Evaluation of the impact of formaldehyde fumigation during the hatching phase on contamination in the hatch cabinet and early performance in broiler chickens.

Commercial hatch cabinet environments promote replication of microorganisms. These pathogenic or apathogenic microorganisms may serve as pioneer colonizers of the gastrointestinal tract (GIT) of poultry. Some of these pioneer colonizers, such as Escherichia coli and Enterococcus spp., are opportunistic pathogens that lead to reduced performance in commercial poultry. Effective hatchery sanitation is imperative to limit contamination of naïve neonatal chicks and poults. Formaldehyde fumigation has been traditionally used to reduce the pathogen load in commercial hatch cabinets. To investigate potential alternatives to formaldehyde fumigation, models to mimic the microbial bloom in a laboratory setting must be utilized. The purpose of the present study was to evaluate the impact of a multispecies environmental challenge model (PM challenge) with and without formaldehyde fumigation during the hatching phase on early performance in broiler chicks. Three experiments were conducted to evaluate microbial contamination in the hatch cabinet environment (air samples, fluff samples), enteric colonization at day-of-hatch (DOH), and 7-day performance. In all experiments, significantly (P < 0.05) more gram-negative bacteria were recovered from the GIT at DOH in the PM challenge control group as compared to the nonchallenged control (NC) group and the formaldehyde-treated group (PM + F). There were no statistical differences in 7-day body weight gain or feed conversion ratio between the PM challenge control group, the NC group or the PM + F group. These data suggest this model could be utilized to evaluate alternatives to formaldehyde fumigation for controlling the microbial load during the hatching phase in a laboratory setting.

Dietary soy galactooligosaccharides affect the performance, intestinal function, and gut microbiota composition of growing chicks.

The objective of this experiment was to investigate the effects of the dietary soy galactooligosaccharides (GOS), raffinose and stachyose, on performance, gastrointestinal health, and systemic stress in young broilers. Birds were fed a GOS-devoid diet based on soy protein isolate (SPI) or the SPI diet with 0.9, 1.8, 2.7, or 3.6% added stachyose and raffinose in a ratio of 4:1 at the expense of corn starch. These 5 treatments were administered to 10 replicate cages of 8 birds. Performance was measured weekly and excreta moisture, N retention, apparent metabolizeable energy, and complete blood cell counts were determined at 14 and 21 d. At 21 d, 2 birds per cage were orally gavaged with fluorescein isothiocyanate-dextran (FITC-d) and serum samples were analyzed for FITC-d as a marker of gut leakage. Additionally, intestinal morphology, crop presumptive lactic acid bacteria (LAB) counts, crop and cecal pH, and cecal microbiota via16S rRNA microbial sequencing were evaluated at 21 d. From 0 to 21 d, feed intake increased linearly (P < 0.01) as dietary GOS increased, whereas BWG increased (P < 0.05) quadratically. Feed conversion ratio increased (P < 0.01) linearly as GOS increased. There were linear increases (P < 0.05) in excreta moisture as dietary GOS increased at 14 and 21 d, as well as dose-dependent responses (P < 0.05) in N retention, AME, and AMEn. There was a quadratic increase (P < 0.05) in crop LAB recovery and a linear decrease (P < 0.01) in ceca pH as GOS increased. At 14 d, a linear increase (P < 0.05) in blood heterophil to lymphocyte ratio was observed as dietary GOS increased. Serum concentrations of FITC-d increased quadratically (P < 0.01) to dietary GOS. Increasing levels of GOS influenced alpha and beta diversities and composition of gut microbiota, including the abundance of Ruminococcus and Bifidobacterium. Results from this trial indicate that soy-derived GOS exert dose-dependent effects on nutrient utilization and intestinal health in young broilers.

Development of an environmental contamination model to simulate the microbial bloom that occurs in commercial hatch cabinets.

Microbial blooms that emerge in commercial hatch cabinets consist of apathogenic and pathogenic microorganisms, including Escherichia coli, Enterococcus faecalis, and Aspergillus fumigatus. Objectives of the present study included the development of a multipathogen contamination model to mimic commercial conditions and optimization of sampling methods to quantify bacterial or fungal presence within the hatch cabinet. The pathogen challenge mix (PM) was recreated from select bacterial or fungal isolates recovered from an egg homogenate (EH) derived from the contents of infertile eggs and late embryonic mortalities. Isolates selected for PM included Enterococcus faecalis (∼108 CFU/egg), Staphylococcus aureus (∼107 CFU/egg), Staphylococcus chromogenes (∼107 CFU/egg), Aspergillus fumigatus (∼106 spores/egg), and 2 Escherichia coli (∼108 CFU/egg) isolates. Challenge (100 µL of PM or EH) was administered using a sterile loop to a 28 mm area on the blunt end of the eggshell at day 19 of embryogenesis (DOE). In 3 experiments, microbiological data were collected from environmental hatcher samples (open-agar plate method), fluff samples, postmortem whole-body chick rinse samples, and gastrointestinal tract (GIT) samples to evaluate select bacteria and fungi circulating within the hatch cabinet and colonization of GIT. Cumulative bacterial and fungal recovery from the PM hatching environment from DOE20 to hatch was higher than the nonchallenged group (NC) and EH group at ∼860 and ∼1,730 CFU, respectively. Bacterial recovery from GIT, fluff, and chick rinse samples were similar for the PM and EH group in Exp. 1. However, Aspergillus fumigatus recovery from fluff and chick rinse samples for the PM group was significantly (P < 0.001) higher than the NC and EH group. In Exp. 2 and 3, PM challenge significantly (P < 0.05) increased Gram-negative bacterial recovery from the GIT, fluff and chick rinse samples compared to both the NC and EH group. These data suggest this innovative multispecies environmental contamination model using PM could be utilized to evaluate strategies to mitigate microbial contamination in commercial hatch cabinets in a laboratory setting.

Research Note: Application of an Escherichia coli spray challenge model for neonatal broiler chickens.

Avian pathogenic Escherichia coli (E. coli) is an opportunistic pathogen often introduced to neonatal chicks during the hatching process. This commensal bacterium, particularly as a pioneer colonizer of the gastrointestinal tract, can have substantial implications in the rearing of poultry because of reduced flock performance. In order to mimic the effects of the natural bacterial bloom present during the hatch, a seeder challenge model was developed to expose neonatal chicks to virulent E. coli. On day 20 of embryogenesis, selected early hatched chicks (n = 18/hatcher) were briefly removed and sprayed challenged with saline (vehicle) or E. coli at 1 × 107 colony-forming unit (CFU)/chick (exp 1) and 2.5 × 107 CFU/chick (exp 2). These challenged chicks were returned to the hatcher to serve as seeders to transmit the pathogen to the indirect challenged or contact chicks (n = 195/hatcher). For two 7-d experiments, the efficacy of transmission was evaluated via enteric bacterial recovery, body weight gain (BWG), and mortality. For exp 1 and exp 2, significantly (P < 0.0001) more gram-negative bacteria were recovered from the seeder and contact gastrointestinal samples than the negative control samples on day of hatch. In addition, there was a reduction (P < 0.05) in 7-d BWG and significantly (P < 0.0001) higher mortality in the contact-challenged chicks than the negative control chicks in both exp 1 and exp 2. These data suggest that this challenge model could be used to evaluate different methods of controlling the bacterial bloom that occurs in the hatching environment.

Development of a wild-type Escherichia coli environmental bloom model to evaluate alternatives to formaldehyde fumigation in broiler chicken hatch cabinets.

Horizontal transmission of opportunistic Escherichia coli during hatch can have detrimental effects on early performance, particularly as pioneer colonizers. Commercially, formaldehyde is often applied in the United States to combat the bacterial bloom that occurs inside of the hatching environment. The purpose of these experiments was to develop a replicable E. coli horizontal challenge model to evaluate alternatives to formaldehyde sanitation applied to the hatching environment. In experiment 1, two trials were conducted for 2 wild-type (WT) E. coli isolates (isolate 1 [I1] or isolate 2 [I2]) to determine the appropriate in ovo challenge dose and day of embryogenesis (DOE) for challenge administration. In experiment 1 trial 1, the most appropriate inoculation dose and time point were determined to be 102 cfu/embryo on DOE 19. Experiment 1 trial 2 evaluated whether placement of seeder (direct-challenged) embryos with contact (indirect-challenged) embryos during hatch affected contact hatchability. Trial 2 showed no differences in hatchability between groups. A 7-day experiment (experiment 1 trial 2) was conducted to evaluate the effects of I1 or I2 on horizontal transmission, gram-negative bacterial (GNB) recovery from the gastrointestinal tract (GIT), and impact on BW gain (BWG). Compared with the negative control, seeder, and contact chicks challenged with I1 or I2, we observed increased (P < 0.05) GNB recovered from GIT on the day of hatch. There was a marked (P < 0.05) reduction in 7-day BWG between the I1 indirect-challenged group and the negative control group. To further validate the model, 2 7-day trials (experiment 2, experiment 3) were conducted to evaluate the effects of formaldehyde fumigation on coliform recovery from the hatching environment and on early performance using I1 for the challenge. Isolate 1 positive control hatchers had increased levels of circulating coliforms compared with the negative control and formaldehyde-treated hatchers, although there was no significant impact on performance induced by challenge or formaldehyde treatment in experiment 2 or experiment 3. These data provide a potential model for investigations related to horizontal transmission of WT E. coli at a low dose on DOE 19 to promote simulated commercially relevant bacterial blooms under laboratory conditions.

Development of a novel in ovo challenge model for virulent Escherichia coli strains.

During the hatching process, chicks are exposed to opportunistic and/or pathogenic organisms, such as virulent or avirulent Escherichia coli. Virulent E. coli strains have not been feasible for induction of neonatal colibacillosis via in ovo challenge due to high embryonic mortality. In this manuscript, we describe the addition and co-administration of the bacteriostatic antibiotic tetracycline to a virulent E. coli challenge culture, improving hatchability and livability of seeder chicks while allowing robust horizontal transmission in the hatching cabinet to contact chicks. Experiment 1 consisted of 3 trials. Experiment 1, trial 1 was conducted to determine an effective ratio of E. coli challenge and tetracycline dose to be utilized in the seeder model. Trials 2 and 3 were conducted to evaluate the transmission of E. coli from seeder to contact chicks. Experiment 2 consisted of 3 independent 7-D trials where body weight gain (BWG), mortality, and selected enteric bacterial recovery were evaluated. In trials 1 to 3, significantly (P < 0.05) more Gram-negative bacteria were recovered from whole gut samples (GIT) vs. negative controls on day of hatch, from both seeder and contact chicks. At day 7 in trial 1, contact chicks had significantly (P < 0.05) more Gram-negative bacteria recovered from the GIT than the negative control, but not in trials 2 and 3. Presumptive lactic acid bacterial recovery was elevated in contact and seeder chicks compared to the negative control in all 3 trials. Contact challenge caused a significant (P < 0.05) reduction in BWG in 2 out of 3 trials at day 7, and there was a significant (P < 0.05) increase in mortality as compared to the negative controls in all trials. These data suggest that co-administration of a virulent E. coli strain with tetracycline allows for hatch of direct challenged chicks and effective horizontal transmission to contact chicks during the hatching process, as evidenced by reduced day 7 performance and altered selected enteric bacterial recovery.

Simvastatin and ML141 Decrease Intracellular Streptococcus pyogenes Infection.

BACKGROUND: Recurrent pharyngotonsillitis due to Streptococcus pyogenes develops regardless of whether infecting strains are resistant or susceptible to first-line antimicrobials. Causation for recurrent infection is associated with the use of first-line antimicrobials that fail to penetrate deep tissue and host cell membranes, enabling intracellular S. pyogenes to survive throughout repeated rounds of antimicrobial therapy. OBJECTIVE: To determine whether simvastatin, a therapeutic approved for use in the treatment of hypercholesterolemia, and ML141, a first-in-class small molecule inhibitor with specificity for human CDC42, limit host cell invasion by S. pyogenes. METHODS: Assays to assess host cell invasion, bactericidal activity, host cell viability, actin depolymerization, and fibronectin binding were performed using the RAW 267.4 macrophage cell line and Human Umbilical Vein Endothelial Cells (HUVEC) infected with S. pyogenes (90-226) and treated with simvastatin, ML141, structural analogs of ML141, or vehicle control. RESULTS: Simvastatin and ML141 decreased intracellular infection by S. pyogenes in a dose-dependent manner. Inhibition by simvastatin persisted following 1 h washout whereas inhibition by ML141 was reversed. During S. pyogenes infection, actin stress fibers depolymerized in vehicle control treated cells, yet remained intact in simvastatin and in ML141 treated cells. Consistent with the previous characterization of ML141, simvastatin decreased host cell binding to fibronectin. Structural analogs of ML141, designated as the RSM series, decreased intracellular infection through non-cytotoxic, nonbactericidal mechanisms. CONCLUSION: Our findings demonstrate the potential of repurposing simvastatin and of developing CDC42-targeted therapeutics for eradicating intracellular S. pyogenes infection to break the cycle of recurrent infection through a host-directed approach.

Characterization and evaluation of lactic acid bacteria candidates for intestinal epithelial permeability and Salmonella Typhimurium colonization in neonatal turkey poults.

The present study evaluated the microbiological properties of three probiotic candidate strains of lactic acid bacteria (LAB) (128; 131; CE11_2), their effect on intestinal epithelial permeability, and their ability to reduce intestinal colonization of Salmonella Typhimurium (ST) individually or as a batch culture in neonatal turkey poults. Isolates were characterized morphologically and identified using 16S rRNA sequence analyses. Each isolate was evaluated for tolerance and resistance to acidic pH, high osmotic NaCl concentrations, and bile salts in broth medium. In vitro assessment of antimicrobial activity against different enteropathogenic bacteria was determined using an overlay technique. In vitro intestinal permeability was evaluated using a stressed Caco-2 cell culture assay treated with/without the probiotic candidates. The in vivo effect of the selected LAB strains on ST cecal colonization was determined in two independent trials with neonatal turkey poults. The results obtained in this study demonstrate the tolerance of LAB candidates to pH 3, a NaCl concentration of 6.5%, and high bile salts (0.6%). All strains evaluated exhibited in vitro antibacterial activity against Salmonella Enteritidis, ST, and Campylobacter jejuni. Candidates 128 and 131 exhibited a coccus morphology and were identified as Enterococcus faecium, and bacterial strain CE11_2 exhibited clusters of cocci-shaped cells and was identified as Pediococcus parvulus. All three candidate probiotics significantly (P < 0.05) increased transepithelial electrical resistance (TEER) in Caco-2 cells following a 3-h incubation period with hydrogen peroxide compared to control and blank groups. The combination of all three candidates as a batch culture exhibited significant efficacy in controlling intestinal colonization of ST in neonatal turkey poults. Evaluation of the combination of these selected LAB strains according to performance and intestinal health parameters of chickens and turkeys are currently in process.

In ovo evaluation of FloraMax®-B11 on Marek's disease HVT vaccine protective efficacy, hatchability, microbiota composition, morphometric analysis, and Salmonella enteritidis infection in broiler chickens.

Three experiments were conducted to evaluate the effect of in ovo administration of FloraMax®-B11 (FM) on Marek's disease (MD) herpesvirus of turkeys (HVT) vaccine protective efficacy, hatchability, microbiota composition, morphometric analysis, and Salmonella enteritidis (SE) infection in chickens. Experiment 1 consisted of 3 trials. In trials 1 and 2, d 18 White Leghorn 15I5x71 embryos were randomly distributed in 4 groups: 1) HVT vaccinated in ovo and no Marek's disease virus (MDV) challenge; 2), HVT + FM vaccinated in ovo and no MDV challenge; 3) HVT vaccinated in ovo and challenge with virulent MDV (vMDV; strain 583A); and 4), HVT + FM vaccinated in ovo and challenge with vMDV. Trial 3 was designed exactly the same as Experiment 1 but chicks were challenged with very virulent MDV (vvMDV; strains Md5 and 612). Birds were monitored until 8 wk of age, and tested for MD incidence. Experiment 2 consisted of 3 trials. In each trial, d 18 broiler embryos were injected in ovo with either saline or FM to measure hatchability and gastrointestinal bacterial composition. In Experiment 3, d 18 broiler embryos were injected in ovo with either saline or FM. All chickens that hatched were orally gavaged with SE at hatch and kept for 7 d to monitor post-hatch BW. No significant difference (P > 0.05) between MD percentage in birds vaccinated with HVT alone or HVT + FM were observed in Experiment 1. In Experiment 2, probiotic did not negatively affect hatchability, but did reduce lactose positive Gram-negative bacteria. Further, increase in BW was associated with higher villi surface area in the ileum in chickens that received the probiotic as well as a significant reduction in the SE incidence in Experiment 3. These results suggest that in ovo administration of FM does not negatively impact the ability of HVT to protect against MD or hatchability of chickens, but improves BW during the first 7 d of life and decreases SE recovery in chickens.

cisRED: a database system for genome-scale computational discovery of regulatory elements.

We describe cisRED, a database for conserved regulatory elements that are identified and ranked by a genome-scale computational system (www.cisred.org). The database and high-throughput predictive pipeline are designed to address diverse target genomes in the context of rapidly evolving data resources and tools. Motifs are predicted in promoter regions using multiple discovery methods applied to sequence sets that include corresponding sequence regions from vertebrates. We estimate motif significance by applying discovery and post-processing methods to randomized sequence sets that are adaptively derived from target sequence sets, retain motifs with p-values below a threshold and identify groups of similar motifs and co-occurring motif patterns. The database offers information on atomic motifs, motif groups and patterns. It is web-accessible, and can be queried directly, downloaded or installed locally.

Treatment of lentigo maligna with topical imiquimod.

A published case report and anecdotal experience suggested that topical imiquimod is an effective treatment for stage 0 melanoma (lentigo maligna). To gauge the efficacy of this therapy, we undertook a trial of topical imiquimod in 30 subjects with histologically confirmed lentigo maligna. Thirty subjects with lentigo maligna were recruited for an open-labelled efficacy trial with daily topical application of imiquimod 5% cream for 3 months. Study subjects were enrolled from the Dermatology service of the University of Oklahoma, the Oklahoma City Veteran's Administration Hospital Dermatology service and from referrals for the study from other practitioners. In order to determine an initial response rate, a four-quadrant biopsy was carried out on all patients 1 month after cessation of treatment, targeting the most clinically and dermatoscopically suspicious areas. Of 28 evaluable subjects who have completed the 3-month treatment phase, 26 (93%) were complete responders and two were treatment failures at the time of the 4-quadrant biopsy. Over 80% of the 28 subjects that completed treatment have been followed for more than 1 year with no relapses. The results of this study demonstrate that topical imiquimod produces a high complete response rate in lentigo maligna when applied daily for 3 months.

Radiologic placement of central venous catheters: rates of success and immediate complications in 3412 cases.

OBJECTIVE: To determine the success and immediate complication rates associated with 3412 central venous catheter placements performed in an interventional radiology suite. METHODS: Success and immediate complication rates were prospectively recorded for 3412 consecutive patients who had central venous catheters radiologically placed at a tertiary care centre between July 1993 and October 2000. The indication for placement and the insertion site were also recorded. RESULTS: The most common indication for both short- and long-term venous access was hemodialysis, and the right internal jugular vein was the most common site for catheter insertion. Placement was successful for 98.8% of tunnelled lines and 99.3% of temporary catheters. The rate for immediate complications (including pneumothorax, air emboli, bleeding and arterial puncture) for tunnelled catheter placements was 3.8% and for temporary catheter placements was 1.6%; no major complications were documented. CONCLUSION: Our results lend further evidence to the claim that the success and immediate complication rates of radiologically placed central venous catheters compare favourably with blind placement and surgical placement of central venous catheters.

Recurrent fetal cystic hygroma with normal chromosomes: case report and review of the literature.

Recurrence of fetal cystic hygroma in subsequent pregnancies is extremely rare. A review of the literature to date revealed only two other reports of recurrence with normal fetal karyotypes documented in at least two of the affected pregnancies. At 11 weeks' gestation, the fetus of a 19-year-old gravida 3 para 0 was discovered to have a large cystic hygroma. Subsequent evaluation during the second trimester revealed increasing size of the septated nuchal mass and ascites. A 46,XX fetal karyotype was noted in her two prior pregnancies, both of which had also been complicated by the development of cystic hygroma and nonimmune hydrops. Cystic hygroma, associated with a normal karyotype, can be inherited as an autosomal recessive trait.

Successful pregnancy outcome in a woman with a gain-of-function mutation of the calcium-sensing receptor. A case report.

BACKGROUND: Gain-of-function mutations of the calcium-sensing receptor gene have recently been identified as a cause of familial hypercalciuric hypocalcemia. There have been no earlier reported cases of pregnancy among patients with this disorder. CASE: A 26-year-old woman, gravida 1, para 0, was diagnosed at age 18 as being a heterozygous carrier of a mutation in the calcium-sensing receptor gene. Stable maternal hypocalcemia was achieved during pregnancy with high-dose calcium and 1,25-dihydroxyvitamin D3 therapy. Prenatal diagnosis was accomplished via amniocentesis at 16 weeks' gestation. The patient underwent cesarean delivery at 35 5/7 weeks' gestation after developing the HELLP syndrome. CONCLUSION: Patients with mutations of the calcium-sensing receptor may have a successful pregnancy outcome. This abnormality may be transmitted to the fetus via an autosomal dominant pattern.

Chromosome duplications and deletions and their mechanisms of origin.

Duplications and deletions of the same gene loci or chromosome regions are known to produce different clinical manifestations and are significant factors in human morbidity and mortality. Extensive cytogenetic and molecular cytogenetic studies with cosmid and YAC probes in two patients with unique mosaicism for reciprocal duplication-deletion allowed us to further understand the origin of these abnormalities. The first patient's mosaic karyotype was 46,XX, inv dup(11) (q23q13)/46,XX,del(11)(q13q23). The second patient had a 46,XY,dup(7)(p11.2p13)/46,XY,del(7)(p11.2p13)/46,XY karyotype. Fluorescence in situ hybridization studies on the first patient placed the two breakpoints near the folate-sensitive fragile sites FRA11A and FRA11B. The presence of repeated sequences responsible for these fragile sites may have been involved in the patient's duplication-deletion. Our investigation leads us to conclude that, in addition to known mechanisms (such as unequal crossovers between homologs, unequal sister chromatid exchanges, excision of intrachromatid loops, and meiotic recombination within a single chromatid), duplication-deletion can also arise by the formation of an overlying loop followed by an uneven crossover at the level of the DNA strand.

T-cell survival.

Like other cells, T cells are dependent on signals from their environment for their survival. Resting T cells are supported in vitro by cytokines such as interleukin (IL)-4, IL-6 and IL-7. The latter two cytokines are made constitutively in animals and hence might affect the lifetimes of their resting T cells. Resting T cells are also kept alive by interaction with an as yet unidentified molecule on the surface of other cells. Activated T cells are also supported in vitro by members of two families of these proteins, the IL-2 family and the interferon-alpha beta family. Members of the latter family may have effects on activated cells in vivo. Thus although both resting and activated T cells require signals to keep themselves alive, the signals are different for the two types of cells. This perhaps allows the immune response to control the numbers of activated cells during infections without compromising its pool of precursor, resting T cells.

Pregnancy complicated by multiple endocrine neoplasia type IIA (Sipple's syndrome).

A patient with preexisting multiple endocrine neoplasia type IIA had normal 24-hour urinary metanephrine and vanillylmandelic acid excretions before and during pregnancy. After a benign prenatal course, the patient had a term spontaneous vaginal delivery. Multiple endocrine neoplasia type IIA antedating pregnancy may be associated with a normal obstetric outcome in the absence of a phenochromocytoma.

Teaching efficacy of a medical education module on genetic testing for cancer.

BACKGROUND: With the identification of the breast cancer susceptibility genes BRCA1 and BRCA2, clinical testing for detection of the mutated genes may be available in the near future. Primary care physicians increasingly serve as full-service providers and gatekeepers and must be aware of presymptomatic testing in order to counsel their patients appropriately. To address this educational need, a new module was incorporated into the genetics course taken by first-year medical students at the Medical College of Virginia. METHODS: The module used small groups, led by genetics faculty and members of the Virginia Breast Cancer Foundation, for discussion of case examples. The medical students' knowledge of and attitudes toward cancer and predictive genetic testing were assessed by a pretest and a posttest. RESULTS: After the module, knowledge scores increased by 27%, and significant changes were seen in the students' attitudes toward issues such as the regulation of testing availability and the psychological effect of testing. Most students consistently felt that predictive genetic testing is beneficial, that they would have the testing themselves, that genetic counseling should be required for testing, and that insurers' access to genetic testing results should be limited. Overall, the module was received favorably by all participants. CONCLUSIONS: Small-group discussion of relevant case examples increases knowledge and awareness of issues regarding presymptomatic genetic testing for breast cancer.

DNA and RNA content analysis by flow cytometry in the pathobiologic assessment of bone tumors.

Studies of simultaneous DNA and RNA contents by flow cytometry in hematologic and some solid neoplasms have been shown to provide information that may be useful in the pathobiological evaluation of these neoplasms. We contend that similar analysis may be equally valuable in assessing bone tumors. Our data revealed significant statistical differences in DNA ploidy and proliferative fraction between benign and malignant bone neoplasms. Benign tumors manifested predominantly DNA diploidy and low proliferative activity, whereas the majority of malignant tumors were DNA aneuploid and showed high proliferation rate. No significant difference in the RNA content between different histopathologic categories was found. We observed, however, a distinct and consistently high RNA content pattern in giant cell tumors, aneurysmal bone cysts, and chondroblastomas that may be useful in their differential diagnosis. Analysis of different prognostic factors in malignant tumors indicated that histologic grade and DNA content are a significant prognostic factors. Further analysis of malignant tumors showed that a correlation between the proliferative activity and the clinical outcome in the low grade category and between RNA content and patients' survival in osteosarcomas. Our study also showed that preoperative treatment significantly impacted on the extent of the proliferative fraction in malignant tumors. We conclude that DNA/RNA analysis of bone tumor may assist in: (1) the differential diagnosis of certain bone tumors, (2) evaluation of treatment response, and (3) the biological assessment of osteosarcomas.