dosimetry of radiation exposures involving uniform whole-body low linear energy transfer (LET) exposures.

Fast-in situ hybridization and immunoenzymatic color pigment detection of mouse bone marrow micronucleus.

The development of a whole mouse genomic DNA probe coupled to color pigment painting detection methodology can accurately verify mouse micronuclei induced by chemicals or drugs leading to a lower probability of potential artifacts. Using color pigment painting detection of probes in conjunction with Wright's Giemsa counterstain instead of the current fluorescence detection technology ensures low cost, high resolution permanent documentation of slides for a particular test compound. The permanent color pigment-detected micronuclei and adjoining counterstain allows slides to be stored for future analysis without enhancing the signal or adding antifading agents that are associated with fluorescence detection. Combining innovative technology such as fast-in situ hybridization of DNA probes with immunoenzymatic color pigment detection provides rapid verification of true micronuclei (DNA containing) within 2-3 hr.

Quantitative assessment of the contribution of clustered damage to DNA double-strand breaks induced by 60Co gamma rays and fission neutrons.

The induction of DNA strand breaks by fission neutrons was studied in aqueous plasmid (pBR322) DNA under aerobic conditions for a wide range of hydroxyl radical (*OH) scavenger concentrations and was compared to the induction of strand breaks by 6OCo gamma rays. Strand breaks were measured using agarose gel electrophoresis coupled with sensitive 32P-based phosphor imaging. Yields are reported for DNA single-strand breaks (SSBs) and double-strand breaks formed linearly with dose (alphaDSBs). The fraction of alphaDSBs that were dependent on the multiply damaged site (MDS) or clustered damage mechanism was also calculated using a model. G values for SSBs and alphaDSBs declined with increasing *OH scavenging capacity. However, with increasing *OH scavenging capacities, the decrease in yields of strand breaks for fission neutrons was not as pronounced as for gamma rays. The percentage of alphaDSBs for gamma rays was dependent on *OH scavenging capacity, appearing negligible at low scavenging capacities but increasing at higher scavenging capacities. In contrast, fission neutrons induced high percentages of alphaDSBs that were approximately independent of *OH scavenging capacity. The levels of alphaDSBs formed by the MDS mechanism after exposure to fission neutrons are consistent with the expected distinctive features of high-LET energy deposition events and track structure. The results also confirm observations made by others that even for low-LET radiation, the MDS mechanism contributes significantly to DNA damage at cell-like scavenging conditions.

Transformation of human osteoblast cells to the tumorigenic phenotype by depleted uranium-uranyl chloride.

Depleted uranium (DU) is a dense heavy metal used primarily in military applications. Although the health effects of occupational uranium exposure are well known, limited data exist regarding the long-term health effects of internalized DU in humans. We established an in vitro cellular model to study DU exposure. Microdosimetric assessment, determined using a Monte Carlo computer simulation based on measured intracellular and extracellular uranium levels, showed that few (0.0014%) cell nuclei were hit by alpha particles. We report the ability of DU-uranyl chloride to transform immortalized human osteoblastic cells (HOS) to the tumorigenic phenotype. DU-uranyl chloride-transformants are characterized by anchorage-independent growth, tumor formation in nude mice, expression of high levels of the k-ras oncogene, reduced production of the Rb tumor-suppressor protein, and elevated levels of sister chromatid exchanges per cell. DU-uranyl chloride treatment resulted in a 9.6 (+/- 2.8)-fold increase in transformation frequency compared to untreated cells. In comparison, nickel sulfate resulted in a 7.1 (+/- 2.1)-fold increase in transformation frequency. This is the first report showing that a DU compound caused human cell transformation to the neoplastic phenotype. Although additional studies are needed to determine if protracted DU exposure produces tumors in vivo, the implication from these in vitro results is that the risk of cancer induction from internalized DU exposure may be comparable to other biologically reactive and carcinogenic heavy-metal compounds (e.g., nickel).

Premature chromosome condensation assay for biodosimetry: studies with fission-neutrons.

Characterization of the premature chromosome condensation assay for radiation quality is needed. To that end, human lymphocytes were exposed in vitro to various doses of 250-kVp x rays (Y(D) = 4 keV microm(-1), Y(D) is the dose-mean lineal energy of the absorbed dose distribution, D(y), where y is defined as the energy deposited in a volume by a single event divided by the mean chord length of the volume) and to fission neutrons (Y(D) = 65 keV microm(-1)). The distribution of prematurely condensed chromosome and fragments following exposure to x rays or to neutrons were non-Poisson after repair at 37 degrees C for 24 h. Dose-response curves were constructed for the yield of excess prematurely condensed chromosome fragments as necessary for biodosimetry applications. The curves were fitted to a weighted linear model by the least-squares regression method. The neutron relative biological effectiveness (RBE) value was estimated to be 2.4 +/- 0.39.

Energy deposition events produced by fission neutrons in aqueous solutions of plasmid DNA.

Using an agarose gel electrophoresis assay, single-strand breaks (ssb) induced by fission neutrons and 60Co gamma-rays in aerobic aqueous solutions of pBR322 plasmid DNA were studied. The energy-deposition events of the two radiations were characterized using a Rossi-type proportional counter to measure lineal-energy spectra. For neutrons, the dose-weighted lineal-energy mean, yD, is 63 keV micron-1--about 30 times that for gamma-rays. With increasing yD, hydroxyl radicals produced within spurs or tracks are less likely to survive due to recombination effects, resulting in decreased ssb yields. In TE buffer solution, the ssb yield induced by gamma-rays is 3.2 +/- 0.66 times that induced by neutrons at the same dose. Since the direct radiation effect is small under these conditions, we can estimate that the previously unknown G for hydroxyl radical production by fission neutrons is 0.088 mumol J-1. For glycerol concentrations that give the solution a hydroxyl radical scavenging capacity similar to that of cellular environments, the ssb yield induced by gamma-rays is about 2.0 +/- 0.24 times that induced by neutrons. Analysis shows that this trend with added scavenger is caused primarily by hydroxyl radical yields.

Application of the premature chromosome condensation assay in simulated partial-body radiation exposures: evaluation of the use of an automated metaphase-finder.

The premature chromosome condensation (PCC) assay has been proposed as a useful and rapid end point for biological dosimetry following accidental high-dose radiation overexposures. A major benefit of the PCC assay is that it does not require cells to divide for evaluation of cytogenetic damage. The PCC assay was performed on isolated human peripheral lymphocytes exposed in vitro to doses from 1 to 9 Gy of 250 kVp x-rays. The dose-response relationships of the frequency distribution and the yield of PCC fragments in cells were determined after one day of repair at 37 degrees C. A Qpcc approach, which involves the analysis of the yield of excess PCC fragments in damaged cells, was used to establish a dose-response calibration curve. This method is identical in concept to the Qdr technique introduced by Sasaki for partial-body exposure dose-estimates using asymmetrical chromosome aberrations (i.e., dicentrics and rings) in metaphase spreads of human lymphocytes. A simulated in vitro test of a partial-body exposure to a 6-Gy dose was performed. The results from this test provided dose estimates of 5.3 +/- 0.6, 4.7 +/- 0.6, 5.0 +/- 0.6 and 4.7 +/- 0.8 Gy for the 20, 30, 50 and 75 percent component of 6-Gy irradiated cells, respectively. An automated metaphase-finding system was evaluated for use with the PCC assay. This system helped to locate PCC spreads among the mitotic inducer Chinese hamster ovary (CHO) metaphase spreads, thereby facilitating rapid scoring of samples.(ABSTRACT TRUNCATED AT 250 WORDS)

Correlation of micronucleus and apoptosis assays with reproductive cell death.

The relationship between ionizing radiation-induced cell killing and DNA damage measured by the micronucleus and apoptosis assays was determined in three established cell lines (L929, HL-60, and Chang). Irradiation experiments revealed a dose-dependent increase of micronucleated cells until a certain dose was reached. Above this dose no further increase of the micronucleus frequency was observed, but in HL-60 and Chang cells additional DNA fragmentation was detected by morphological criteria, characteristic of apoptosis. This change was detected at different doses for the three cell lines examined, suggesting the existence of a cell-type-dependent upper limit for the employment of the micronucleus assay. However, the sum of both kinds of cellular DNA damage (e.g. micronucleation and morphological-like apoptosis) led to a significant cell-type-independent correlation with cell survival, even above the dose where micronuclei levels saturated. Therefore, a total cell damage assay, involving the inclusion of micronuclei and morphological-like apoptotic events, should be considered when evaluating the use of a predictor assay for ionizing radiation-induced cell killing, especially in conditions when apoptosis (-like) processes may occur.

Simplified and optimized kinetochore detection: cytogenetic marker for late-G2 cells.

Cytogenetic detection of kinetochore proteins using the CREST antibody coupled with secondary antibodies labeled with different fluorescent probes has been optimized for several in vitro mammalian cell lines. This study investigated selected parameters including the influence of common fixatives (methanol, ethanol, methanol:acetic acid (3:1)), detergents (Triton-X100, Tween), fluorescent probes (CY3, BODIPY, FITC), washing protocols, and an antifading agent (paraphenylenediamine) on the detection of kinetochore proteins in control and X-ray (240 kVp)-irradiated cells. Utilizing an optimized fixation and staining protocol, a brilliant visualization of kinetochores in interphase cells was obtained in control as well as X-ray-irradiated interhase cells. Application of this improved kinetochore staining methodology readily permits discriminating cells containing either single or paired kinetochores, the latter of which are characteristic of late-G2 phase and prophase cells.