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Abstracts of Presentations at the Association of Clinical Scientists’ 123rd Meeting, in St. Petersburg, Florida, on 7 to 11 May 2003

The congenital myopathies are an heterogeneous group of primary muscle diseases characterized by unique pathologic changes, early onset, slow progression, and genetic predisposition. Definitive diagnosis requires muscle biopsy, often with enzyme histochemistry, electron microscopy, and biochemical analysis. In nemaline myopathy there is a subsarcolemmal accumulation of rod-like inclusions that are seen ultra-structurally as clumps of abnormal Z-band material. "Ragged-red" fibers are present in modified Gomori trichrome-stained sections of mitochondrial myopathies, and abnormal mitro-chondria with paracrystalline inclusion are demonstrated by electron microscopy. Other myopathies with characteristic features include central core disease, centro-nuclear myopathy, myotubular myopathy, and fiber type disproportion. Muscle biopsy is necessary to distinguish congenital myopathies from other pediatric neuromuscular disorders such as muscular dystrophy, glycogen storage disease, inflammatory myopathies, and neuropathic disorders. Making an exact diagnosis is critically important for planning treatment, assessing the prognosis, and genetic counseling.

[2] The role of the cytoskeleton in dilated cardiomyopathy. Neil E. Bowles, Baylor College of Medicine, Houston, TX.

In this presentation, data are described that highlight the importance of the integrity of the cytoskeleton in cardiac function. These include the identification of gene defects in patients with inherited cardiomyopathies and the observation of reverse remodeling of dystrophin in patients undergoing assist-device therapy for heart failure. Congestive heart failure, a significant cause of morbidity and mortality worldwide, is characterized by poor prognosis, increasing incidence, and high economic cost. Regardless of the underlying myocardial insult, progressive myocardial dysfunction occurs at a time that is distant from the initial injury; it is typically characterized by cardiac enlargement and decreased contractile function. Dystrophin was identified as the gene responsible for cardiomyopathy in patients with X-linked cardiomyopathy (XLCM). Typically, mutations in the N-terminus of dystrophin have been reported in cases of XLCM. Dystrophin is thought to provide structural support for the myocyte and cardiomyocyte membrane. It serves as a link between actin at the N-terminus, the dystrophin-associated protein complex (DAPC), the sarcolemma at the C-terminus, and the extracellular matrix of muscle. Importantly, mutations in dystrophin or DAPC subcomplexes result in a wide spectrum of skeletal myopathy and/or cardiomyopathy in humans and animal models in mice and hamsters. Because of the structural importance of dystrophin in cardiac function, we hypothesized that acquired abnormalities of the integrity of dystrophin and other components of the cyto-skeleton may provide a "final common pathway" for the progressive dysfunction observed in heart failure, irrespective of etiology. Over the past five years, a number of genetic defects have been identified in components of the cytoarchitecture in patients with dystrophin cardiomyopathy (DCM). Further, we have observed reversible changes in dystrophin in patients with end-stage cardiomyopathies.

[3] Clinical diagnosis and evaluation of heart failure. Richard V. Milani, Ochsner Clinic Foundation, New Orleans, LA.

As newer therapeutic avenues become available for treating heart failure, the time has never been more important to develop cohesive strategies for the early and more precise diagnosis of this entity. Generically, heart failure is a syndrome characterized by cardiac dysfunction that leads to inability of the heart to meet the metabolic demands of the tissues. However, this definition clinically steers us in the wrong direction, since hemodynamically directed therapy has been associated with worse survival. On the other hand, appropriate clinical diagnosis of heart failure must center not only on detection of the underlying entity, but also on the clinical expression exemplified by decreased effort tolerance, propensity for increased ventricular arrhythmias, and reduced life expectancy. Several newer modes of diagnostic evaluation are now available. These include dynamic echocardiography, metabolic stress testing, and proteomic biomarkers. In this regard, a proteomic marker, B-type natriuretic peptide (BNP), an index of ventricular wall stress, is gaining wide acceptance. This bio-marker has been shown not only to correlate with severity of ventricular dysfunction, but also to aid in the differential diagnosis of heart failure. Metabolic stress testing offers an exciting and accurate avenue to predict disease severity and also to assist in the differential diagnosis of conditions that mimic heart failure. Newer advances in specialized techniques of echocardiography using dynamic cardiac maneuvers are allowing physiological stratification of disease risk. Despite the recognition of familial contributions to the etiology of cardio myopathy, genetic testing is an academic exercise at this time.

[4] Correlation of B-type natriuretic peptide and left ventricular function, determined by echocardiography. Matthew Glover and Anthony J. Girgenti, St. Joseph’s Hospital, Tampa, FL.

Retrospective review was performed of 125 consecutive medical records of inpatients for whom blood B-type natriuretic peptide (BNP) was assayed within 48 hr of echocardiography. The subjects were 70 women and 55 men. The age of all patients was 33 to 96 yr (mean ± SD, 69.1 ± 14.8 yr). A cardiologist interpreted each echocardiogram without prior knowledge of the corresponding BNP result. Blood BNP concentration (mean ± SE) for these unselected hospitalized patients whose echocardiograms were judged to be normal for systolic and diastolic function averaged 367 ± 48 pg/ml (n = 68). Patients with abnormal systolic function and normal diastolic function had significantly higher (p <0.01) blood BNP concentrations, 765 ± 76 pg/ml (n = 28). The highest mean values for BNP were obtained for the patients with abnormal systolic and diastolic function of the restrictive type (1012 ± 138 pg/ml, n = 9). Intermediate mean concentrations of BNP were obtained for those patients with normal systolic function and pseudonormal diastolic dysfunction (544 ± 289 pg/ml, n = 4), or restrictive diastolic dysfunction (513 ± 123 pg/ml, n = 6). Patients with normal systolic function and diastolic dysfunction of the impaired relaxation type had BNP’s with a mean of 275 ± 151 pg/ml (n = 9). In conclusion, BNP correlates with left ventricular function as determined by echocardiography. However, BNP concentrations may be higher than expected in hospitalized elderly patients due to confounding physiologic variables. BNP is considered a valuable adjunct for the diagnosis of congestive heart failure and should be used in combination with established diagnostic procedures and sound clinical judgement.

[5] B-type natriuertic peptide (BNP) as a biomarker of myocardial failure. Philip R. Foulis, Pathology and Laboratory Medicine, Tampa Veterans Administration Hospital and University of South Florida, Tampa, FL.

The Central Florida Healthcare System comprises a 307-bed acute-care hospital, two nursing home-care units, three multi-specialty outpatient clinics with comprehensive ancillary support, and five community-based outpatient clinics. The Cardiology Service requested BNP assays to assess patients who presented to the emergency room. BNP is a neurohormone secreted from the cardiac ventricles in response to volume expansion and pressure overload; BNP assay is able to differentiate congestive heart failure (CHF) from other causes of dyspnea. This retrospective study describes the experience with BNP utilization at our facility. During one year (February 25, 2002, to February 25, 2003), our laboratory processed 1985 BNP assays from 1237 patients. Males were 96.7% of the population. The ethnicity of the population was: Caucasian (58.4%), African-American (5.3%), unknown (31.9%), and other (4.6%). During the study, 12.9% of the population died. There were 3155 admissions, with the greatest number to the medical service (39%); 31.2% were admitted to medical cardiology or vascular surgical services. There were 126,158 outpatient visits. CHF was the diagnosis in 5.5%, coronary artery disease in 9.4%, and dysrhythmias in 4.8%. Requests for BNP assays came from outpatient locations (49.9%), the coronary care unit (16.2%), other hospital locations (24.1%), and emergency room (9.8%). The majority of BNP assays (77.6%) were accompanied by requests for other tests. The associated panels and/or tests included the basic metabolic panel (36.9%), comprehensive metabolic panel (23.9%), urinalysis (9.4%), CBC (46.9%), coagulation testing (17.5%), and troponin assay (17.7%). Assay of BNP, which was initially implemented for triage of patients in the emergency room, has expanded its utilization to the evaluation and follow-up of patients in all inpatient and outpatient settings.

[6] Laboratory evaluation of the B-type natriuretic peptide (BNP) test for left ventricular dysfunction. Cynthia Schandl, Beverly Horne, and Christine Papadea, Medical University of South Carolina, Charleston, SC.

A major advance in the diagnosis of congestive heart failure (CHF) has been the discovery of BNP, a hormone secreted from the left ventricle in response to excess tension. BNP measurements are used to monitor CHF therapy and to aid in the diagnosis of symptomatic and asymptomatic patients. We evaluated the analytical performance of a rapid test for measuring BNP in plasma by a fluorescent immunoassay device (Triage, Biosite Diagnostics). We also examined the correlation of BNP values with clinical diagnosis by retrospective review of 150 patient charts. The BNP testing is performed by a 25-min immunoassay; after EDTA-anticoagulated plasma (0.25 ml) is introduced into a single-use cartridge, BNP molecules bind with fluorescence-labeled antibodies. The cartridge is inserted into a miniature fluorescence reader that displays the result; the suggested cutoff is 100 pg/ml. Analytical evaluation consisted of 4 studies: (a) within- and between-day reproducibility (CV) of bi-level controls; (b) accuracy verification using samples of known concentrations (31 to 3,890 pg/ml); (c) reportable range verification using patient sample dilutions; and (d) tests for lipemia interference. The CV’s were 8% (within-day) and 16% (between-day). Results of accuracy samples processed by linear regression analysis produced the equation: measured BNP = -18 + 0.928 (assigned BNP), r = 0.99. The reportable range was 20 to 4000 pg/ml. Elevated triglyceride levels (680 mg/dl) did not affect BNP results. Correlation of BNP results with clinical diagnoses revealed the BNP test has 77% positive predictive value and 76% negative predictive value. In conclusion, the BNP test has performance and clinical correlation (predictive values) features that are characteristic of a screening test. The 4-fold increase in testing volume (currently 320/month) that has occured during the 10 mo since the BNP test was implemented indicates that the assay has been well received by our physicians as an aid in diagnosis and management.

[7] BNP versus NTproBNP in patients with congestive heart failure. Alan H.B. Wu, Department of Pathology and Laboratory Medicine, Hartford Hospital, Hartford, CT.

B-type natriuretic peptide is released from cardiac myocytes in response to volume overload and ventricular stretching. It originates from preproBNP (134 amino acids, aa), which is cleaved to proBNP (108 aa plus a 25 aa signal peptide). Both BNP (32 aa) and proBNP (108 aa) are released into blood (as monomer and trimer). Assays have been developed for BNP on a point-of-care platform (POCT) (Triage, Biosite Diagnostics), central laboratory immunochemistry analyzer (Bayer Centaur, prototype assay), and radioimmunoassay (Shionoria), and for NTproBNP (Roche Elecsys). While both markers are approved for use in congestive heart failure (CHF), there are subtle differences in the biochemistry and clinical performance of these tests. BNP has a biological half-life of 20 min in blood compared to 1–2 hr for NTproBNP. This results in some differences in intra-individual biological variability (40–60% for BNP vs 30% for NTproBNP). The critical difference required for serial results to change significantly is 130% vs 90%, respectively. This difference may have an impact if these peptides are used for monitoring the efficacy of CHF therapy. Due to cross-reactivity, BNP cannot be used during infusion with nesiritide (recombinant BNP). However, the short half-life of BNP enables monitoring of residual BNP secretion soon after the end of drug infusion. BNP is stable for 4 hr at room temperature (Biosite) and 24 hr at 2–8°C and it requires collection in EDTA tubes. NTproBNP is stable for up to 3 days at 2–8°C and can be measured in serum or plasma. As expected, precision data of the central laboratory BNP and NTproBNP tests are superior to those of the POCT. However, high precision is unnecessary for tests that have high intra-individual biological variation. Many clinical studies have been published concerning the use of BNP for diagnosis, management, screening, and prognosis. Time will reveal if these clinical indications will prove to be valid for NTproBNP as well.

[8] Organic acid disorders. Piero Rinaldo, Department of Laboratory Medicine, Mayo Clinic, Rochester, MN.

Organic acids are intermediate metabolites of amino acids, lipids, carbohydrates, nucleic acids, and steroids. With increasing demand in clinical practice, organic acids are analyzed in urine to seek a biochemical diagnosis of a highly heterozygous group of inborn errors of metabolism, the so-called organic acidemias. Informative results include an accumulation of either metabolites that are not present under physiological conditions, or pathological amounts of normal metabolites. The incidence of individual inborn errors of organic acid metabolism varies from 1:10,000 to >1:1,000,000 live births. Collectively, however, the incidence of conditions where informative organic acid profiles could be detected in urine is much higher, approaching 1:1000 live births. Most of these disorders share a common natural history, which is the occurrence of either acute life-threatening illness in early infancy or unexplained developmental delay. The biochemical diagnosis of individual disorders ultimately relies on urine organic acid analysis by gas chromatography/mass spectrometry. Informative profiles may not always be detected in disorders where the excretion of diagnostic metabolites is a function of the residual enzyme activity, the dietary load of precursors, and the anabolic status of a patient. Interpretation is based on pattern recognition and correlation of positive and negative findings. When abnormal results are detected, a detailed interpretation should be provided, including quantitative results, correlation to available clinical information, elements of differential diagnosis, recommendations for additional testing and in vitro confirmatory studies, name and telephone number of key contacts who may provide these procedures, and a telephone number to call if the referring physician has additional questions. In summary, assay of organic acids in urine is a difficult test to perform, one that requires reliable procedures for sample preparation, chromatographic separation, and an adequate mass spectra library. Clinical and biochemical interpretive skills are essential.

[9] Mitochondrial dysfunction in cardiomyopathies. Egil Fosslien, Department of Pathology, College of Medicine, University of Illinois at Chicago, Chicago, IL.

Many new genes for mitochondrial proteins have recently been sequenced and gene knockout models have been developed to study their function. The objective of this paper was to review the role of such genes in the dysfunction of mitochondrial bioenergetics in cardiomyopathy and cardiac failure. Evidence of dysregulation of mitochondrial function in cardiomyopathies was derived from embryology, studies on cardiac tissues from patients with cardiomyopathy, autopsy findings, animal models, and in vitro studies. Over one third of the cardiac mass consists of mitochondria. The normal adult heart relies on mitochondrial oxidative phosphorylation (OXPHOS) to provide the majority of the adenosine triphosphate (ATP) required for the contractile apparatus of the heart. During experimental hypertensive cardiac hypertrophy, the heart energy metabolism reverts from the adult type, which generates the majority of the ATP from metabolism of fatty acids, to the fetal type that metabolizes glucose and lactate instead. By comparison, genetic mitochondrial dysfunction is caused by inherited or acquired mutations of either mitochondrial (mt) or nuclear (n) deoxyribonucleic acid (DNA). The nDNA-encoded mitochondrial transcription factor A (Tfam, mtTFA) controls mitochondrial replication and transcription. Tfam-null mouse embryos have no mitochondria and fail to develop a heart. Mitochondrial dysfunction enhances the generation of radical oxygen species (ROS) and can damage mtDNA, nDNA, proteins, and lipid membranes. Mice lacking the mitochondrial antioxidant enzyme manganese-superoxide dismutase (Mn-SOD) develop dilated cardiomyopathy. In conclusion, inherited or acquired cardiac mitochondrial dysfunction induces cardiomyopathy and cardiac failure. Mitochondrial palliative therapy consists of coenzyme Q10 and L-acetyl-carnitine. By comparison, cure is only achievable by gene therapy, which repairs aberrant genes by the use of vectors to insert replacement mitochondrial genes into either the mitochondrial or the nuclear genome.

[10] Metabolic and molecular forms of cardiomyopathy in children. Enid Gilbert-Barness, Department of Pathology, Tampa General Hospital, Tampa, FL.

Metabolic cardiomyopathies include amino acid, metabolism, lipid, and mitochondrial disorders, as well as storage diseases. A number of metabolic disorders are associated with both myopathy and cardiomyopathy. These include the glycogen storage diseases: acid maltase deficiency, infantile, childhood and adult onset, and McArdle disease, debrancher and brancher deficiencies. Disorders of lipid metabolism include: systemic carnitine deficiency, carnitine palmitoyl-transferase (CPT), long-chain acyl-CoA dehydrogenase, multiple acyl-CoA dehydrogenase, and multiple acyl-CoA dehydrogenase. Disorders of mitochondrial metabolism include complex I, II, III, IV, and V, multiple respiratory chain defects. These may be either hypertrophic or dilated cardiomyopathy. In addition, cardiomyopathy is frequently a component part of the storage disorders. These include mucopolysaccharidosis, mucolipidosis, Fabry disease, gangliosidosis, and neuronal ceroid lipofuscinosis. Primary hypertrophic cardiomyopathy is caused by mutations in one of the several genes that encode proteins of the cardiac sarcomere. Mutations in different genes each carry a different prognosis and a different risk of sudden death. Myosin binding protein C (MBP-C) and tropomyosin mutations have low penetrance and are mild forms of the disease. Troponin T and ß-myosin mutations carry a worse prognosis. Conduction disorders result in arrhythmias that may be fatal. Histiocytoid cardiomyopathy is usually an autosomal recessive disorder that results in the presence of abnormal Purkinje cells that interfere with normal cardiac conduction. Other conduction defects include arrhythmogenic right ventricular dysplasia (ARVD), congenital heart block, non-compaction of the left ventricle, and long Q-T syndrome (LQTS). The genetic loci for LQTS reside usually in the potassium channel, and, less frequently, in the sodium channel (channelopathies). Although the histological appearance of some of these disorders may be diagnostic, molecular analysis may be necessary to define clearly the particular type of cardiomyopathy.

[11] The impact of automation on laboratory productivity. Stephen M. Mastorides, James A. Haley Veterans Hospital, Tampa, Fl.

This presentation describes the implementation and impact of modular automation in the chemistry laboratory of the James A. Haley Veterans Hospital. As laboratory managers, we have been challenged to develop better systems that improve processes that reduce costs while still providing test results that are accurate, available, and precise. This must be accomplished while laboratory workloads are steadily increasing due to an aging population, but with decreased availability of trained laboratory specialists. The US Bureau of Labor Statistics estimates that 5300 new medical technologist (MT) and licensed medical technician positions will be created annually through 2008. However, the number of MT training programs has dropped 66% during the last decade. Coinciding with the labor shortage is heightened concern about medical errors and improving the procedures and processes that cause them. Many laboratories have turned to automation to relieve workflow problems, reduce errors, and utilize personnel better. However, automated instruments come in several forms, and it is imperative to assess the specific needs of the lab and the requirements for each type of automation before making a choice. The James A. Haley VA Medical Center, opened in 1972, is a 324-bed Level III tertiary care teaching hospital that serves 350,000 veterans in central Florida. The hospital treats >12,000 inpatients annually, with outpatient care anticipated to be >770,000 visits. Our laboratory performs >2.5 million chemistry tests and 0.4 million immunochemistry tests per yr. The goals of automation for our laboratory include (a) increasing efficiency to accommodate increased test volume with present staff, (b) consolidating automated and manual workstations, (c) decreasing the footprint of the laboratory with minimal expenditure, (d) reducing errors by supporting uniformity, and (e) improving post-analytical specimen archiving.

[12] Claude P. Brown Memorial Lecture: Genes and ice cream: the role of cholesterol in development. John M. Opitz, University of Utah, Salt Lake City, UT.

The insights obtained from a study of the RSH (so-called Smith-Lemli-Opitz) syndrome are paradigmatic of the new biomedicine, which continues to respect clinical methods and approaches to the patient but has been immensely expanded and enriched through methods of molecular genetics and developmental biology. Thus, at the same time that the Madison team was defining (clinically) and delineating (genetically) the RSH syndrome, French teratologists (Charles Roux and collaborators) created an animal model of the syndrome with inhibitors of cholesterol synthesis given to the pregnant rat. When it was discovered that the RSH syndrome is a simple Garrodian error of metabolism [defective cholesterol synthesis from its precursor 7-dehydrocholesterol (7DHC) due to deficient activity of 7DHC-reductase], it was astonishingly simple to create not one, but two "knock-out" mouse models through methods of homologous recombination in ES cells. Prenatal diagnosis is easy and reliable; the DHCR7 gene is cloned and sequenced (over 100 known mutations), allowing genetic characterization and carrier detection. Prognosis and treatment success correlate with the initial serum cholesterol level (the lower the serum cholesterol, the worse the prognosis and responsiveness to treatment). High carrier (4%) and gene frequencies (2%) predict a birth prevalence of 1/2500; the actual prevalence (~1/20,000) suggests a >85% prenatal mortality rate; stillborn RSH babies are being found with increasing frequency. Newborn screening is highly reliable and economical. Thus, we now have the methods to address the issues of carrier advantage, prenatal death and development, and treatment approaches.

[13] The role of dietary, adipose tissue, and plasma lipids in atherogenesis in children. Robert E. Olson, University of South Florida, Tampa, FL.

The natural history of atherosclerosis in humans shows that only the first stage of the disease, the fatty spot or streak, occurs in children and this process appears to be insensitive to the serum lipid changes that occur during the childhood period (2–15 yr). The more harmful atheromatous plaque does not appear until after puberty in boys, and much later in girls. The adult pattern of serum lipids with the ratio of LDL/HDL cholesterol of about 2.6 in males compared to 1.8 in females is not established until about age 18 yr. Furthermore, it is clear that the early lesions seen in the arteries of healthy children bear no relationship to the risk of atherosclerosis in later life. Attempts to intervene in childhood to alter serum lipids with low-fat, low-cholesterol diets have been largely ineffective. Furthermore, data show that children respond more poorly to lipid-lowering diets than do adults, and that the emphasis on low-fat diets for children leads some parents to overdo the guidelines and unwittingly push their children into malnutrition. The program adopted by Health Canada on the advice of pediatricians in that country, which is also supported by the European Society of Pediatrics, Gastroenterology and Nutrition, recommends that the fat content of diets for children should be tapered gradually from 40% of energy at 2 yr of age to 30% of energy at the conclusion of linear growth (late adolescence). Apart from serum lipids in childhood, which have negligible effect on atherosclerosis in adulthood, it is becoming clear that obesity in childhood can lead to type II diabetes in adolescence and to increased risk of atherogenesis in adulthood.

[14] Incorporation of tandem mass spectrometry in state newborn screening. Ursula Nawab and Louis Bartoshesky, Thomas Jefferson University, Philadelphia, PA.

With significant advances in analysis and laboratory evaluation of metabolic diseases, our ability to screen and identify affected infants has changed the field of newborn screening. Since screening practices are governed at a state level, there exists a wide range of capacities to adopt, regulate, and follow-up testing with tandem mass spectrometry (MS/MS). To assess the impact on newborn screening programs in the United States of the incorporation of MS/MS, we surveyed newborn screening directors of 50 states through a mail questionnaire. The survey contained 20 questions designed to ascertain each state’s current practices as well as the incorporation of MS/MS, specifically focusing on the changes in infrastructure. Literature searches and review of national databases were performed. All states currently test for congenital hypothyroidism and phenylketonuria (PKU). There exists a wide range of variation beyond these, specific to each state. Within the year, a majority of states are planning to add testing with MS/MS for various metabolic disorders. One-fourth of the states currently perform or offer this screening test. Increase in fees was the major challenge. For most states, the current infrastructure could accommodate the follow-up and counseling. A majority of the states support national data sharing. There would be little change in informed consent, most practicing a parental non-dissent. In conclusion, MS/ MS offers a unique, comprehensive screening test for a variety of metabolic disorders. Public opinion and commercial marketing have sparked controversies regarding its current lack of availability to all infants. The survey respondents believe that it is clinically important and are directing appropriate resources. Though the MS/MS assay would lead to increased expense, states agree it is a necessity and will work to make MS/MS testing available in all 50 states.

[15] Hyperglycemic damage to the central nervous system. John I Malone, University of South Florida, Tampa, FL.

Children with diabetes onset at <5 yr of age have neurocognitive dysfunction attributed to hypoglycemia. Hyperglycemia damages peripheral nerve function but has been ignored as an offending agent for the central nervous system. Hyperglycemia was induced in 4-wk-old Wistar rats (n = 12) by ip administration of Streptozotocin (50 mg/kg). The animals received enough insulin to maintain normal weight gain but sustained hyperglycemia (HbA1c 10.9%) when compared to age-matched controls (HbA1c 5.2%). A third age-matched group of Wistar rats (n = 12) without diabetes received regular insulin (10 U/kg, 3 x each wk), which produced blood glucose levels <60 mg/dl for 3 hr. Each group of animals was provided ad libitum food and water in the same environment for 4 wk. Then each animal was beheaded; the brain was removed and stored in liquid nitrogen, and samples were prepared later for measurements of protein, DNA, cholesterol, and taurine by colorimetric methods. Group means ± SE were compared by ANOVA with Bonferroni T-test, after the group data were normalized by log transformation. The blood HbA1c was higher (10.8 ± 0.41 %) in the diabetic animals than in the control (5.2 ± 0.37) and hypoglycemic (5.3 ± 0.19) animals. The cell size (protein/DNA) was less in the diabetic than in the control and hypoglycemic animals (p <0.03). The cholesterol/DNA content of brains of the diabetic rats was reduced (p <0.02), compared to control and hypoglycemic rats. The neurotransmitter taurine level was also less in the diabetic brains (0.78 ± 0.14) than in control (1.33 ± 0.12) or hypoglycemic (1.35 ± 0.19) animals (p < .002). Taurine deficiency has been reported to reduce brain development in the neonate. Chronic hyperglycemia significantly alters normal brain growth while intermittent hypoglycemia has no measurable effect. The chronic hyperglycemia of diabetes may be a major contributor to the delayed neurocognitive development of young children with diabetes mellitus.

[16] Fetal laryngoscopy and lung biopsy for congenital cystic adenomatoid malformation of the lung. Ruben A. Quintero and Craig S. Kalter, Florida Institute for Fetal Diagnosis and Therapy, Tampa, FL.

The sonographic appearance of laryngeal atresia is characterized by universal hyperechogenic lungs and downward displacement of the fetal diaphragm. We describe a case of massive cystic adenomatoid malformation of the lung, mimicking laryngeal atresia, confirmed by percutaneous direct fetal laryngoscopy and ultrasound-guided fetal lung biopsy. The patient was 19-yr-old, gravida 2, para 0, referred at 25 wk gestation. Ultrasound showed polyhydramnios, hyperechogenic right lung, and left mediastinal deviation. A rim of left lung appeared to be present behind the heart. Amnio-centesis revealed a normal 46, XY karyotype. The differential diagnosis included laryngeal atresia, right main bronchial obstruction, and large congenital cystic adenomatoid malformation of the lung (CCAM). After informed consent, a percutaneous direct fetal laryngoscopy was performed. Under ultrasound guidance, lung biopsies were performed. The trachea and carina appeared patent on endoscopy, without evidence of obstruction or tracheo-esophageal fistula. Lung biopsy confirmed the diagnosis of CCAM. The patient had spontaneous rupture of membranes at 29 wk and delivered a 2232 g male fetus at 32 wk. Computerized tomography scan confirmed bilateral lung involvement with residual normal left lung tissue. Neonatal death occurred on day 10 of life. In conclusion, direct fetal laryngoscopy and ultrasound-guided fetal lung biopsy may be useful in the differential diagnosis of hyperechogenic fetal lungs.

[17] Iron overload: a case report including clinical and postmortem findings. Frank M. Taylor, III, South Florida Baptist Hospital, Plant City, and St. Joseph Hospital, Tampa, FL.

Iron overload implies a surplus of total body iron, which results from iron stores that exceed iron requirements. Since humans lack a physiological mechanism to excrete surplus iron, a state of iron overload can result from (a) inappropriate sustained increase of intestinal iron absorption, (b) recurrent parenteral administration of iron, or (c) a combination of both. The first mechanism, increased iron absorption, has been related to a recently discovered gene (HFE) and its gene product, which greatly enhances iron absorption from the intestine. The second mechanism often relates to chronic disorders of erythropoiesis in which iron supplements and blood transfusions are mainstays of therapy. This presentation reports the case of a 69-yr-old man whose state of iron overload manifested clinical features that were consistent with acquired hemochromatosis. While the dominant source of the patient’s iron load appeared to be the parenteral route and related to an underlying disturbance of erythropoiesis, intestinal mechanisms could not be excluded as contributory factors. The patient’s clinical manifestations included endocrine, cardiac, and hepatic dysfunctions. Post-mortem examination displayed major end-organ disruption, related to parenchymal cell damage by excess iron deposition.

[18] Iron overload and cardiac abnormalities. Ernest M. Walker, Jr., and Christopher P Epling, Joan C Edwards Medical School, Marshall University, Huntington, WV.

Iron excess and associated oxygen free-radical production are probably causative, contributory, or complicating factors in ischemic heart disease, vascular strokes, and at least 60 other medical or disease conditions in humans. These iron-accumulating conditions or diseases are divided into primary hemosiderosis (hereditary hemochromatosis) and secondary hemochromatosis or hemosiderosis (due to repeated blood transfusions, ineffective erythropoiesis, or other causes). Some degree of iron excess is seen in at least 50 to 100 million people worldwide. Severe iron overload and storage most commonly causes adverse functional and structural changes in the heart, pancreas, skin, liver, and endocrine systems, including the gonads. Serious, potentially lethal myocardial involvement is seen in at least one-third of severe iron-overload cases and is probably detectable in many other cases. Myocardial iron accumulations in primary or secondary hemochromatosis may cause a variety of common cardiac abnormalities including supraventricular and ventricular arrhythmias, conduction abnormalities, cardiac hypertrophy, congestive heart failure, dilated/restrictive cardiomyopathy, interstitial fibrosis, and a number of additional structural and functional changes. Primary hemochromatosis patients may be treated by a long series of weekly phlebotomies and secondary hemochromatosis (hemosiderosis) cases may respond favorably to treatment with the iron-chelating agent, desferrioxamine (Desferal®). Cardiac and/or liver transplants may be beneficial in selected cases. None of these treatment modalities are ideal, so that better treatment approaches and more effective iron-chelating agents are needed. One major problem, especially regarding white males with hereditary hemochromatosis, is that while iron accumulations begin early in life, diagnosis of the condition is often not made until the individual is in his/her thirties or forties, at which time iron accumulations may be severe and very difficult to remove or treat. PCR tests are currently available to detect the HFE gene of hereditary hemochromatosis at an early age. Additional, reliable laboratory tests are available to detect iron overload from whatever cause. Early diagnosis allows treatment of these individuals at an early age to reduce or prevent excess iron accumulation. Electrocardiogram and echocardiographic cardiac studies plus periodic laboratory testing can assess the effectiveness of preventive therapy and response of more advanced iron-overload cases to therapeutic interventions. Thus, iron-induced cardiovascular and other organ/tissue complications can be prevented or controlled. Earlier diagnosis and treatment of iron overload offer a great medical breakthrough since iron-induced complications are so difficult to control, treat, or reverse once they have reached advanced stages.

[19] Echocardiographic evaluation of iron-induced cardiac damage in gerbils. Ernest M. Walker, Jr., Christopher P. Epling, Cordel Parris, Imran Arif, Silvestre P. Cansino, Romaine R. Perdue, Sayyed Rameez, Paulette Wehner, Elsa I. Mangiarua, and Gary L. Wright, Joan C. Edwards School of Medicine, Marshall University, Huntington, WV.

Our major objectives were to induce iron overloading in a gerbil model and to compare the abilities of representative agents and combinations to prevent or reverse iron-induced cardiotoxicity. Male gerbils were divided into groups of 10: negative (saline) controls, positive iron controls (iron injections only), iron overloaded, treated with EX, EY, Desferal® (DF), or combinations of EX plus DF or EX plus EY. Gerbils designated to receive iron were iron overloaded by a series of ip injections of iron dextran complex during a 2-mo period. Treatments were injected 2–4 hr after each iron injection and continued for 4 wk after completion of iron overloading. Echocardiograms were done on gerbils in the control and iron-overloaded groups 2 mo after completion of iron overloading (1 mo after completion of treatment) and again on animals in all groups 2 mo later. Echocardiographic studies included 9 parameters to evaluate internal cardiac structure, plus 7 parameters to evaluate cardiac function. Gerbils were autopsied after completion of the second series of echocardiograms and resulting tissue slides evaluated by light microscopy. Weighed whole hearts and liver samples were acid-digested and measured for total iron content. Echocardiographic comparisons of hearts in control versus iron-overloaded, untreated gerbils revealed iron-induced adverse cardiac structural (increased thicknesses of intraventricular septal, left ventricular, and right ventricular walls) and functional changes (decreased left ventricular ejection fractions and decreased maximum blood flow velocities through aortic, pulmonary, tricuspid, and mitral valves). Of the treatment agents or combinations the best positive, statistically significant results were obtained with EX only, which appeared to prevent or reverse iron-induced deleterious structural and functional changes and restore values to those similar to saline controls. Desferal® (DF), the only iron-chelating agent approved for human usage in the USA, provided only minimal protection against iron-induced cardiac damage. EY produced no statistically significant protection. Combinations of EX and EY, or EX and DF, produced less impressive protective results than seen with EX only. In conclusion, EX, probably through the removal of excess iron, provided protection against iron-induced cardiac damage in gerbils. The gerbil model closely simulates the human pattern of body iron accumulation, distribution, progressive toxicity, and iron-induced cardiac damage. Iron-induced echocardiographic changes are very similar. Iron-induced tissue damage is probably mediated through the production of oxygen free radicals. Iron excess and associated oxygen free radical production are probably causative, contributory, or complicating factors in ischemic heart disease, vascular strokes, and at least 60 other medical or disease conditions in humans. Some degree of iron excess is seen in at least 50 to 100 million people, worldwide. It can be speculated that EX may provide benefits in the many iron-related diseases or conditions in humans.

[20] Echocardiogram evaluation of homocysteine-induced cardiac dysfunction in rats. Ernest M. Walker, Jr., Cordel Parris, Jason E. Black, Christopher P. Epling, Silvestre P. Cansino, Lisa D. Hunt, Elizabeth C. Bryda, Gary L. Wright, and Mark A. Studeny, Joan C. Edwards Medical School, Marshall University, Huntington, WV.

Major objectives were to inject adult male rats with homocysteine (hcy) or its metabolite homocysteine-thiolactone (hct-tl) and to determine cardiotoxic effects. Twelve-wk-old male Sprague-Dawley rats were divided into groups of 8 including controls (no injections given), homocysteine (hcy), and homocysteine-thiolactone (hcy-tl). Homocysteine rats were injected ip with 3 ml of a hcy solution (0.083 mg/ml) resulting in 0.25 mg administered to each rat. The homocysteine-thiolactone group received 3 ml of hcy-tl solution (0.083 mg/ ml) resulting in 0.25 mg per rat. The injections were given once daily for a total of 14 days. Echocardiograms (ECHO) and electrocardiograms (EKG) were performed on the 7th and 14th days of injections for all animals. ECHO studies included 9 parameters to evaluate internal cardiac structure plus 7 parameters to evaluate cardiac function. ECHO comparisons of control hearts versus homocysteine and homocysteine-thiolactone hearts revealed adverse structural (changes in thicknesses of intraventricular septal, LV and RV walls) and functional changes (decreased LV ejection fractions and decreased maximum blood flow velocities through cardiac valves). Marked weight losses were seen in hcy and hcy-tl groups. ECHO abnormalities, cardiotoxicity, and weight losses were more severe in hcy than in hcy-tl rats. Histopathological comparison of cardiac sections from control and treated animals suggested that the treatment agents produce direct cardiotoxicity. In conclusion, several animal and human studies have suggested that injections or body accumulations of homocysteine, possibly through the production of homocysteine-thiolactone, have the ability to affect arterial contractility adversely. Few, if any, studies have addressed the effects of these agents on the heart. Our study suggests that ip injections of hcy or hcy-tl to rats adversely affect cardiac contractility and are cardiotoxic. The fact that hcy produced more severe cardiac abnormalities than hcy-tl in our rat model suggests that even though conversion of hcy to hcy-tl may be one mechanism of cardiotoxicity, additional toxic mechanisms also may be involved.

[21] Use of high-dimensional vectors with cluster analysis for optical-based query and automated classification of metabolic disease in the liver. Ulysses J. Balis, Massachusetts General Hospital and Harvard Medical School, Boston, MA.

Recent advances in digital imaging and the concomitant advent of wide-field microscopy now enable the digitization of complete histologic sections atdiffraction-limited resolution. The availability of resultant large surface area datasets allows for statistical and stochastic data extraction processes previously excluded from consideration. These resultant data may be further modeled in an N-dimensional vector tree for rapid optical-based query and automated differential diagnosis generation. One interesting area of histopathologic diagnosis, where automated feature classification may be of benefit, is metabolic liver disease, where features span both the cellular length scale and the tissue-level meso-scale. An Aperio Technologies ScanScope^TM was used to fully digitize liver biopsies with known metabolic diagnoses along with a cohort set of normal controls. N-dimensional Vector Quantization classification was performed on these cases with the creation of a highly compressed vocabulary of vectors which spatially correlated with reproducibly identifiable features of diagnostic merit. These vectors were then entered as a training set into a stochastic-reiterative classifier model for selection of the most predictive vector sets. General vector cluster members were seen to be widely dimorphic between normal histology and cases with known metabolic diagnoses. Cluster patterns were highly significant (p <0.001) for findings of sinusoidal dilatation, ballooning degeneration, rosette formation and Mallory’s hyalin. Ductular injury was noted to classify along three major cluster designations, also with statistical significance (p <0.02). Cluster profiles were noted to generally segregate themselves into classifications matching with established diagnoses of storage disease, obstructive cholangiopathy, chemical etiologies and a final indeterminate pattern (or patterns). The ability to derive unique N-dimensional cluster profiles for major branches of hepatic metabolic disease classification is a useful first step in the ultimate goal of developing automated image-based diagnostic classification platforms. It is anticipated that as vector-based vocabularies increase in size and diversity, the full diagnostic selective potential of this approach will be further understood and ultimately, available for clinical use as a diagnostic tool.

[22] Clinical science at the University of South Florida/All Children’s Hospital Children’s Research Institute. Robert D.Christensen, Darlene A. Calhoun, John W. Sleasman, Maureen M. Goodenow, Gary W. Litman, Lisa A. Simpson, Robert J. Boucek, James C. Huhta, and Kersti K. Linask, University of South Florida/All Children’s Hospital, Children’s Research Institute, St. Petersburg, FL.

The Children’s Research Institute (CRI) was created by collaborative efforts of All Children’s Hospital, the University of South Florida, and the State of Florida. The 54,000 sq ft research facility was completed in 2000 and serves as the central research facility on the All Children’s Hospital Campus. Basic and translational researchers have been recruited to the CRI, and have been awarded endowed chairs or endowed programs to enhance their extramural research funding and perpetually support their programs. The following projects are among those currently being conducted at the facility: Dr. Christensen (Barness Chair) and Dr. Calhoun (Rothman Chair) study problems in neonatal hematology. Funded projects include cytokine production during human fetal development, transplacental passage of hematopoietic growth factors, high-dose erythropoietin as a neuroprotectant, and mechanisms causing thrombocytopenia among SGA infants (HD-01180, HD-42326, HL-69990, HL-61798, HD-42308). Dr. Sleasman (Good Chair in Immunology) and Dr. Goodenow (Good Chair in Immunodeficiency) study pediatric HIV infection, HIV viral mutation, and optimization of chemotherapy for the treatment of HIV (AI-28571, AI-47723 AI/HD-39015). Dr. Litman (Hines Chair) studies the development of immunity using a zebra fish model and other models (AI-12338). Dr. Simpson (Guild Chair), recently recruited from the Agency for Healthcare Research Quality in Washington, DC, studies pediatric health policy and quality outcomes research. Dr. Boucek (Raymond Endowment), who directs the cardiac transplantation service at All Children’s Hospital, studies myocardial dysfunction resulting from chemotherapeutics (American Heart Association). Dr. Huhta (Andrews/Daicoff Chair) and Dr. Linask (Mason Chair) study the origins of congenital heart disease, and the early detection and in utero treatment of fetuses with heart defects (HL-67306, HL-64347, and American Heart Association). The basic work of these investigators is translated into clinical science and clinical trials through the Pediatric Clinical Research Center (PCRC), a federally-funded center directed from the CRI, providing infrastructure, ancillary cost support, research nurses, biostatistical support, and data management services, integrating basic and clinical science with patient-oriented research performed at All Children’s Hospital. The development of the CRI, the endowed chair program, and the PCRC have resulted in the University of South Florida climbing from a reputational score (NIH funding to departments) considerably below average in 2000 to within the top one-third of medical school pediatric departments in 2003.

[23] Current options for physiological amino acid analysis in the USA. Stephen Benford, All Children’s Hospital, St. Petersburg, FL.

Ion-exchange liquid chromatography with post-column ninhydrin detection has been the standard method for physiological amino acid analysis for over twenty-five years. With the withdrawal of a major US manufacturer of dedicated amino acid analyzers, our Biochemical Genetics Laboratory has been discussing what options are available to provide this important testing. In the USA there are currently two companies that provide fully supported clinical turnkey systems. These analyzers are essentially modern HPLC systems that have been modified to incorporate post-column ninhydrin detection and the latest column technology. These systems are expensive but their advantage is the full support of both the hardware and the application. For those with HPLC experience, a less costly alternative would be to build a system using a basic HPLC system and adding the necessary hardware and columns. These systems would require more start-up time for validation, and though the hardware can be supported, the applications may not be. For laboratories using a MS/MS system, it is conceivable that amino acid analysis could be adapted to that system, provided labeled standards were available and reasonably priced.

[24] Genomics and proteomics of inherited cardiac disorders. O. Thomas Mueller, All Children’s Hospital and University of South Florida, St. Petersburg, FL.

The recent publication of the completed human genome sequence has dramatically changed the prospects for unraveling the etiology of complex disorders. Many inherited or congenital disorders of the heart are recognized either as distinct clinical entities or as part of recognized clinical syndromes. This report summarizes recent findings on the effects of identified gene mutations in inherited syndromes that have cardiac involvement. Several genes implicated in familial hypertrophic cardiomyopathy and inherited dilated cardiomyopathies have been identified and are applicable to clinical diagnosis. Another group of disorders known collectively as transthyretin amyloidoses include familial amyloid cardiomyopathy, familial amyloid polyneuropathy Types I and II, leptomeningeal amyloidosis, and familial oculoleptomeningeal amyloidosis (FOLMA). These disorders are caused by mutations in the transthyretin (TRR) gene and genetic screening for mutations in the gene detects mutations in 99% of cases. The disorders collectively identified as 22q11 deletion syndromes include Shprintzen syndrome, DiGeorge syndrome (DGS), velocardiofacial syndrome (VCFS), and conotruncal anomaly face syndrome. Most patients have a very similar deletion of about 4 Mb in the 22q11 region containing at least a dozen genes, although rare patients have partial deletions. We characterized the extent of the deletion in a series of 24 patients using molecular markers in an attempt to identify genes associated with particular clinical characteristics of 22q11 deletion syndrome. Several candidate genes suspected to be involved in the etiology of this syndrome have been identified from this consensus deletion region. Some have been found to contain deleterious mutations in non-deletion patients and are promising candidates for genes involved in essential cardiac development or function in view of the high rate of heart defects in these disorders.

[25] Slide seminar on neuropathology. Atilano G. Lacson, Department of Pathology, All Children’s Hospital, St Petersburg, FL.

This session will touch on the central nervous system (CNS) neoplasms, developmental disorders, and neuro-muscular diseases of infancy and childhood that one encounters in a tertiary pediatric neuropathology practice. The cases were encountered during the past four years at All Children’s Hospital. Some of these represent unusual challenges that required combined serendipity, persistence, and technology in reaching a diagnosis, while others are cases that illustrate newer diagnostic concepts. All provide unique opportunities in gaining additional insight into the nature of childhood CNS neoplasms, developmental CNS abnormalities, and primary neuromuscular diseases. Relevant details of each case are provided in the handout in advance of the conference to allow participants to review the material. The 1-hr session will be divided into 10-min segments per case to allow interactive discussion among the participants. At the end of the session, the participants will be able to: (a) describe the essential elements of certain CNS neoplasms that arise in early life; (b) list the differential diagnoses of certain myopathic conditions in children; and (c) discuss the pathologic approach to certain developmental CNS abnormalities to reach an embryological explanation.

[26] Workshop on congenital heart malformations. Enid Gilbert-Barness and Diane D. Spicer, Tampa General Hospital, Tampa, FL.

This 1-hr session will demonstrate approximately 30 heart specimens that represent a spectrum of congenital heart malformations that practicing pathologists or pediatric cardiologists may encounter. Each carefully dissected specimen will be presented with an accompanying illustration and diagram to indicate the defects in the heart. Three instructors will be available for specific demonstration of the specimen on an individual basis. At the end of this workshop, the participants will be able to: (a) describe the essential features of the types of cardiac defect; (b) understand the hemodynamics and blood flow patterns of each malformation; and (c) correlate the clinical features of congenital cardiac defects with pathological specimens.

[27] Workshop on molecular cytogenetics in the investigation of cardiomyopathy. Maxine J. Sutcliffe, All Children’s Hospital and University of South Florida, St Petersburg, FL.

Congenital cardiovascular malformations have an incidence of 5–8/1000 live-born infants with 6% showing obvious chromosome aberrations such as Turner and Down syndromes. With the morphogenesis of the heart focussed around the 5–8th wk of embryonic development and more than one-third of conceptions having a chromosome abnormality, this is likely an underestimate. Molecular cytogenetics is one of the technologies that attempts to define better the 85% of congenital heart defects (CHD) of unknown etiology. It is known that aneuploidy (gain or loss of entire chromosomes) results in CHD. It is also known that cardiac malformations have been associated with structural abnormalities of chromosomes 3, 4, 5, 9, 10, 11, 13, 15, 18, 21, 22, and X, sometimes as submicroscopic deletions or duplications. In addition, a number of unidentifiable "marker" chromosomes are found in patients with CHD. Fluorescence insitu hybridization (FISH) defines the localization of a region on a chromosome spread or cell nucleus on a slide by molecular hybridization of a labeled DNA probe. FISH originally used 2 major haptens, biotin and digoxigenin, that bind either of 2 fluorescent labels FITC (green) or Rhodamine/Texas (red) to the DNA probe with DAPI (blue) counterstaining. Now there are more than 400 commercially available probes to detect constitutional chromosome abnormalities such as rearrangements and microdeletions and acquired oncology-specific gene rearrangements. The workshop will demonstrate a number of these applications, together with the most recent pantelomere FISH (telomere probes to all 46 chromosomes simultaneously) and multi-color FISH (simultaneous 24-color application), as examples of recent advances that enable the investigation of extremely small changes in chromosomes that were previously unresolvable in patients with congenital heart defects.

[28] Workshop on point-of-care testing: continuing challenges and new advances. Diane Davis and Joseph P. Laurino, St. Petersburg and Tampa, FL.

The creation, implementation, and maintenance of a successful point-of-care testing program requires a cooperative effort involving laboratory scientists, healthcare providers, administrators, and manufacturers. This workshop will allow the participants to view and use point-of-care testing instruments, including the LifeScan SureStep Flex, the iSTAT analyzer, the Hemotec ACTII, and the Hemocue hemoglobin analyzer. Participants will learn about the various hardware and software features, such as the control of instrument access and data release, employed to assist non-laboratory-based testing programs. Instrument-specific quality control and assurance safeguards, as well as infection and contamination control, will also be discussed. Members of the laboratory and healthcare staff of All Children’s Hospital will address specific implementation and maintenance issues.

[29] Thrombophilia in the pediatric population: is this a concern in the neonate? John Lazarchick, Medical University of South Carolina, Charleston, SC.

Thrombophilia is defined as any state, either hereditary or acquired, that results in an increased tendency toward venous and/or arterial thrombosis. Although the relative risk of thrombosis is well defined in the adult population for individual thrombophilic states, few studies have defined these risks in the neonatal period. Thrombosis in the neonatal period is an uncommon event, estimated to occur in 0.24–0.5/10,000 live births. Perhaps because of its rarity, little is understood about the underlying pathological mechanisms involved, nor is there is a large data base about diagnostic criteria or appropriateness of therapeutic interventions. We shall first review "normal" hemostasis in the newborn period and examine the differences vis-à-vis adult hemostatic mechanisms. Within this framework, both hereditary and acquired thrombophilic states will be discussed, including deficiencies of protein C, protein S, anti-thrombin III, presence of factor V Leiden, prothrombin G20210A, and methylenetetra-hydrofolate reductase mutations in the former and primary and secondary anti-phospholipid antibody syndrome related to the latter; the limitations in this age group of diagnostic assays commonly used in the adult population will be noted; therapeutic interventions will be briefly discussed.

[30] Ovarian carcinoma. Santo V Nicosia, Univ. of South Florida College of Medicine; Moffitt Cancer Center, Tampa, FL.

Ovarian carcinoma (OC) represents the leading gynecologic malignancy in American women, with an estimated annual incidence of over 23,000 cases and 13,500 deaths. The pathobiology of OC is still incompletely understood due to a number of factors, including the lack of an animal model and an overall late stage presentation. Experimental data suggest that tissue injury related to ovulation, hormones, sun exposure, or other yet unknown factors and familial predisposition (~10% of OC) may activate genomic, paracrine/autocrine mechanisms and intracellular signal transduction pathways that then drive Müllerian-oriented cell growth, cell survival, and morphogenesis in the ovarian surface epithelium (OSE), the putative source of OC. This cancer may arise de novo, from precursor lesions, or from a dual pathway with wild type K-ras rich, allelically imbalanced, high-grade carcinomas arising de novo and mutated K-ras rich carcinomas arising from precursor lesions including low malignant potential neoplasms. The latter developmental modality suggests a wider preclinical window of opportunity for screening intervention. A number of currently investigated biomarkers may add detection power to CA 125 and vaginal ultrasound, two currently prevailing but not completely reliable diagnostic modalities for OC. These include: newer imaging endpoints, fluorescence spectroscopy, lysophosphatidic acid, c-erbB-2, mesothelin, c-Kit, telomerase, homeobox genes, and angiogenic and proteomic profiling, as well as brushing cytology. It is anticipated that identification of phenotypic and molecular abnormalities associated with early OC and high risk tumor progression and the development of scientifically sound and accurate OC detection programs will lead to earlier diagnosis, targeted therapies, and ultimately better survival in women affected by this most insidious and lethal disease. (Supported by DAMD 17-02-1-0670.)

[31] Meningioma: a dominant neoplasm in dural-based tumors. Joseph C. Parker, Jr., University of Louisville School of Medicine, Louisville, KY, and John R. Parker, Vanderbilt University School of Medicine, Nashville, TN.

With neuroimaging, dural-based tumors may be incidental findings or can present with various neuropsychiatric disorders and headaches. Evaluation of these tumors requires histomorphology, since they may be benign or malignant neoplasms, cystic lesions, or even inflammatory reactive lesions that may be infectious. Dural-based masses include meningiomas as well as other primary and metastatic tumors. Dural-based cysts may be arachnoidal, epidermal, dermoid, or enterogenous cysts. The inflammatory reactive dural-based masses can be caused by tuberculosis, collagen vascular disorders, fibrovascular proliferations, and reactions to intracranial hypotension. Evaluation of 36 autopsied cases over a 5-yr period demonstrated 7 incidental primary brain tumors (20%), including 3 meningiomas; 2 subependymomas; and 2 well-differentiated astrocytomas. Meningiomas are the most frequent dural-based tumors occurring throughout the neuraxis. They may be genetically and hormonally induced, but have been associated with previous radiation and trauma. Estrogen and progesterone receptors are demonstrated in most. Various histomorphologic types of meningioma, and malignant (WHO Grade III) including papillary and rhabdoid meningioma. Mitoses are used to separate atypical from the anaplastic meningioma (more than 20 mitoses/10 HPF). Proper recognition of these dural-based tumors will allow appropriate use of combination therapy to assure a natural life span.

[32] Molecular profiling of hormone-receptor-negative breast carcinoma: templates for consultative proteomics. Robert E. Brown, Geisinger Medical Center, Danville, PA.

Objective: To develop templates for consultative proteomics based on the molecular profiling of hormone-receptor-negative breast carcinoma. Methods: Three human breast carcinoma cell lines (SKBR-3, MDA-175, and MDA-231) provided as part of the DAKO HercepTest and slides from a patient with hormone-receptor-negative breast carcinoma were reacted in immunohistochemical procedures to detect the following categories of protein analytes: signaling ligands (transforming growth factor [TGF]-, platelet-derived growth factor [PDGF]-AB, interleukin [IL]-6, IL-11, stem cell factor [SCF], IL-1); signal transducers (HER-2/neu, epidermal growth factor receptor [EGFR], PDGFR-, PDGFR-ß, gp130, c-kit [CD-117], protein kinase C [PKC]-, PKC-ßII, phosphorylated [p]-PKC-/ßII); transactivators (angiotensin system [cathepsin D, angiotensin-converting enzyme, angiotensin II type 1 receptor] and isoprenylation pathway [p21ras, a subunit of farnesyl transferase and geranylgeranyl transferase]); downstream effectors (p-c-Jun N-terminal kinase, p-p38 mitogen-activated protein kinase, c-Jun, cyclin D1); genomic impact (Ki-67); proapoptotic/growth inhibitors (TGF-ß1 [latency-associated peptide], TGF-ßRII); and antiapoptotic/tumorigenic factors (COX-2, bc1-2). Immunoreactivities and cellular compartmentalization were scored from 0–3+ positivity using bright-field microscopy. Results: Constitutive expressions of protein analytes in the 3 cell lines revealed commonalities and selective differences and provided for functional grouping of proteins that defined distinctive templates for consultative proteomics that take into account such variations in the molecular pathways controlling tumoral growth. The application of such customized profiling with a standardized reporting format is illustrated utilizing the aforementioned patient case material. Conclusion: Molecular profiling of hormone-receptornegative breast carcinoma reveals several templates of molecular pathways that can be applied to patient material so that individual tumors can be characterized accordingly (consultative proteomics).

[33] Merkel cell carcinoma: a case series and examination of prognostic factors. Michael B. Morgan, University of South Florida College of Medicine, St Petersburg, FL.

Merkel cell carcinoma (MCC) is an uncommon but deadly cutaneous neuroendocrine carcinoma of which little is known regarding the etiopathogenesis or prognostic factors. We examined the clinical and pathologic attributes of a series of 30 MCC to determine the influence of demographic factors such as age or anatomic location and pathologic factors such as depth of invasion on survival. Statistical analysis was determined by univariate and multivariate analysis with Cox proportional hazards modeling. Of 30 patients, 26 were available for follow-up. Of these, 5 patients died of unrelated disease and 14 of metastatic disease. The average survival was 37 mo and follow-up ranged from 30 to 62 mo. Demographic factors such as age at presentation, gender, and anatomic location had no influence on survival. Pathologic factors such as vascular permeation, ulceration, and lymphoid infiltrate had no influence on survival. The mitotic rate (>3/HPF) and depth of invasion were statistically associated with outcome. MCC is a rare deadly cutaneous malignancy. Consideration should be given to an assessment of the depth of invasion and mitotic rate of the tumor at the time of initial diagnosis, as these factors might influence prognosis and therapeutic decisions.

[34] IGF1-receptor protein expression correlates with tumor grade of renal cell carcinoma. Connie Keehn, Nazeel Ahmad, Domenico Coppola, Moffitt Cancer Center-USF, Tampa, FL.

Recent reports have shown significant correlation between Furman’s nuclear grade of renal cell carcinoma (RCC) and patient survival. However, no specific gene alteration has yet been described to account for this correlation. In this study we investigated the expression of the insulin-like growth factor 1 receptor (IGF1-R) in RCC and searched for correlation of the results with tumor grade. Formalin-fixed, paraffin-embedded sections from 68 cases of RCC were stained using the immunohistochemical avidinbiotin-peroxidase method. We used an anti-human IGF1-R rabbit polyclonal antibody (dilution 1:100, microwave antigen retrieval with EDTA, Neomarkers, Freemont, CA). The stains were semiquantitatively evaluated using the Allred score system, assessing intensity of stain and percentage of positive tumor cells. Strong and diffuse cytoplasmic IGF1-R stain (Allred score 7–8) was identified in 25/25 (100%) of RCC grade 3 and 4. Grade 2 RCC had a median IGF1-R Allred score of 4. Grade 1 RCC was negative in 10/10 (100%). Even in the positive high-grade tumors, areas of low nuclear grade, when present, were IGF1-R negative. Statistical analysis using the Kruskal-Wallis test showed significant correlation between high nuclear grade and IGF1-R Allred score (p <0.0001). In conclusion, we report found significant correlation between IGF1-R protein expression and Furman’s nuclear grade of RCC, and consequentially with patient survival.

[35] Performance of the FocalPoint slide profiler on conventional and liquid cervical cytology specimens. Jenny L. Boyle and Myra L. Wilkerson, Geisinger Medical Center, Danville, PA.

The goal of this study was to evaluate the FocalPoint slide profiler (formerly AutoPap; TriPath Care Technologies, Burlington, NC) in the primary screening of conventional and thin-layer cervical cytology slides versus manual screening. A total of 8,136 slides was included, consisting of 6,162 conventional smears and 1,974 thin-layer specimens collected using the SurePath test pack (formerly CytoRich; TriPath) and processed with the PrepStain slide processor (formerly AutoCyte PREP; TriPath). All slides were screened by the FocalPoint, which ranked the slides into quintiles, or assigned them to either a "no further review (NFR)" or a "process review (PR)" category. Quintiles indicate the need for manual screening and range from 1 (Q1), or most likely to contain an epithelial cell abnormality, through 5 (Q5), or least likely to contain an abnormality. The NFR category indicates the slide is unlikely to contain an epithelial cell abnormality. The PR category indicates a technical problem. All FocalPoint screened slides were manually rescreened by a cytotechnologist. A pathologist reviewed slides with epithelial cell abnormalities or slides designated unsatisfactory (U). Of the 8136 slides, the FocalPoint instrument ranked 6555 (80.6%) into quintiles; 1196 (14.7%) as NFR; and 385 (4.7%) as PR. Of the 6555 ranked slides, 218 (3.3%) were interpreted as having a squamous or glandular epithelial cell abnormality and 469 (7.2%) as having benign inflammatory changes. Of the 1196 NFR slides, 11 (0.9%) had an epithelial cell abnormality and 28 (2.3%) had inflammatory changes. One high grade squamous intraepithelial lesion (HSIL) was classified NFR by FocalPoint. All other HSILs, squamous, and adenocarcinomas were ranked in Q1-Q3, with most in Q1. When compared with manual screening as the "gold standard," the FocalPoint instrument sorted the cases appropriately for manual review or for archiving without review.

[36] Viral escapology and cytomegalovirus (CMV). M. Kent Froberg, University of Minnesota Duluth, School of Medicine, Duluth, MN.

Escapology has recently been used to describe the complex biochemical methods by which viruses avoid host immune detection and destruction It implies a long, intimate period of co-evolution in which host genes may become incorporated into the virus genome. Cytomegalovirus (CMV) is a ubiquitous virus that causes little disease in immune competent individuals, but may cause serious disease when the immune system is suppressed. Besides latency, CMV escape techniques include camouflage and sabotage. Camouflage includes methods by which the virus hides from immune detection (invisibility); sabotage includes disruption or manipulation of host inflammatory/immune responses. A number of in vitro studies demonstrate that CMV produces host cytokine homologues (virokines), soluble chemokine receptors (cytokine binding proteins), and dummy receptors that bind cytokines to the cell surface (viroreceptors) to facilitate camouflage and sabotage. CMV intereferes with both MHC I and MHC II expression by interfering with metabolism and trafficking of these molecules, thus leading to decreased expression of viral antigens on the cell surface. CMV immediate early genes induce proliferation of infected cells, while interfering with apoptosis, thus increasing the chances of virus survival. CMV upregulates host IL-10 and secretes its own IL-10 homologue, inducing a TH1/TH2 switch, hence decreasing cell mediated immunity, which is thought to be more effective at killing intracellular pathogens. CMV produces a G protein-coupled receptor that binds the chemokine monocyte chemoattractant protein-1 (MCP-1) with high affinity. This may dampen the host response and enhance virus survival. We recently infected MCP-1 transgenic mice with murine CMV or mock infection and collected serum on day 6 post-infection. Mock infected mice had higher levels of serum MCP-1 than MCMV infected mice, suggesting that this chemokine receptor can sequester MCP-1 in vivo and may contribute to virus survival.

[37] The National Prion Disease Pathology Surveillance Center. Clive R. Hamlin and Pierluigi Gambetti, Case Western Reserve University School of Medicine, Cleveland, OH.

Neurodegenerative disease research has been conducted at this medical school under the leadership of Pierluigi Gambetti for >20 yr. In October 1997, the National Prion Disease Pathology Surveillance Center was officially established here. A brief description of normal and pathologic prion proteins will be followed by our classification of human prion diseases. Cases received since 1997 will be summarized and compared to the experience of the United Kingdom center. A description of the services available will supplement information listed on the center’s web site (www.cjdsurveillance.com). To date, only a single case of new variant Creutzfeldt-Jakob Disease has been identified in the United States, and this is thought to have been acquired in the United Kingdom. In addition, there are 7 cases of prion disease in hunters. This presents a challenge to the center, as a detailed classification of sporadic prion diseases has been established. However, additional tools must be used to definitely detect cases caused by exogenous prions, especially those that might be related to consumption of contaminated elk/deer meat.

[38] Human papilloma virus DNA testing: a new tool for cervical cancer screening. Maura Pieretti, Anita O’Connor, Arsalan Ahmed, Elliot Carter, Carole Boudreaux, and Sally Holmes, University of South Alabama, Mobile, AL.

This report delineates the implementation of human papilloma virus (HPV) DNA testing as a clinical tool to guide management of patients with abnormal Pap test results. Following the guidelines of the American Society for Colposcopy and Cervical Pathology for the management of women with cervical cytology abnormalities and cervical cancer precursors, we established reflex HPV DNA testing for all patients with a Pap test diagnosis of ASC-US (atypical squamous cells of unknown significance). It has been firmly established that the presence of HPV DNA in cervical cells is a major risk factor for the development of cervical cancer; therefore patients with an ASCUS diagnosis who are positive for HPV DNA should be referred to colposcopy, while HPV negative patients can be spared this invasive and costly procedure. Thirteen major oncogenic HPV types have been associated with cancer risk, while other low-risk HPV types are associated with benign lesions. HPV DNA testing is conducted on residual cells remaining after completion of the Pap test, utilizing a commercially available method that involves RNA:DNAhybridization, hybrid capture and signal amplification (Digene Hybrid Capture II). A cocktail of 13 probes corresponding to the oncogenic HPV types is utilized. The method is semi-quantitative; however, results are released in a qualitative format (positive/negative). We validated this methodology on residual cells from the liquid-based SurePath test (TriPath Imaging), the method of choice for the Pap test at our institution. We devised a simple but efficient system for (a) monitoring patients with an ASC-US diagnosis; (b) retrieval of specimens; (c) ordering of reflex testing (prior physician order requested); (d) processing of samples for longer storage; and (e) DNA testing in cost-effective sample batches. We are currently evaluating the accuracy of borderline positive results, as well as their cytological and clinical correlates. We are also evaluating the utility of HPV DNA testing for the triage of patients with an AGC (atypical glandular cells, endocervical type) diagnosis.

[39] Diagnosis of babesiosis using an immunoblot assay. Raymond W. Ryan and Peter J. Krause, University of Connecticut Health Center, Farmington, CT, and Connecticut Children’s Medical Center, Hartford, CT.

Babesiosis is a tick-borne disease caused by a protozoan parasite, Babesia microti. This parasite is transmitted to humans by the same Ixodes ticks which transmit Lyme disease and human granulocytic ehrlichiosis. Although the current indirect immuno-fluorescent assay (IFA) used to detect specific antibody in human babesiosis is sensitive and specific, an immunoblot antibody test may be less subjective, and easier to standardize and perform. Our objective was to determine the utility of, and develop interpretive criteria for, an immunoblot antibody test for diagnosing human babesiosis. We compared the reactivity of sera to a B. microti immonoblot assay in 24 human subjects experiencing clinical symptoms and having laboratory evidence of babesiosis, 28 patients with confirmed Lyme disease, 12 patients with human granulocytic ehrlichiosis, and 51 individuals who reported no history of any of these diseases and whose sera did not react against B. microti antigen in the IFA test. The sera of all subjects who experienced babesiosis reacted against the B. microti antigen in the IFA and against at least 1 of 9 immunoblot protein bands specific to B. microti. None of the sera from individuals who appeared not to have experienced this disease reacted against the B. microti antigen in the IFA. When 2 reactive bands were considered as definitive, immunoblot sensitivity was 96%, while specificity was 99% and predictive positivity and predictive negativity were 96 and 99%, respectively. This B. microti immunoblot procedure shows promise as a sensitive, specific, and reproducible assay for routine laboratory diagnosis of babesiosis.

[40] Self-staining slides used for fluorescence microscopy to detect urinary infection. Andrew E. Lorincz, Birmingham, AL.

More than 140 specimens submitted for urinalysis and culture in a community hospital were blindly screened by fluorescence microscopy. Two types of slides were used by each of 2 microbiology laboratorians and an experienced fluorescence microscopist. The slide screening results were compared to quantitative urine culture results, as well as to urine dipstick readings. Slide preparation technique #1 requires a standard glass microscope slide to which 35 µl of uncentrifuged urine is added and an equivalent volume of buffered acridine orange fluorochrome. A glass cover slip is added and the edges of the coverslip are sealed with melted paraffin. An epi-fluorescence microscope using a 100x oil objective is used for viewing. This is the traditional supravital microscopic fluorescence technique previously described by the author. Slide preparation technique #2 requires a novel plastic, self-staining slide developed for use with epi-fluorescence. After exposing a target viewing area by peeling back an adhesive tape, 35 µl of uncentrifuged urine is added and the slide resealed with adhesive tape. Viewing by epi-fluorescence microscopy was performed as in slide preparation #1. If the microscopist observed >1 microorganism per high powered field or >1 WBC per 10 high powered fields, the specimen was considered to need microbiological culture. An arbitrary definition of a colony count >100,000 CFU/ml was considered positive for significant bacteriuria, while a count <100,000 CFU/ml was considered negative. Using slide preparation #1, the 3 microscopists were able to detect significant bacteriuria with a specificity of 93–96%. Using slide preparation #2 (ie, the self-staining slide), specificity was 93–96%. The ability of fluorescence microscopy to determine accurately, rapidly, and on-site, which urine specimens require microbiological culture could eliminate millions of unnecessary urine cultures that are performed annually in the United States.

[41] The laboratory diagnosis of paroxysmal nocturnal hemoglobinuria. Jonathan S. Krauss, Medical College of Georgia, Augusta, GA.

Paroxysmal nocturnal hemoglobinuria (PNH) is an uncommon acquired stem cell disorder associated with periodic hemolytic events. This clonal process is caused by abnormalities of the X-linked phosphatidyinositol glycan class A (PIGA) gene, which lead to cytopenias and thrombotic manifestations. The trilineage of bone marrow elements is affected; however, the red blood cell (RBC) line is the most prominently involved due to its abnormal sensitivity to complement mediated intravascular hemolysis. Abnormal blood cell membrane proteins include decay accelerating factor (DAF, CD55), membrane inhibitor of reactive lysis (MIRL, CD59), homologous restriction factor (HRF, C8-binding protein), and other proteins affixed to the glycophosphatidyinositol (GPI) spine. Stem cell transplantation can be curative. Diverse laboratory abnormalities observed in patients with PNH include bone marrow hyperplasia, hematologic cytopenias, micro- and macrocytosis, decreased leukocyte alkaline phosphatase (LAP), hemoglobin- and hemosiderinuria, as well as associated iron deficiency. The more definitive laboratory tests consist of older biochemical (hemolytic) and newer immunological (flow cytometric) techniques. The former group includes the sucrose hemolysis test utilizing hypotonic complement activation for screening, followed by the complete Ham acid hemolysis test for confirmation. Recently, flow cytometric tests for CD55 and CD59 have begun to replace the Ham acid hemolysis test. Finally, quantitation of the specific binding of fluorescently labeled inactive variant of the protein aerolysin (FLAER) to GPI anchors may be more accurate than quantitation of antibody-binding to CD59 for diagnosis of PNH.

[42] Use of recombinant factor VIIa in diverse massive uncontrollable surgical hemorrhage. John Theus, Steven Hughes, James Flick, Louis Fink, and Alex Pappas, University of Arkansas for Medical Sciences, Little Rock, AR.

Recombinant factor VIIa (rFVIIa,) is an effective and approved hemostatic agent for managing hemorrhage in hemophiliacs with inhibitors. As a universal hemostatic agent, rFVIIa potentially can be used to manage severe uncontrollable surgical hemorrhage in patients without pre-existing coagulopathies. Recombinant FVIIa was used in 8 adult (median age = 47 yr [range, 19 to 67 yr]) patients with massive uncontrollable bleeding requiring multiple transfusions (median = 32.5 units [range, 2 to 44 units] of packed red cells) in diverse clinical settings (2 with gunshot wounds, 2 with cardiac surgery, 1 with severe trauma, 1 with abdominal aortic aneurysm, 1 with ectopic pregnancy and 1 end-stage myeloma patient). Prior to administration of rFVIIa, consultation with clinical pathology was provided to assist in management of possible preexisting hypofibrinogenemia (median = 188.5 mg/ dl [range, 88 to 296 mg/dl]), thrombocytopenia (median = 98 K/ul [range, 21–243 K/uL]) and to recommend appropriate dosage of rFVIIa (median = 16,800 micrograms [range 4800–30,000 µg]). The prothrombin time had a median of 21.8 sec (range = 11.5 to >120 sec) and the partial thromboplastin time had a median of 42.8 sec (range = 32 to >140 sec). Four of the 8 patients survived, 2 died of reasons other than bleeding or thromboembolism, and 2 died because of continued bleeding. These preliminary findings for rFVIIa are encouraging for use in patients with severe hemorrhage that does not respond to conventional hemostatic therapy.

[43] Effect of protein on hemoglobin and hematocrit results obtained with point-of-care testing devices: comparison to core laboratory results. Sidney M. Hopfer, Francesca L. Nadeau, and Gregory S. Makowski, University of Connecticut Health Center, Farmington, CT.

Point-of-care testing (POCT) for hemoglobin and hematocrit (H&H) provides rapid patient assessment including transfusion need in ERs and ORs. Although methodology can vary, conductivity-based methods are influenced by protein concentration. To assess this limitation we performed H&H testing using 2 methodologically different POCT instruments. Lithium-heparin anticoagulated whole blood (Vacutainer, Becton-Dickinson) was centrifuged (3200 rpm, 10 min); the plasma was carefully removed and combined to serve as source of protein for mixing studies. The RBC pellets were washed by resuspension in 2 vol of 0.85% (w/v) saline, centrifuged (1000 rpm, 10 min), and the wash was removed. RBC pellets were inverted several times to suspend homogeneously. Mixing studies were performed using RBCs, plasma, and 0.85% saline or Ringer’s lactate to obtain a series of samples containing variable RBC and protein concentrations. The samples were analyzed on 2 POCT instruments [iSTAT-1 (conductivity method) and Hemocue (optical method)] and compared to core laboratory GenS hematology analyzer (Beckman/Coulter). The samples were then centrifuged and protein determined in the supernatant (LX-20, Beckman/Coulter). Our results indicate that the Hemocue instrument correlated exactly with GenS at protein concentrations from 0.7–6.2 g/dl, using hemodilution with 0.85% saline (r = 0.999) or Ringer’s lactate (r = 1.000). In contrast, H&H results obtained using the iSTAT-1 demonstrated less correlation, with results obtained with the GenS instrument (r = 0.978–0.980) over this protein range. In general, iSTAT-1 results were lower in comparison to the GenS instrument with discrepancies of up to 2 g/dl hemoglobin and 4% hematocrit at the lowest protein concentrations. To avoid inappropriate transfusion, it is recommended that H&H testing in patients with suspected hypoproteinemia or following hemodilution should be performed using a non-conductivity-based method.

[44] Molecular diagnostics and clinical laboratory testing. Donald J. Cannon, Quest Diagnostics Inc., Teterboro, NJ.

The promise and impact of molecular diagnostic testing on clinical laboratory medicine has been slow to materialize. Complexity, the need for trained personnel, and the expense of the techniques have created a lag between potential and reality. That may be changing. We are in a post-genomic period, the technologies are being simplified, the need for and the volume of certain molecular tests are evident, and the availability of reimbursement is building. As a consequence, physicians and healthcare workers must become familiar with this new mode of testing, laboratories need to implement it, and the general public, especially patients subjected to molecular testing, needs to understand what it means to them. This review will summarize the opportunities in this substantial market; the terminology that describes molecular diagnostics; and the main laboratory areas that are immediately affected (ie, infectious diseases, genetics, and cancer). Specimen handling, specific methods, and unique testing variables will be covered. Attention will also be focused on other areas that are impacted by recent advances in molecular diagnostics.

[45] Beaumont Reference Laboratory: 1993–2002. Frederick L. Kiechle and Joseph E. Skrisson, William Beaumont Hospital, Royal Oak, MI.

The growth of Beaumont Reference Laboratory (BRL) was reviewed from its inception in 1993 to 2002. In 1994, there were 307,403 BRL procedures (14.5% of all clinical pathology (CP) procedures, compared to 2,292,672 in 2002 (46.2% of CP procedures). In 2000, liquid-based cytology was 21% of the total, which increased to 79% in 2002. Growth has ranged from 5–20% per yr. Nine subspecialties are reviewed each yr to determine requisition/physician, procedures/ requisition, and procedures/physician. In 2002, BRL send-out tests represented 46% of the total. Financial outcome is monitored in 4 major areas (test volume, supply cost, personnel costs and revenue cycle) using a variety of monitors. Improvements have been implemented in operating efficiency, computer-assisted courier routing, online test directory, client interaction tracking and report system, and software to identify new clients. Affiliation with the Joint Venture Hospital Laboratory has contributed to the success. The key factor for success in this network affiliation is the ability of the laboratory network to gain provider status for its members with the major managed care organizations.

[46] Alzheimer’s disease and Down syndrome. Huntington Potter, Medical College, Univ of South Florida, Tampa, FL.

Despite a common set of hallmark neuropathological lesions and clinical symptoms, Alzheimer’s disease has an apparently complex etiology. The disease can be caused by autosomal dominant mutations in at least 3 genes, can be influenced by certain allelic variants of at least 3 "risk factor" genes, or may arise""sporadically" with no evident genetic component. In the end, as many as 30–40% of individuals over the age of 85 yr may have some symptoms of Alzheimer’s, which underscores the fact that age itself is the strongest risk factor for the disease. It has been known for almost 20 yr that individuals with trisomy 21 (Down syndrome) exhibit Alzheimer neuropathology by the time they are 30–40 yr old. Because the gene for amyloid precursor protein (APP) resides on chromosome 21, its consequent overexpression in trisomy 21 cells presumably contributes to the development of Alzheimer’s disease in Down syndrome individuals. We have obtained evidence in favor of the hypothesize that many cases of classical Alzheimer’s disease, including both the genetic and late-onset, sporadic forms, might similarly be caused by chromosome mis-segregation, leading to a small number of trisomy 21 cells developing during the life of the affected individual. These findings indicate a new function for some Alzheimer’s disease genes and new methods of diagnosis.

[47] Cellular mechanisms of neurodegeneration and Alzheimer’s disease. John Savory, Othman Ghribi1, and Mary M. Herman, Departments of Pathology, Biochemistry, and Molecular Genetics, University of Virginia, Charlottesville, VA, and IRP, NIMH, NIH, Bethesda, MD.

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by the deposition of extracellular neuritic plaques (NPs), the formation of neurofibrillary tangles (NFTs), and massive neuronal and synaptic loss. Beta-amyloid (Aß) is the major component of the NPs, with hyperphosphorylated tau protein being the primary constituent of NFTs; both of these aggregates are generally accepted as being key players in the pathogenesis of AD. Whether Aß or tau actually represents the primary cause of neuronal loss in AD, and which of the two abnormal changes is generated first and is presumably the more important, remains a matter of debate. Furthermore, the mechanisms by which Aß and/or tau kill neurons in AD are not fully understood. However, whether apoptosis or necrosis is the major route of cell death remains to be clarified, although an apoptotic mechanism is gaining increasing support. We have developed a unique animal model where the intracisternal administration of aggregated Aß(1–42) peptide into rabbit brain induces neuronal death and hyperphosphorylation of tau protein. Death of neurons is mediated by endoplasmic reticulum (ER)-specific apoptosis, as is evident by the activation of caspase-12, a specific marker for ER stress, and by activation of the pivotal inducer of apoptosis, caspase-3, which we show to be primarily localized in the ER. These results implicate Aßas the trigger of tau phosphorylation with the ER is a direct target for Aß-induced neurotoxicity. We further demonstrate that neurotoxic effect of Aßcorrelates with the activation of the JNK and ERK MAP kinases along with the activation of the transcription factors, c-jun and c-fos. Treatment of the experimental animals with lithium or glial cell neuronal-derived factor (GDNF) prevents induction of ER stress but not hyper-phosphorylation of tau. These results suggest that 2 distinct pathways are involved in the apoptosis cascade activation and in tau phosphorylation following Aß administration. While the effects of lithium and GDNF on caspase-3 and tau are similar, they differentially regulate the MAP kinases, JNK, and ERK pathways. The results of these experiments add extended insight into intracellular molecular signaling pathways that could form the basis underlying neurodegeneration, particularly that resulting from the neurotoxic effect of Aß. (Supported by grants from the US Department of the Army and the Virginia Center on Aging, Virginia Commonwealth University.)

[48] Applied cytogenetics 2002–2003. Judith D. Ranells and Boris G. Kousseff, University of South Florida, Tampa, FL.

Following the rediscovery of human chromosomes in 1956, essential knowledge about numerous congenital disorders and syndromes emerged. Among these, Prader-Willi (PWS) and Angelman (AS) syndromes were particularly informative; they not only turned out to be cytogenetic disorders but proved that imprinting is relevant to the human species as well. FISH (fluorescence in situ hybridization) and DNA studies (methylation and microsatellite analysis) illustrated that. Since 1992, we have incorporated FISH and DNA analysis in a systematic fashion in the clinical studies/diagnosis of PWS and AS. From 2 January 1982 to 31 December 2002, we evaluated 105 probands with a referral diagnosis of suspected or clinically confirmed PWS. The probands were part of the 42,795 patients/families evaluated during the period. Forty-three of 105 had the condition and in 27/105 the syndrome was ruled out. The rest remained as suspected PWS patients in need of re-evaluations and further diagnostic testing. Three were no-shows and 8 were normal with positive family history. In 42/ 43 a karyotype was done. Eighteen of 42 had 15q deletions and 1 of these was mosaic. Another had a karyotype of 45,X,t(Y;15)(q12;q12)/46,X,t(Y;15),dic(15)(15pter->15q12::15q12->15pter)/47,X,t (Y;15),dic(15),dic(15) (Am J Med Genet 1987;28:803–811). FISH study was done in 7/43 probands and was positive in 3 of these. DNA studies were done in 7/43 and 6 of these were positive. PWS and AS are clinically distinct disorders resulting from microdeletion, uniparental disomy (UPD), or imprinting center defect involving chromosome 15q11-q13. In PWS, the abnormality is on the paternally derived chromosome 15, and in AS the defect is on the maternally derived chromosome 15. The maternally inherited genes in the PWS region are imprinted, that is, they are rendered inactive via methylation. The contributions of epigenetic phenomena to human disease are now being identified.

[49] The polymorphisms of PSA promoter genes determined on filter paper collected cord blood. Jie Liu, Jin-San Zhang, Charles Y. F. Young and Pai C. Kao, Guangdong Hematopoitic Stem Cell Therapy Technology Center, Guangzhou, China, and Mayo Clinic and Mayo Foundation, Rochester, MN.

It seems a mystery that the incidence of prostate cancer in African-Americans is 4-fold to 5-fold greater than in Asian-Americans, but 50- to 60-fold greater than in Asians who reside in Asia. Many polymorphisms that affect androgen function or potency may explain the ethnic difference, since androgens play a major role in prostate cancer. Polymorphisms that most influence androgen activity include the androgen receptors that mediate androgen functions and 5-alpha reductase that converts testosterone to dihydrotestosterone (DHT). Large numbers of healthy controls and prostate cancer patients were compared in Shanghai, China. However, none of the known polymorphisms of androgen receptor or the reductase were statistically significant biomarkers of prostate cancer, because the diseased and control subjects had similar polymorphisms. Recent studies identified another polymorphism as a risk factor that Asians may not have. It is a promoter gene for prostate specific antigen, ie, androgen receptor element 1 (ARE-1) gene. Portugese males with prostate cancer have high (43%) AA polymorphism of the gene (41%AG, 16%GG). Healthy Japanese subjects have very low (5%) AA polymorphism. We studied 94 specimens of cord blood from Chinese infants collected onto filter paper to demonstrate that Chinese also have very low (5%) AA polymorphism; there were 26%AG and 69%GG. Low percentage of AA is a unique polymorphism of Asians that may reduce their risk for prostatic cancer. Since migration to America increased the incidence of prostate cancer in Asians, the unique diet/life-style of Asians may possibly play an important role.

[50] Translocation (11;16)(q23;p13) in acute myelo-genous leukemia and myelodysplastic syndrome. Armand B. Glassman and Kimberly J. Hayes, The University of Texas M. D. Anderson Cancer Center, Houston, TX.

The purpose of this study is to examine the relationship of t(11;16)(q23;p13) to the type of myeloproliferative disorder noted by hemopathology. Previously, t(11;16) has been reported in <20 patients, all with the diagnosis of therapy related (secondary) acute myelogenous leukemia (sAML) or myelodysplastic syndrome (MDS). Putative involved genes are the MLL on 11q23 and CBP at 16p13. Data from the UTMDACC cytogenetics laboratory revealed 3 patients with t(11;16) reported during the past 4 yr. Two of these patients had a prior diagnosis of non-Hodgkin’s lymphoma (NHL) and had been treated with chemotherapy, which included cyclophosphamide. One patient presented with de novo AML and no history of cancer or chemotherapy. Two of the 3 patients had t(11;16) as the sole cytogenetic abnormality; the third patient had a t(11;16) clone that included t(9;21) and t(10;21).

Patient I (age 37 yr, female): NHL treated by CHOP (2001); refractory anemia; karyotype: 46,XX,t(11;16) (q23;p13)[12] and 46,XX[8].

Patient II (age 53 yr, female): NHL treated by CHOP (1997), AML M5; karyotype: 46,XX,t(9;21) (q31;q22), t(10;21)(q22;q22),t(11;16)(q23;p13)[11] and 46,XX[5].

Patient III (age 63, male): AML M4; karyotype: 46,XY,t(11;16)(q23;p13)[20].

Abnormalities of 11q23 have been associated with AML. Translocation (11;16) has been reported only as being therapy-related. This fusion gene has been identified in monocytes, granulocytes, and erythroblasts, but not in lymphocytes. The associated disorders have been M2, M4, CMML, and RAEBT. In this study, t(11;16) was seen in 2 patients with previous lymphomas treated with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP). The patient with apparent de novo AML represents the first reported instance of non-treatment-associated t(11;16) AML.

[51] Anatomy of a laboratory error. Roger L. Bertholf, Shawna Perry, and Robert L. Wears, University of Florida Health Science Center, Jacksonville, FL.

Malfunctions involving an automated chemistry analyzer and the laboratory information system went undetected for > 48 hr, during which time erroneous acetaminophen results were reported for several patients, some resulting in inappropriate treatments. When there is clinical suspicion of acetaminophen toxicity, the window of therapeutic opportunity is narrow and precedes frank biochemical signs of hepatic damage. Therefore, clinicians will initiate antidotal therapy solely on the basis of serum acetaminophen measurements. In this incident, a quality control failure prompted recalibration of the acetaminophen method, following which control values for acetaminophen were acceptable. Subsequent acetaminophen controls, however, generated an error code indicating a result above the linear limit of the method. Since the error code did not cross the interface to the laboratory information system, it went unnoticed. When a patient’s specimen produced this same error code, the technologist concluded that a high concentration of acetaminophen was present. When a high acetaminophen result was eventually questioned by the emergency department staff, the error was finally discovered. Subsequent investigation revealed that a sequencing algorithm in the instrument control software created the possibility of generating satisfactory control results after an unacceptable calibration. Overall, the error resulted from several contributing factors: human, instrumental, and operational. Modern healthcare involves the interplay of numerous complex systems and skilled professionals. As medical devices become more technically complex, the demands on the training and experience of healthcare professionals increase, and opportunities for errors are created. Automation simplifies the operation of highly complex devices, but less user interaction also creates the potential for errors when technologists are shielded from critical operational parameters that affect the integrity of analytical data. This incident is instructive because it illustrates how complex systems can fail due to the unanticipated synergistic consequences of multiple independent events.

[52] Detecting and reducing interferences in tandem mass spectrometry in the clinical laboratory. Alan L. Rockwood and Mark M. Kushnir, ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT.

We report strategies to detect and reduce or eliminate interferences in clinical samples analyzed by liquid chromatography-tandem mass spectrometry (LC-TMS). Although LC-TMS is notable in its specificity and is therefore rapidly gaining importance in clinical laboratories, it is not entirely free from interferences when used in its simplest form. In our laboratory we have identified several significant interferences or potential interferences, including fenofibrate in urinary free cortisol, prednisolone in plasma with cortisone and cortisol, and succinic acid in plasma methyl malonic acid. The usual approach to reducing interferences in LC-TMS occurs at the method development stage where the LC method is chosen to minimize known interferences, and the parent/ daughter ion transition in the tandem mass spectrometry is chosen to be as specific as possible. These approaches often lead to compromises in run time or detection limits, and even then may still be subject to interferences from sources not previously identified. We have developed an alternative strategy, adapted from ideas widely used in drug testing laboratories, and have applied the approach to LC-TMS. Rather than relying exclusively on eliminating interferences at the method development stage, our approach seeks to detect interferences at run time by monitoring at least 2 parent/daughter ion transitions. If the intensity ratios of the transitions are outside the limits measured with authentic standards, then an interference is detected, in which case one of several approaches may be applied to eliminate the interference. These approaches may include reverting to a secondary parent/daughter ion transition for quantitation, application of mathematical methods to deconvolute the target compound signals from the interfering signals, or re-analysis of the problem sample by an alternative method. All these methods have been successfully applied in our laboratory. This technique of monitoring multiple parent/daughter ion transitions relies on the unique power of tandem mass spectrometry to monitor simultaneously several highly specific sample properties (different parent/ion transitions) and to use this information to detect and eliminate sample interferences.

[53] Urinary 8-OH-dG: development of an ELISA for estimating oxidative stress. James T. Wu, University of Utah Health Sciences, Salt Lake City, UT.

Urinary 8-hydroxy-2-deoxyguanosine (8-OHdG) is a major marker of oxidative DNA damage excreted in urine after DNA repair. Elevated urinary 8-OHdG has also been detected in cancer, cardiovascular disease, and diabetes. 8-OHdG is most frequently measured by HPLC, a tedious and complicated procedure. We want to develop a simple enzyme-linked immunosorbent assay (ELISA) for urinary 8-OHdG to be used in routine clinical laboratories. The ELISA was established by coating BSA-conjugated 8-hydroxyguanosine (8-OHG) on microplates. Quantification of 8-OHdG was based on the competition between urinary 8-OHdG and coated antigen for the monoclonal anti-8-OhdG antibody added (Trevigen). Final quantification was performed by color development with the addition of HRP-conjugated sheep anti-mouse antibody. The calibration curve of the ELISA covers the concentration of 8-OHdG from zero to 100 ng/ml. The sensitivity of this assay is 0.5 ng/ml. Both within-day and day-to-day precision were <10%. This in-house assay compared well with a commercial kit from the Japan Institute for the Control of Aging (correlation coefficient = 0.8). We have also found that urine from patients with bladder and prostate cancers had approximately 20% (70.5 ± 38.2 ng/mg creatinine, mean ± SD) and 31% elevation (58.8 ± 43.4 ng/mg creatinine) in urinary 8OHdG, respectively, which were significantly higher than in urine of healthy control (36.1 ± 24.5 ng/mg creatinine). The results suggest that the cancer patients suffered more oxidative stress. This ELISA for urinary 8-OHdG appears to be a useful, simple assay to evaluate the extent of oxidative stress, and might conceivably be used to estimate risks for cancer.

[54] Clinical utility of a serum light chain assay in Bence-Jones disease. Brent Staggs, Leeja Joseph, and Alex Pappas, University of Arkansas for Medical Sciences. Little Rock, AR.

In Bence-Jones disease or free (f-) light chain (LC) multiple myeloma (MM), serum f-LCs are not easily detected by routine nephelometric methods. We prospectively and concurrently evaluated the utility of an ultra-sensitive quantitative serum f-LC (Freelitetm) assay in 23 patients with a previous diagnosis of f-LC MM, using bone marrow biopsy (BM-Bx) as the "gold standard". Data for serum calcium (Ca2+), and beta-2-microglobulin (ß2µ) were collected at the same time. The median patient age was 58 yr (range = 44–74 yr). The median serum f-kappa concentration was 150 mg/dl (range = 0.04 to 1230 mg/dl) and the median serum f-lambda concentration was 124 mg/dl (range = 1.82 to 927 mg/dl). Of 23 BM-Bxs, 14 were positive for MM and 9 were negative by routine microscopy; 14 BM-Bx were involved with MM and all had elevated f-LC (100%); 8 BM-Bx, which were negative, had elevated f-LC (89%). Only one BM-Bx that was negative morphologically had negative f-LC (11%). Serum Ca²⁺ was "normal" (median = 9.1 mg/dl; range = 8.7 to 10.5 mg/dl) in all patients and the serum ß2µ was elevated in 21 of 23 patients (median = 8.5 mg/dl; range = 1.4 to 60.2 mg/dl). The sensitivity, specificity, and positive-, and negative-predictive values of Freelitetm using the bone marrow biopsy as the "gold standard" are: sensitivity 100%, specificity 11%, ppv 63%, and npv 100%, respectively. Ultra-sensitive quantitative serum f-LC appears to be a sensitive indicator of involvement of the bone marrow by MM. The potential of this ultra-sensitive method needs to be further evaluated as an early indicator of relapse and monitoring of therapy in patients with f-LC MM.

[55] Peak shape and concentration effects on migration in zone electrophoresis. Zakariya K. Shihabi, Elma Wilson, and Mark Hinsdale, Department of Pathology, Wake Forest University School of Medicine, Winston-Salem, NC.

Electrophoretic migration is a characteristic for each compound and is used in compound identification. It is constant under a specified set of analytical conditions. However, occasionally it does not match well with the standards. A good example is in the analysis of hemoglobinopathies, which can be confusing in clinical analysis. Three factors have been identified as affecting or modulating the "apparent" migration time or distance in zone electrophoresis (both gel and capillary): peak shape, solute concentrations, and component interactions in the sample. Very high sample concentration can influence the migration, due to differences in field strength. As the front edge of the band moves from a concentrated sample into lower ionic strength buffer, it can experience a higher field strength, which leads to accelerated ion migration in that region compared to the rest of the band. Thus the leading edge of the band exhibits increased diffusion. Non-symmetrical peaks, which can arise due to several factors, are more affected by sample concentration and thus exhibit changes in migration. In addition, the migration of a protein can be influenced by the presence of another high concentration of a slowly migrating protein in the sample. An interaction between the 2 can occur during the separation, leading to a decreased velocity. This was confirmed by observation of a new small band formed during analysis of HBF by capillary electrophoresis upon the addition of HBS to the separation buffer.

[56] Analysis of Tamm-Horsfall protein by HPLC with native fluorescence. Zakariya K. Shihabi; Mark Hinsdale, and A . J. Bleyer, Departments of Pathology and Nephrology, Wake Forest University School of Medicine, Winston-Salem, NC.

Tamm-Horsfall, a large glycoprotein, is an abundant urinary protein that originates in the thick ascending limb of the loop of Henle of the kidney. It is thought to be involved in stone formation and binding uropathogens. Recently it was found that the amino acid composition of this protein is altered in medullary cystic kidney disease and familial juvenile hyperuricaemic nephropathy. This protein has been measured mainly by immunoassay (ELISA). Here we describe a different analytical approach, based on HPLC using a molecular size-exclusion column with native fluorescence detection in the UV range. This method has the advantages of speed, sensitivity, and the ability to detect some changes in the aggregation. Urine, 1 ml, was mixed with 100 µl of 20% NaCl and left at 37°C for 1 hr. The urine was centrifuged (12,000 rpm, 20 sec). The supernatant was discarded and the precipitate was vortex-mixed for 20 sec in 1 ml of triethanolamine buffer (15 mmol/L, pH 7.5 containing EDTA, 4 mmol/L). A 20 µL aliquot was injected onto the column, (macrosphere GPC 500 7 U, 250 x 4.6 mm, Alltech Associates, Deerfield, IL). The column was eluted with phosphate buffer (7 mmol/L, pH 5.7) and the effluent was monitored by a fluorometer (280 nm excitation; 325 nm emission). Tamm-Horsfall protein elutes as the first peak in ~2.5 min. Since the protein has a very large molecular weight compared to other urinary proteins, we did not experience any interference. The protein precipitates rapidly in <1 hr. It also precipitates better at 37°C, compared to 4°C. The UV detection is very sensitive for this protein down to 1 mg/L in absence of concentration steps. The method was linear from 1 to 100 mg/L. The CV was 10.4 % (mean 62 mg/L, n = 10). The mean level in 42 controls was 31 mg/g creatinine; in 30 patients with various renal disorders, it was 23 mg/g creatinine.

[57] Hoechst 33342-induced apoptosis alters glycosphingolipid composition. Frederick L. Kiechle and Xinbo Zhang, William Beaumont Hospital, Royal Oak, MI.

Hoechst 33342 (H342) inhibits topoisomerase I and induces apoptosis in a human promyelocytic leukemia cell line (HL-60). (Arch Pathol Lab Med 1999;123:1151–1153). To investigate the effect of H342-induced apoptosis on glycosphingolipid (GSL) concentration, high-performance thin layer chromatography was used. Three neural GSLs (ceramide monohexoside [CMH], ceramide dihexoside [CDH], and ceramide trihexoside [CTH]) and 4 gangliosides (GM3, GM2, GM1 and GD1a) were detected in untreated HL-60 cells. During H342-induced apoptosis, no new GSLs were noted. Distinct changes in GSL composition were observed: (a) decrease in GM3 and GD1a after 5–15 µg/mL H342 treatment for 24 hr; (b) decrease in CTH after 10–15 µg/mL H342 for 24 hr; (c) decrease in CMH, CDH, and CTH after 15 µg/mL H342 for 24 hr. In conclusion, H342-induced apoptosis is associated with changes in GSL composition, indicating that these changes might represent a marker for apoptosis, and that alteration of GSL composition may enhance or prevent H342-induced apoptosis.

[58] Fibrin-binding of type IV collagenases from inflammatory synovial fluid: implications for calcium phosphate crystal induced arthropathy. Gregory S. Makowski and Melinda L. Ramsby, University of Connecticut Health Center, Farmington, CT.

Basic calcium phosphate (BCP) and calcium pyrophosphate dihydrate (CPPD) participate in crystal-induced erosive joint disease, but the mechanisms of their actions remain unclear. We previously demonstrated that the amorphous form of calcium phosphate (CaP), a hydoxyapatite precursor and BCP aggregate, increased fibrin binding of matrix metalloproteinase-9 (MMP-9), a type IV collagenase. Plasmin-induced fibrinolysis resulted in activation of latent MMPs and subsequent collagenolysis. Here we evaluated the ability of phosphate and pyrophosphate (PPi) to complex calcium and mediate the binding of two distinct type IV collagenases, ie, matrix metalloproteinase-2 and –9 (MMP-2 and MMP-9). CaP and CaPPi formation were monitored turbidometrically (405 nm). Scatchard analysis (Ca/P ratio) revealed that the CaPPi aggregate (1.96) was similar in chemical composition to calcium pyrophosphate dihydrate (CPPD) (2.00) and distinct from BCPs: amorphous (1.50) or crystalline (1.66) CaP, ie, hydroxyapatite. CaP and CaPPi interactions with MMPs and fibrin binding activity were monitored by gelatin zymography. CaP actively bound MMP-2 (72-kDa) and MMP-9 (92-, 130-, 225-kDa), forming a precipitable high Mw complex with low electrophoretic mobility. CaP:MMP complexes readily bound fibrin. CaPPi was inactive in binding MMPs and did not promote fibrin uptake. Electrophoretic analysis (8.5% SDS-PAGE) revealed that fibrin matrices formed with CaP and CaPPi contained characteristic – dimers, ß-monomers, and higher Mw -polymers. Artifactual fibrin trapping was minimized using diluted plasma and a washing step. Inflammatory synovial fluid MMPs aspirated from 3 patients also demonstrated fibrin binding consistent with CaP mediation. Synovial MMPs were found to precipitate upon centrifugation consistent with CaP mediation. CaP may influence localization of MMPs at sites of fibrin deposition in crystal arthropathies such as BCP disease.

[59] Improving pancreatic islet cell viability and function during the peri-transplant period. Magali J. Fontaine, Christine Papadea, Inderjit Singh, and Lyndon Key, Medical University of South Carolina, Charleston, SC.

The success of islet cell transplantation to cure type I diabetes is hampered by the early loss of islet cell viability and function from cytokine-mediated injury. Release of inflammatory cytokines is regulated by NF-kappa B transcription factor, which can be inhibited by the drug statin. We tested mouse pancreatic islets from two donor groups, A and B. Group A, C57/B6 mice, were preconditioned with statin injections (1 mg/kg) 24 hr and 12 hr prior to pancreas harvest and islet isolation. Group B (control) C57/B6 mice received no preconditioning regimen. We compared cell viability and function of isolated islets from either donor group A or B. Islets were cultured in supplemented CMRL Medium-1066. At 0, 6, 24, and 48 hr, medium culture supernatants from samples of 20 islets from both groups A and B were analyzed for lactate dehydrogenase (LDH) to quantitate cytolysis. Additionally, samples of 20 islets from both groups A and B were stimulated with high glucose solution (20 mmol/L) to measure insulin release using a radioimmunoassay. At 0, 6, and 24 hr, insulin response varied from a 1.2- to 2-fold increase from base line (no difference between groups A and B). At 48 hr, the insulin response was increased (3-fold) in the statin treated islets from group A, compared to group B (no response). At 48 hr, the LDH release was increased (3-fold) in the islet culture supernatant from group B (control) compared to group A (1.5-fold). In conclusion, the islets treated with statin (group A) showed better insulin response to glucose with least cytolysis at 48 hr compared to control islets, untreated (group B), suggesting that statin drug may have a protective effect on islet function and viability during the early phase following islet cell isolation.

[60] X- ray synchrotron radiation microprobe analysis of the beta cell from the islet of Langerhans. Lahsen Assoufid, Stefan Vogt, Dan Legnini, Jorg Maser, and Magali J. Fontaine, Argonne National Laboratory, Argonne, IL, and Medical University of South Carolina, Charleston, SC.

A hard X-ray microprobe at the 2-ID-E beamline of the Advanced Photon Source (APS) (Argonne National Laboratory, IL) is used to map and quantify trace element distributions of the beta cell of the islet of Langerhans. Mouse islets are isolated after pancreatic harvest and cultured overnight. Individual islets are then trypsinized and stained with dithizone (DTZ). DTZ stains zinc in the insulin granules resulting in a characteristic red stain, allowing the distinction between beta cell and nonbeta cell. Identified beta cells are then mounted on electronmicroscope grids and transferred to the hard x-ray microprobe. The APS hard X-ray microprobe uses Fresnel zone plates to focus x-rays with a photon energy of 10,000 electron volts into a spot of approximately 300 nm on the specimen. Characteristic X-ray fluorescence radiation (XRF) excited in the focal spot is detected using an energy dispersive solid-state X-ray detector. Due to low background signal in X-ray–excited X-ray fluorescence, trace amounts on the order of tens of attograms can be detected. Using spectral analysis, the spatial distribution of elements from phosphorus, selenium, and chloride to copper and zinc can be extracted from a single scan. We found that zinc, copper, nickel, and cobalt are evenly distributed throughout the sampled beta cell, while iron, potassium, sodium, chloride, calcium and phosphorus showed an uneven distribution inside the beta cell. Ongoing studies are now correlating the beta cell elemental map with beta cell morphology, viability and function. (Supported by US Dept. of Energy, Office of Science, contract no. W-31-109-Eng-38.)

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