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Breast and prostate cancers are the two most common malignancies occurring among women and men and are among the leading causes of cancer deaths in the United States. Breast cancer is not only a systemic disease with major physical impairment, but it is also associated with significant psychosexual problems. The psychological and physical consequences of breast cancer have a far-reaching social impact on relatives and friends of patients suffering from this disease. It appears that no one is entirely unaffected. It is estimated that 192,000 women will be diagnosed with breast cancer this year and approximately 25% of them will die of this disease within the next 10 years. Similarly, for men, prostate cancer has become a silent epidemic. The American Cancer Society predicts one man in six will be diagnosed with prostate cancer during his lifetime. Reports indicate that 198,000 men in this country have been diagnosed with prostate cancer just this year. African-American males are about 34% more likely to develop the disease than Caucasian males and more than twice as likely to die from it. To improve quality of life and to reduce mortality from these malignancies, health care providers must now give attention to health protection and promotion, risk prevention, and quality of health status among all populations, regardless of age, color, social status, and ethnicity. A combined strategy of early detection and access to high quality treatment is the key in reducing mortality from breast and prostate cancers. Early detection and quality therapy require a better understanding of the pathogenesis of these two malignancies. Currently there is ample evidence in the literature to suggest that there are common features between breast and prostate cancers. The evidence includes epidemiology, dietary factors, steroid hormones, genetic alterations, growth factors, theories of breast and prostate cancer development, and pathology. The rationale for finding common features of breast and prostate cancers is to trigger interest in finding common pathology in these two cancers. This knowledge may lead to strategies for common diagnostic procedures, therapy, prevention, monitoring, and cure.
[2] Morphoproteomic confirmation of an overexpressed angiotensin system in invasive breast carcinoma. RE Brown, N Siegelmann-Danieli, PL Zhang, GC Wood, V Khandelwal and ID Bucaloiu. Geisinger Medical Center, Danville, PA.
Low activity genotypes of angiotensin-converting enzyme (ACE), which converts angiotensin (Ang) I to Ang II, have been associated with a reduced risk of breast cancer (
Cancer Res 2003;63:573–578
[3] Immunocytochemical (ICC) detection of hormone receptors in breast carcinoma by quantitative image analysis (QIA). JW Prichard, H Liu, F Lin, Geisinger Medical Center, Danville, PA.
Recent analyses of estrogen receptor (ER) using a 5% cutoff value reported 98.3% agreement between cytologic and surgical specimens ( Anal Quant Cytol Histol 2004;25: 323 ). Another study comparing hormone receptors to outcomes showed that an all-or-nothing cutoff for ER is preferable to the 5% cutoff ( Cancer 2005;103:164[Medline] ). Cytologic studies using a 1% cutoff and including progesterone receptor (PR) have not been reported. This study evaluates the expression of ER and PR on matched cytology and surgical specimens using QIA to provide information on the reliability of ICC on cell blocks. In 48 consecutive cases of invasive breast carcinoma with matching cytology and surgical specimens, formalin fixed, paraffin-embedded sections were stained with monoclonal antibodies to ER and PR. The ER and PR staining was measured using quantitative ACIS system at 1% cutoff. Tumor cells in selected areas with carcinoma were counted on histology and corresponding cytology specimens. Our histology gold standard resulted in 70% positivity, similar to previously published rates. The concordance between cytology and surgical specimens was 70% for ER and 82% for PR, respectively, with either ER or PR staining resulting in concordance of 88%. False positive cases were not noted. The false negative rate at 5% cutoff was 27% for ER and 15% for PR; it improved to 23% and 10% at 1% cutoff. The specificity and positive predictive value was 100% for ER and PR, whereas the negative predictive value for ER, PR, and combined ER-PR at 5% cutoff was 52%, 73%, and 63% respectively, which improved at 1% cutoff to 56%, 78%, and 67%, respectively. In conclusion, these data indicate that ICC detection of ER and PR on cell blocks using QIA is a reliable test when the result is positive. Using combined results of ER and PR assays and cutoff of 1% rather than 5% increases the sensitivity and negative predictive value. Different methods of preparation and fixation, scant material, and frequent absence of a positive internal control in cytologic material may contribute to the false negative rate for cytologic material. Therefore, repeat testing of subsequent surgical specimens is recommended if the cytology result is negative.
[4] High concordance of expression of cytokeratin cell origin markers, ER-
, PR, Her-2/neu, and EGFR in co-existing in situ and invasive ductal carcinoma of the breast. S Steinman, J Wang,* P Bourne, Q Yang, L Schiffhauer, Xi Wang, SI Hajdu, P Tang, University of Rochester Medical Center, Rochester, NY, and *RTI Health Solution, Research Triangle Park, NC.
We have previously shown that pure ductal carcinoma in situ (DCIS) of the breast can be divided into 3 subtypes (luminal, basal/stem, and null) based on the expression patterns of five cytokeratin (CK) cell origin markers (CK5/6-stem, CK8 and 18-luminal, and CK14 and 17-basal), and their distribution is associated with nuclear grade and expression of estrogen receptor-alpha (ER-), progesterone receptor (PR), Her-2/neu, and epidermal growth factor receptor (EGFR). We further explored the relationship of expression of these CKs, ER-, PR, Her-2/neu, and EGFR between co-existing DCIS and invasive ductal carcinoma (IDC) and determined the differential expression patterns between co-existing DCIS with IDC and pure DCIS that we studied earlier. Immunohistochemical stains were performed on 52 non-high-grade and 44 high-grade breast ductal carcinoma with coexisting DCIS and IDC with these molecular markers. The results show that: (i) There is high a degree of concordance of expression of these markers between coexisting DCIS and IDC, suggesting that DCIS is frequently the precursor lesion for its coexisting IDC. (ii) The rate of discordant expression of all these markers is low, and seems more frequently associated with high-grade carcinoma, indicating that DCIS is not an obligate precursor for IDC; IDC may either evolve de novo or through altered pathway(s). (iii) Similar expression patterns of CKs, ER-, PR, Her-2/neu are observed between co-existing DCIS and IDC and pure DCIS, suggesting that pure DCIS in most instances is an earlier lesions than co-existing DCIS and IDC during breast carcinogenesis. (iv) The differential expression pattern for EGFR that is observed between co-existing DICS and IDC and pure DCIS suggests that at least some pure DCIS is molecularly distinct from co-existing DCIS and IDC.
[5] Clinical and laboratory investigation of ambiguous genitalia. WE Winter, University of Florida College of Medicine, Gainesville, FL
Gender is classified as male, female, or ambiguous (eg, ambiguous internal or external genitalia are present at birth). In utero, genes on the Y chromosome are predominantly responsible for stimulating the development of the testes from the medulla of the undifferentiated gonad. In the absence of a Y chromosome, the cortex of the undifferentiated gonad develops into an ovary. Testosterone (T), dihydrotestosterone (DHT), and mullerian inhibiting hormone (MIH) are responsible for expression of a normal male phenotype. In the absence of T, DHT, and MIH, the female phenotype develops in utero. Ambiguous genitalia are classified as male pseudohermaphroditism (ie, testicular tissue is present), female pseudohermaphroditism (ie, an ovary or streak gonad is present) and true hermaphroditism (ie, there is histologic evidence of ovarian and testicular tissue). While gender is a complex concept, the best single guide to the biologic gender of an individual is the histologic appearance of the gonads. Histology reveals the expression of the genes and their products (eg, enzymes, hormones, receptors, etc). The most common etiology of ambiguous genitalia is female pseudohermaphroditism from exposure of the female fetus to excessive concentrations of adrenal androgens in utero caused by 21-hydroxylase deficiency. Male pseudohermaphroditism is much less common and can result from failure of gonadotropin secretion or response, testicular dysgenesis of any cause, inborn errors in testosterone biosynthesis or response (eg, androgen resistance).
[6] Microarray technology: bringing the big picture to clinical decision making. ST Lott, University of Florida Health Science Center–Jacksonville, Jacksonville, FL
The completion of the Human Genome Project and unprecedented advances in nucleic acid based technologies have ushered in a new era of scientific inquiry into the molecular basis of human disease. With its potential ability to query the entire transcriptome, genome, or proteome in a single assay, DNA microarray or chip technology has become an inescapable term in the current scientific vernacular. Unfortunately, its adoption in the clinical setting has been slow with only one instrument having received FDA approval in late 2004. As with any new technology, considerable hurdles remain on its path to acceptance as a diagnostic and/ or prognostic assay; however, the promise of microarray technology demands that we continue to improve and validate this platform. This presentation will explore the variety of microarray platforms, the types of tests that can be performed, and the difficulties associated with data analysis. In addition, the ability of this technology to analyze thousands of parameters simultaneously will be discussed in the context of its direct impact on clinical decision making
[7] Advanced mutation detection for hereditary sensorineural hearing loss through a comprehensive microarray. I Schrijver, J Rodriguez-Paris, P Gardner, Stanford University Medical Center, Stanford, CA.
The recently implented Universal Newborn Hearing Screening (UNHS) is a pivotal first step in a successful and cost-effective program towards prompt diagnosis and management of children with hearing loss (HL), but implementation and follow-up testing guidelines vary greatly among states. It is expected that UNHS will be coupled with guidelines for molecular follow-up, because >50% of congenital HL is genetic. Non-syndromic (isolated) HL accounts for >70% of these cases. Inherited HL is characterized by impressive genetic heterogeneity and many mutations escape diagnosis due to limited molecular analyses. We have developed a stepwise and practical testing algorithm based on the currently available molecular diagnostic tests. In addition, however, we created a research and diagnostic microarray that may revolutionize genetic HL testing and can help elucidate the genetics and biology of the inner ear through better characterization of mutations in involved genes. Through arrayed primer extension (APEX, Asper Ltd), 198 mutations in 8 genes (the connexin genes GJB2, GJB6, GJB3, GJA1; SCL26A4, SLC26A5; mitochondrial genes 12S rRNA and tRNA Ser) can be evaluated simultaneously. The currently available assays mainly concentrate on the GJB2 gene, and most evaluate only a single gene at a time. Our pilot study with the APEX array, by contrast, demonstrates easy detection of multiple mutations in multiple genes that contribute to syndromic and non-syndromic HL, excellent sensitivity and specificity, low cost, and robust performance. This approach can save diagnostic time and resources in an otherwise difficult to categorize group of disorders. It markedly increases the sensitivity of genetic HL testing, and is easily modified. Early identification of mutations impacts counseling, management, and treatment of children with HL and their families.
[8] Tissue processing 2006–a new look for an old friend. SE Vernon, University of Miami, Miller School of Medicine, Miami, FL.
Tissue processing methods in histopathology have remained essentially unchanged for nearly a century, while the demands for more detailed, complex diagnoses have accelerated at an astonishing rate. While the tried and true methods of formalin fixation, overnight processing through graded alcohols followed by paraffin infiltration and embedding, and hematoxylin and eosin (H&E) staining continue to form the basis for diagnosis, newer treatments, including target directed therapies, have stimulated investigation into alternative methods of tissue processing. Our laboratory has pioneered a continuous throughput, rapid tissue processing system (CTRTP) that allows for traditional histopathologic techniques as well as molecular analyses from the same paraffin embedded tissues. A molecular friendly fixative (not formalin) is required, as formalin fixation essentially denatures many macromolecules beyond the point of useful recovery. Carefully controlled microwave energy is delivered to the tissues, accelerating additional fixation and reagent penetration. After vacuum infiltration, paraffin blocks and subsequent routine diagnostic slides are prepared in traditional fashion. The instrument can process samples in about 60 min, and traditional embedding, microtomy, and staining may add another 1–2 hr, depending on staffing and workloads. Extensive studies have shown that special histo-chemical stains and immunohistochemistry are improved or unaffected by the technique. Thus a diagnosis can be reported on the same day as the surgical procedure in many, if not most cases, which can be advantageous for patients and clinicians alike. Other benefits include the elimination of toxic chemicals from the workplace, notably formaldehyde and xylenes. Studies in our laboratory have shown that macromolecules are well preserved, not just DNA, but also large RNAs and proteins. Paraffin sections can be micro-dissected for PCR analyses, eliminating the need for a frozen tissue bank. Last but not least, turnaround times for the completion of surgical pathology reports have been considerably shortened.
[9] Role of mitochondrial dysfunction in the etiology of neoplasia. E Fosslien, College of Medicine, University of Illinois at Chicago, Chicago, IL.
The primary objective of this paper is to examine the role of altered mitochondrial energetics in the etiology of neoplasia, due to germ-line or somatic mutations of mitochondrial (mt) or nuclear (n) deoxyribonucleic acid (DNA) coding for mitochondrial proteins, or resulting from aberrant morphogen signaling. A secondary objective is to clarify the role of abnormal mitochondrial energetics in determining tumor morphology, as suggested in my prior publications. The pathogenesis of neoplasia caused by primary mitochondrial dysfunction was analyzed and compared with secondary mitochondrial dysfunction in tumorigenesis resulting from aberrant morphogen signaling. This showed that cancer cells exhibit high glycolytic rates, increased lactate production, and mitochondrial dysfunction in spite of increased transcription of mtDNA- and nDNA-encoded components of the mitochondrial oxidative phosphorylation system (OXPHOS). OXPHOS-dependent Complex V ATP generation is insufficient to maintain the inner mitochondrial membrane potential (
m), which instead is supplemented from glycolytic ATP via reversed proton flux through Complex V. Mutations of OXPHOS Complex I and Complex II are associated with basal cell carcinoma and pheochromo-cytoma and paraganglioma, respectively. Mutation of fumarate hydratase is associated with uterine leiomyomas; the activity of aconitase, important in prostate metabolism, declines with age, and aging is a risk factor for neoplasia. Restoring aconitase function and thereby mitochondrial energy metabolism using transgenic frataxin normalizes growth behavior of colon cancer cells. Signaling by bimodal morphogens of the transforming growth factor (TGF)-beta family is often impaired in neoplasia. TGF-beta1 can regulate OXPHOS by regulating expression of adenine nucleotide translocator (ANT) and cyclooxygenase (COX)-2. Arachidonic acid, the substrate for cyclooxygenase, inhibits OXPHOS. Prostaglandins (PG) J2 and 15-deooxy- 12,14-J2 (15d-PGJ2) show bimodal effects, stimulating or inhibiting Complex I; PGJ2 induces proliferation at low and inhibits growth at high concentrations. In conclusion, mutations of components of the mitochondrial energy metabolism system can cause neoplasia. Secondary mitochondrial dysfunction caused by chronic inflammation or by altered bimodal growth factor signaling can also result in tumor formation. Experimental evidence and theoretical considerations advocate that restoring proper mitochondrial energetics may normalize tumor metabolism and restore normal tissue architecture.
[10] Workshop on real-time integration of pathology coagulation consultations into the practice of transfusion medicine. RF Reiss, RE Brown. Columbia University, New York, NY; Geisinger Medical Center, Danville, PA.
The charge to the transfusion service is to provide patients with blood component and derivative and recombinant therapies that are specific to their needs, as determined by clinical and laboratory parameters, and in a dosage and unit type that is appropriate and efficacious, while minimizing the risk of transfusion-associated reactions and adverse effects. The pathologist can play a central role in this by integrating coagulation testing with the clinical situation and discussing with the patient’s physician, in a proactive fashion, the therapeutic options in the context of the immediate clinical situation. This workshop illustrates such an approach and invites open discussion and participation by the attendees at a luncheon session, sponsored by the Association of Clinical Scientists’ scientific section on hematology and transfusion medicine. Five case studies describing clinical hemostasis problems that are frequently encountered by transfusion services are posted on the Association’s website (www.clinicalscience.org). Discussion of these clinical scenarios will be geared toward their rapid diagnosis utilizing immediately available screening tests that are performed by most laboratories. A table reflecting the prothrombin and activated partial thromboplastin times with increasing dilutions of pooled normal plasma will also be provided. A subsequent posting on the website will propose recommended approaches to the diagnosis and management of these cases. These will form the basis for an interactive discussion and an exchange of alternative approaches at the luncheon session. Finally, one of us (REB) will illustrate how the Geisinger Medical Center has utilized component triggers to provide immediate (24 hr/7 da) interactions between the blood bank and pathologist and the pathologist and the patient’s physician, to establish a dialogue regarding additional coagulation testing in guiding transfusion therapy. The presentation will illustrate the impact of such an approach on blood component utilization and clinical outcomes.
[11] Unique automation systems in a high volume clinical reference laboratory. C Hawker, W Roberts, T Ring, B Choi, E Bamberg, B Corbin, A DaSilva, ARUP Laboratories, Salt Lake City, UT, and Motoman, Inc., Irvine, CA.
ARUP Laboratories provides esoteric testing to hospitals and other clients in the 50 states. We receive 30,000 patient specimens each day and have implemented unique automation systems to track, transport, sort, store, and retrieve these large volumes. These include an automated track and sorter system that can transport and sort up to 7000 specimens per hr, two robotic storage sorters connected to the track that can each store more than 1000 specimens per hr in stainless steel trays, and an automated storage and retrieval system (AS/RS) in a two-story freezer that can store up to 2.3 million specimens with robotic retrieval of any specimen in <2.5 min. We have now started a 4-year endeavor in our largest laboratory section to connect up to 15 automated analyzers to the track. This section performs 7–8,000 tests daily on 5–6,000 specimens over a range of 140 chemistry and immunochemistry tests. This endeavor requires the development of several unique technologies, not previously attempted in a clinical laboratory. These include: a robotic thawing and mixing workstation (half of the specimens for this section arrive frozen), an automated spectrophotometric detection system for interferences such as clots, fibrin, lipemia, icterus, and hemolysis that scans through the side of a closed tube covered with labels, and a machine vision system for automated inspection of tubes to assure the tube is correct for the ordered tests. At the time of this conference, the first and third systems will have been developed, although not yet installed, while the interference detection system is still being developed.
[12] Novel approaches for collection of DNA for genetic and epidemiological studies. SM Hopfer, CJ Kolakoski, LA Avery, MC Senh, RW Ryan. University of Connecticut School of Medicine, Farmington, CT.
Collection, transportation, and storage of suitable amounts of DNA for clinical studies have changed significantly over the past few years. Whole blood, with subsequent separation of white cells, remains the gold standard for PCR-based testing. The collection of blood and the process of extracting DNA from white cells is laborious and time-consuming, with the final product utilizing significant freezer space for storage. We describe a simple procedure for collection, transportation, and concentration of adequate numbers of buccal cells for 100% successful amplification. Ten ml of alcohol-based mouth rinse (concentrations of 15, 22, and 50%), flavored non-alcoholic mouth rinse, sterile saline solution (NaCl), or tap water were vigorously swished in the mouth for 60 sec and then expectorated into a 50 ml conical polypropylene centrifuge tube. Specimens were analyzed immediately or left at room temperature for one week to simulate time and conditions during transportation. Samples were then centrifuged at 2000 x g for 10 min in the original conical tube. Immediately following centrifugation the supernatant was discarded and 100 µl of PCR wash buffer was added to the pellet. The tube was vortexed and 300 µl of liquid placed directly into the PCR extraction tube or onto Whatman No. 903 filter paper. Roche Gold reagents for identifying mutations associated with cystic fibrosis were used as an indicator system. No significant differences were observed between any of the rinses whether amplified immediately or after storage for 7 days at room temperature. The DNA placed on the filter paper was stable for at least 1 m0, but may be stable indefinitely. This collection procedure is simple and requires no specialized equipment or skills. Specimens can be easily transported without refrigeration since DNA is stable on the filter paper.
[13] Acid-extractible nuclear antigen (ENA) procedure suitable for a small laboratory–II. CR Hamlin, Case Western Reserve University School of Medicine, Cleveland, OH.
In May 2002, I described an ENA procedure that was well suited for my laboratory. In September 2005, the company ceased production of the kit. I now describe a replacement procedure. Most ENA assays performed by immunofluorescence or enzyme assays require a specific slide or well for each antibody measured, with appropriate calibration and quality control. The replacement kit detects anti-ENA antibodies by counter-current immuno-electrophoresis and is available from The Binding Site (San Diego, CA). Serum and antigens are applied separately to an agarose gel. When voltage is applied, the antibody and the antigen migrate towards each other and produce an immunoprecipitation line where they meet. Up to 10 samples can be evaluated with each gel. Positive findings require antibody identification and quantitation by a separate procedure. The only disadvantage of this screening procedure is that low-titer antibodies (~1.0 – 1.3) sometimes do not give a discernable precipitin arc. A major advantage is that the procedure is cost-effective.
[14] Comparative evaluation of two assay systems for automated measurement of hemoglobin A1c. C Papadea, Medical University of South Carolina, Charleston, SC.
Glycated hemoglobin, measured as HbA1c, is a standard tool for monitoring glycemic control in individuals with diabetes mellitus. Emphasis on intensive medical and educational interventions to improve outcomes and reduce hospital length-of-stay required our laboratory to provide HbA1c test results faster than once/weekday by our batch-process cation-exchange chromatography method (CE). In addition to CE, the most commonly used automated assays in clinical laboratories are based on immuno-inhibition (IA) or boronate affinity chromatography (BA) techniques. Hb variants S, C, or elevated F, which are prevalent in our patient population, can falsely increase or decrease HbA1c results, depending on the assay. We selected IA for random-access processing, rapid (<2 hr) continuous turnaround, and easy sample preparation. Our study examined whether the reproducibility, analytical measuring range (AMR), and patient results by IA were equivalent to CE. Both methods are certified for accuracy and reproducibility (CV <5%) by the National Glycohemoglobin Standardization Program. Patient samples (n = 150) tested by IA and CE contained normal and abnormal Hbs and increased Hb F. Those with highly elevated S, C, or F were also referred for BA testing, which is unaffected by hemoglobinopathies. Bi-level controls were assayed daily (n = 25) for reproducibility. IA results: CV<4%; AMR, 4–18%; regression analysis of paired results (IA [y] versus CE [x]) for 120 samples containing HbAA, AS, or AC produced the following statistics: slope 0.95; intercept –0.09; bias –0.5; and r 0.984. In 30 samples containing Hb S, C, or SC >60% or Hb F >8%, HbA1c was underestimated by IA and overestimated or not recognized by CE. In conclusion, when compared with CE, IA is faster and has similar performance characteristics for the majority of samples. Interference by elevated Hb variants confounds both methods, but this limitation did not offset the advantages of IA to replace CE for HbA1c testing by our laboratory.
[15] Serum zinc and diabetes. R Rosebrock, P Laborde-Lahoz, C Ahn, M Bhattacharjee, University of Texas Medical School, Houston, TX and Beth Israel Deaconess Medical Center, Boston, MA.
This study resulted from the chance observation of significant hyper- instead of hypozincemia in the investigation of a patient exhibiting poor wound healing. This observation led to the systematic examination of zinc levels in patients with unexplained anemia, peripheral neuropathy, and immune dysfunction. The original protocol sought to evaluate the causes and manifestations of hyperzincemia, and potential toxic effects of zinc. It was subsequently found that an error in laboratory reporting of zinc values had exaggerated the true serum levels of zinc in the population of patients being studied. After correction of the units of measurement, however, a subset of patients was found to have actual hyperzincemia. Serum zinc levels were obtained using flame atomic absorption spectrometry. The reported reference range for normal zinc levels from this laboratory was 580–1060 µg/ L. Six of 24 patients were found to have hyperzincemia; 5 of the 6 hyperzincemic patients also had diabetes. Thus a trend toward elevated zinc levels was observed for patients known to have type 2 diabetes mellitus. We divided the patients into two single-variable groups: one with known diabetes mellitus (n = 14), and another without known diabetes (n = 10). The observed values for each patient in the cohort were compared using the exact Wilcoxon rank-sum test; a p-value of 0.042 showed that the zinc levels were significantly higher in the diabetic cohort. In type 2 diabetics, the effect of higher extracellular zinc on pancreatic islet cells may be physiologically relevant, as suggested by the mineral’s potential involvement in a negative feedback mechanism via its action on KATP channels.
[16] Diabetes alters pressure-induced regulation of MAPKs in the rat inferior vena cava. KM Rice, D Desai, DL Preston, EM Walker, ER Blough, Marshall University, Huntington, WV.
Autologous vein grafts remain the only surgical alternative for many types of vascular reconstruction. Diabetic patients are often the recipients of vascular interventional procedures, including vein grafts. Compared to non-diabetics, vein grafts in the diabetic population are often characterized by increased vein graft neointimal hyperplasia along with significantly higher vein graft failure rates. The reasons underlying this phenomenon are not entirely clear; however, recent data suggest that diabetes influences endothelial and smooth muscle cell signaling and protein expression. The route whereby these cellular changes may influence graft failure is not known. We and others have recently demonstrated that phosphorylation of the mitogen activated protein kinase (MAPK) is regulated by mechanical stimuli in the vasculature. It is thought that the MAPK proteins are important as regulators of protein synthesis and cell differentiation, as mediators of apoptosis and the inflammatory response, and as key players in the control of load-induced alterations in protein expression. Here, using lean and obese Zucker rats, we investigated how diabetes may affect inferior vena cava (IVC) MAPK content and the ability of the IVC to induce MAPK phosphorylation (activation) in response to a loading (120 mmHg) stimulus. Immunoblotting demonstrated that extracellular signal-regulated kinase (ERK) 1/2-MAPK expression increased in the diabetic IVC, while p38- and c-Jun NH2 -terminal kinase (JNK)-MAPK content remained unchanged. Using an ex vivo IVC preparation, we found that ERK 1/2-MAPK and JNK-MAPK load-induced phosphorylation was unaffected by diabetes while p38-MAPK phoshorylation in the diabetic IVC was diminished. These observations confirm previous data showing that MAPK proteins are mechanically regulated, and, in addition, suggest that ERK 1/2- MAPK expression and p38-MAPK activation are controlled differently in patients with diabetes. Taken together, these data may help explain why diabetes may be associated with increased incidence of vein bypass failure.
[17] Organization and delivery of molecular diagnostics in a hospital laboratory. FL Kiechle, Memorial Healthcare System, Pathology Consultants of South Broward, Hollywood, FL.
For the past 15 years the Department of Clinical Pathology at William Beaumont Hospital in Royal Oak, MI, has offered molecular diagnostic testing. The initial Southern blot assays have been replaced by real-time amplification methods. In 2005, 60,000 assays were performed and 33 billable procedures were offered. The strategy for selecting tests requires the evaluation of market need, cost for performing the assay, reimbursement based on the potential payer mix, availability of trained medical technologists, and administrative support. Only 6 molecular assays currently lose money: hepatitis C viral load, HIV viral load, hepatitis C quantitation, hepatitis B viral load, mycobacterium (TB), and cytomegalovirus. Tracking physician ordering patterns aids in identifying market segments where focused sales efforts may increase volume. In 2003, the extended net revenue in molecular diagnostics was $1,750,294 with a margin of 58.5%. At that time 26 tests were offered and 6 of them lost money. Therefore with careful planning it is possible to generate a profit in a molecular diagnostics laboratory in a hospital.
[18] Primary Burkitt lymphoma of the testes in an HIV+ patient. J Lazarchick, Medical University of South Carolina, Charleston, SC.
A 47-year-old male with a history of HIV and hepatitis C positivity was first seen in 2005 with painless right testicular swelling. He underwent a right inguinal orchiectomy. Microscopic examination of the lesion demonstrated a diffuse infiltrate of intermediate-sized lymphocytes with round nuclei and clumped chromatin with a "starry sky" background. The cells were positive on immunostaining for CD20 and >99% of the cells were positive for Ki67. Kappa light chain restriction was evident. Only scattered CD3+ cells were noted. These findings were consistent with Burkitt lymphoma. Staging of the lymphoma showed no evidence of nodal or extranodal disease in any other site. A discussion of primary testicular lymphoma will be presented, including the incidence, morphologic subtypes, therapeutic options and prognosis of this lymphoproliferative disorder.
[19] Heparin-induced thrombocytopenia. D Seckinger and D Andrews, University of Miami School of Medicine, Miami, FL
Clinically significant heparin-induced thrombocytopenia (HIT) is an immune-mediated clinicopatholologic syndrome that often results in life-threatening venous and arterial thrombosis. A clinically insignificant from of HIT is characterized by reversible thrombocytopenia and is not immune-mediated. This benign form of HIT (called type I HIT) usually occurs shortly after starting an intravenous infusion of unfractionated heparin in a patient and is not associated with clinical sequelae. Type II HIT (hereafter referred to as HIT) is clinically significant and develops after an immune response has mounted, several days after the initiation of heparin (unfractionated or low molecular weight). While thrombocytopenia is a key feature of this disorder, HIT is a thrombotic disorder and is not associated with bleeding. The diagnosis of HIT is difficult, which explains why a large proportion of HIT cases are diagnosed only after a thrombotic event, often when disease has progressed to life- and limb-threatening levels of severity. It is essential that when HIT is suspected on clinical grounds, heparin therapy should be stopped and the patient should be switched to alternative anticoagulants. The mortality from HIT not treated by alternative anticoagulation is approximately 30%. The target antigen in HIT is platelet factor 4 (PF4), a small protein within the alpha granules of platelets that, when released, binds to heparin. Antibodies to a complex of PF4 and heparin are thought to trigger a cascade of events that leads to platelet and endothelial activation and a hypercoagulable state. In conjunction with clinical criteria, antigen-based assays designed to detect anti-PF4 antibodies are the mainstay of laboratory diagnosis of HIT. Functional assays that assess the ability of anti-PF4 antibodies to activate platelets in vitro are employed, but these assays are not available widely and are difficult to standardize. Recently, the presence of anti-PF4 antibodies has been shown to indicate a poorer outcome in clinical settings other than classic thrombosis-associated HIT. It seems likely that the spectrum of diseases associated with anti-PF4 antibodies will broaden.
[20] Tacrolimus (FK 506)–induced thrombotic thrombocytopenia purpura after lung transplant. F Boctor, M Marques, H Burford, J Reardon, University of Alabama, Birmingham, AL.
Thrombotic thrombocytopenia purpura (TTP) is a syndrome characterized by microangiopathic hemolytic anemia and thrombocytopenia. Autoantibodies binding to ADAMTS-13, a metalloprotease responsible for cleaving von Willebrand multimers, have been implicated in many cases. However, TTP can also occur in association with infections, drugs, and organ transplantation. In such cases, antibodies to ADAMTS-13 are usually absent and other causes of endothelial damage may play a role. A 56-year-old female with a history of a lung transplant was admitted with fatigue, confusion, and abdominal pain. She had been discharged from a local hospital with severe epistaxis two days prior. The bleeding stopped with local measures and the discontinuation of warfarin, which she had been taking for a deep venous thrombosis. Her immunosuppression regimen consisted of tacrolimus (FK 506), sirolimus, and prednisone. Physical exam was notable for multiple ecchymosis on her extremities. On laboratory evaluation, she had anemia (hemoglobin 9.7 gm/dl), thrombocytopenia (platelet count 26.8 x 103), and elevated lactate dehydrogenase (LDH), 607 IU/L (normal 125–220 IU/L). Her serum creatinine level was 1.8 mg/L, which was slightly increased from baseline. Peripheral blood smear revealed a moderate number of schistocytes. The FK-506 was discontinued, and after 13 plasma exchanges her platelet count improved to 113 x 103. Platelet count at two week follow-up was 141 x 103. ADAMTS-13 activity collected prior to plasmapheresis was 80% (normal >67%), and no inhibitor was detected. There are reports of TTP related to FK-506 in bone marrow and kidney transplants. In most cases, symptoms occurred shortly after the offending drug was initiated. Our case is the first described case of FK-506-induced TTP in a lung transplant patient after 3 years of therapy.
[21] Paradoxical behavior of pore-forming proteins in paroxysmal nocturnal hemoglobinuria (PNH). JS Krauss, Augusta, GA.
Paroxysmal nocturnal hemoglobinuria (PNH) is a rare condition due to an acquired X-linked recessive mutation of the PIG-A gene leading to a lack of the glycophosphatidyl inositol-anchor proteins (GPI-APs) in the blood cell membrane. Certain GPI-APs inhibit complement and their lack leads to non-immune hemolysis in PNH. Paradoxically, possibly due to preferential immune destruction of normal blood cells, the PNH clone predominates vs normal blood cells, despite the heightened susceptibility of the PNH cells to complement-mediated lysis. Here, another paradox is noted for PNH: 3 pore-forming proteins, complement, perforin, and aerolysin, which might be expected to have consistent effects upon the set of both normal and PNH cells, because all three proteins cause cell lysis, have been shown to have differing effects upon the combination of both normal and PNH cells. In the case of (a) complement, PNH cells are more vulnerable to complement-mediated lysis than normal cells, whereas (b) both PNH and normal cells are equally susceptible to lysis by free-perforin, whilst (c) normal and PNH cells are less sensitive to lysis by aerolysin than are normal cells. The susceptibility of PNH cells to complement has been employed diagnostically in Ham’s test for acid hemolysis and the sucrose lysis test, whereas the fluorescent inactivated proaerolysin variant (FLAER) test has utilized the preferential binding of normal, as opposed to PNH, cells by aerolysin. Because free-perforin does not differentiate between normal and PNH cells, it is not diagnostically useful, however. Thus, the paradoxical effects of the pore-forming proteins, complement, and aerolysin, upon the set of both normal and PNH cells, remain useful for laboratory diagnosis of PNH.
[22] The utility of eluate testing following microscopically positive direct antiglobulin tests with anti-IgG (micro-positive DATs). E Richa, G Benidt, C Tauscher, J Stubbs, Mayo Clinic, Rochester, MN.
Serologic data from eluates prepared from red cell samples with micro-positive DATs were analyzed for the period of January-June 2005. Ninety-nine (50.8%) of 195 elution procedures followed micro-positive DATs. The eluates from micro-positive DAT samples yielded the following:
On the basis of this analysis, it was concluded that routine preparation and testing of eluates in the setting of a micro-positive DAT with anti-IgG is unwarranted at our institution.
[23] The laboratory evaluation of antiphospholipid syndrome. SM Abo, VA DeBari, School of Graduate Medical Education, Seton Hall University, South Orange, NJ.
The antiphospholipid syndrome (APS) was first described in 1986. The original association of this hypercoagulable state with anticardiolipin antibodies (aCL) resulted from the synthesis of evidence stemming from laboratory findings in systemic lupus erythematosus (SLE), ie, the frequent occurrence of false-positive VDRL tests and the paradoxical observation of the so-called lupus anticoagulant (LAC), an increase in phospholipid (PL)-dependent clotting times. By the early 1990s, it was becoming clear that a co-factor was involved in the reaction of antibodies to PL(aPL) in SLE patients with secondary APS and that this was a hitherto obscure protein, beta-2 glycoprotein I (ß2GPI). In the intervening years, it has been established that ß2GPI and other PL-binding proteins such as prothrombin (Pro) are the relevant antigens in APS and assays for these antigens have been developed, standardized, and widely applied to subjects with both primary and secondary APS. Measurement of LAC activity is based on multiple coagulation tests (screening and confirmation) with varying sensitivity and specificity due to the heterogeneous nature of aPL. A positive test requires prolongation of PL-dependent coagulation times and confirmation of PL-dependence. Enzyme immunoassays, primarily ELISA, for aCL continue to be widely used. Although standardization of aCL dates back to the late 1980s, commercial preparations vary widely in their reactivity to autoantibodies. Standardized ELISA for anti-ß2GPI has been available since 1996 and has been shown to substantially increase specificity for APS. More recently, anti-Pro and anti-Pro-phosphatidylserine ab assays have become available, and recent studies have suggested their utility in the diagnosis of APS. In conclusion, the diagnostic armamentarium for APS has been substantially augmented by developments within the past decade.
[24] Resilience and resilience engineering for health care. RI Cook, University of Chicago, Chicago, IL.
The low rate of catastrophic failures in healthcare is puzzling. Indeed, what is most remarkable about medical accidents is not that there are so many but that there are so few. Opportunities for catastrophic failure are endemic, even omnipresent, throughout hospitals, clinics, and pharmacies. Virtually every study conducted in a healthcare setting has reported a high observed or inferred error rate during patient care. Both the tempo and complexity of healthcare work are high and both are known to contribute to accidents. Clinical experience supports the idea that the accident rate should, given the circumstances, be higher. Clinicians frequently acknowledge that the nature and conditions of their work could produce many more accidents than actually occur. Yet accidents are rare. Why are there so few failures in a failure prone system? How do we explain the relatively robust performance of a system that appears to always be so precariously perched on the brink of catastrophe? We believe the answer is a systemic property called resilience. Resilience is the source of a system’s ability to withstand potentially failure-producing stresses without failing. Although we are now in the early stages of understanding the nature of resilience and its progenitors, we are convinced that it is a critical component of many successful systems. It appears to us that resilience is largely found in the people who occupy sharp-end positions in the system but it is clearly enhanced and eroded by technology and organizations. We believe that resilience is dependent on specific system features and that it can be enhanced or eroded, an activity called resilience engineering. The response to a bus bombing in Israel or to a sudden demand for an operating room are examples of resilience but there are many more. This presentation will characterize resilience and offer a vision of what resilience engineering may make possible for healthcare.
[25] Patient safety: "Ship of Fools"–all rowing hard in different directions. RL Wears, SJ Perry, University of Florida Health Sciences Center–Jacksonville, FL.
Safety in healthcare requires viewing organizations as macro-systems composed of highly interdependent and variably coupled micro-systems. As in other 24/7, high-risk, complex, hazardous industries, successful operations require a myriad of workers performing what to them seem to be unrelated functions that cumulatively meet organizational goals. Against this backdrop, seemingly isolated changes or improvements, especially of information technology, may demonstrate only benefits within the area of change but have wholly unintended yet life-threatening reverberations in other parts of the system, including direct patient care. This session will use case studies to illustrate the opaque and frequently unrecognized interfaces that exist within healthcare organizations. The cases will also demonstrate the problem of suboptimization where safety improvements in components can inadvertently undermine safety of the overall process in surprising ways.
[26] Banquet Address: A clinical eye at the Metropolitan Museum of Art, HA Johnson, New York, NY
For the past 12 years, I have been a walking tour guide in the Metropolitan Museum of Art. After practicing and teaching pathology for 40 years, a habit of looking for signs of human disease is not easily shed, and that fascination has been carried into the world of art. There is, not surprisingly, a fair amount of pathology on view in the museum, much of which has escaped the notice of curators and art historians. We will make our medical rounds in the galleries of American, European, Ancient Egyptian, and Ancient Greek Art.
[27] Issues in prostate cancer. WM Murphy, Consultant in Urologic Pathology, Gainesville, FL.
Issues in prostate cancer (PCa) are discussed under 3 categories: detection, precursors, and prognostic factors. Each is manifested by clinical factors but changes in pathologic features have also appeared. At least 8 sites are biopsied currently. This approach increases detection but creates problems in diagnosis, evaluation, and reporting. The very small tumors often revealed tend to increase the use of immunohistochemistry for confirmation and create diagnostic uncertainty, leading to confusing terms like atypical small acinar proliferation (ASAP). Non-neoplastic tissues (para-ganglia, seminal vesicles, and Cowper’s glands) that confound diagnosis are more frequent as are calls for a second opinion. Increased sampling decreases the clinical value of prostatic intraepithelial neoplasia (PIN) while it introduces other putative precursors, like post-inflammatory atrophy (PIA). Non-surgical therapies (x-ray, cryotherapy, hormonal therapy) can alter the morphology of PCa and confound accurate diagnosis in follow-up biopsies. Pathologic prognostic factors for PCa remain problematic, especially since definitions vary and treatment options are limited. These subjects are important for pathologists and their periodic review is appropriate.
[28] Pediatric small round blue cell tumors: diagnostic challenges resolved by molecular studies and electron microscopy. N Tatevian, J Hicks, D Lopez-Terrada, M Bhattacharjee, Texas Children’s Hospital and Baylor College of Medicine, Houston, TX.
Small round cell tumors (SRCT) constitute approximately 20% of the solid tumors in children. The usual spectrum includes neuroblastoma (NB), Ewing’s sarcoma/peripheral primitive neuroendocrine tumor (ES/PNET), rhabdomyo-sarcoma (RMS), and non-Hodgkin’s lymphoma (NHL). An extended spectrum adds desmoplastic small round cell tumor (DSRCT), small cell (undifferentiated) types of osteosarcoma (UOS), and hepatoblastoma (UHB), blastemal Wilms’ tumor (BWT), small cell variants of malignant peripheral nerve sheath tumor (MPNST), and synovial sarcoma (SS), and unclassified SRCTs. Diagnostic challenges are mainly encountered in the less differentiated tumors, represented by sheets of small-to-medium cells with large nuclei with inconspicuous nucleoli and minimal amount of cytoplasm. While conventional immunohistochemistry is invaluable as a first line in differential diagnosis, it is not always sufficient to differentiate these tumors. We found electron microscopy to be extremely important in recognition of PNET (occasional pleomorphic neuroendocrine granules, neurite processes, and glycogen accumulations), NB (membrane bound dense-core neurosecretory granules, neurites), RMS (thick and thin myofilaments [66% of our cases], external lamina, mono-particulate glycogen), DSRCT (neurosecretory granules, neurites, keratin-like intermediate filaments, myofilaments), and NHL (lack of intercellular junctions). Molecular characterization and identification of tumor-specific genetic rearrangements on a subset of these tumors (ES, alveolar RMS, DSRCT, SS) has led to the development of a number of molecular diagnostic tests, widely applied today for the differential diagnosis and correct classification of these neoplasms. We have successfully applied microarray comparative genomic hybridization to differentiate unusual metastases from second primary tumors in treated children. We have also demonstrated the value of laser capture micro-dissection in combination with BAC array CGH analysis to delineate the most aggressive cellular component of histologically favorable neuroblastomas with unusually aggressive behavior.
[29] New developments in the diagnosis of pulmonary and pleural neoplasms: immunohistochemistry-based strategies for resolving diagnostic questions. DS Zander, J Liu, D Tan, University of Texas Health Science Center at Houston Medical School, Houston, TX.
Accurate diagnosis of malignancies in the lung and pleura is crucial for determination of appropriate therapy and assessment of prognosis. In some cases, however, tumor classification may be challenging due to low tumor representation in the sample, tumor cell necrosis, tumor heterogeneity, and morphologic features that overlap between entities. Immunohistochemistry has become a frequently used tool for the evaluation of lung and pleural neoplasms, and recently, introduced antibodies have further extended the applications of this technique. Properly selected immunohistochemistry panels can offer assistance in several settings. Differentiation between pulmonary neoplasms and mesotheliomas, lung primary and metastatic neoplasms, and small cell carcinomas and non-small cell carcinomas can be facilitated by use of appropriate stains. This presentation will discuss recent approaches to these sometimes challenging diagnostic questions, and provide strategies that aid in the accurate evaluation of pulmonary and pleural neoplasms.
[30] Atypical pathological findings in a case of persistent hyperinsulinemic hypoglycemia of infancy with new mutation in the ABCC8 gene. N Brunetti-Pierri, O Olutoye, N Tatevian, Texas Children’s Hospital and Baylor College of Medicine, Houston, TX.
Persistent hyperinsulinemic hypoglycemia of infancy (PHHI), also known as familial hyperinsulinism or nesidiodysplasia (MIM 256450 [OMIM] ), is a rare inherited disorder characterized by dysregulation of insulin secretion resulting in severe, often life-threatening hypoglycemia. Genetically heterogenous PHHI is caused by mutations in at least four genes. About 50% of the patients have mutations in one of the two subunits of the ß-cell ATP sensitive K+ channel, ABCC8 and KCNJ11. Two morphologic forms of PHHI, diffuse and focal, are recognized. We report a patient who developed severe hypoglycemia almost immediately after birth and presented with inappropriately high insulin levels with low levels of blood glucose. The hypoglycemia was unresponsive to medical treatment and required 85% pancreatectomy. The pancreatic disease was best classified as a diffuse form, with, however, unusual pathological findings of decreased amount of insulin positive cells, absence of large islets, predominance of scattered small clusters and single insulin producing cells, aberrant increase and distribution of glucagon positive cells, and distinct adenoma, by immunohistochemistry and electron microscopy proven to be non-insulin producing. Molecular analysis showed that the patient is a compound heterozygous for two mutations in the ABCC8 gene: a nonsense mutation Arg841X (this mutation is reported for the first time) and a missense mutation, Asp1471Asn.
[31] Hyalinizing clear cell carcinoma of salivary gland: Report of a case. F Mohammadi, M Ionesco, J Fantasia, P Farmer, North Shore–Long Island Jewish Health System, Manhasset, NY.
Hyalinizing clear cell carcinoma (HCCC), also referred to as clear cell carcinoma of salivary gland origin, is distinct from biphasic epithelial-myoepithelial carcinoma. Although both tumors contain clear cells and express epithelial markers, HCCC exhibits stromal fibrosis and lacks myoepthelial differentiation. HCCC is a low-grade carcinoma that typically behaves less aggressively than most other salivary gland malignancies. It arises primarily in the minor salivary glands. Of 35 HCCC cases reported since its introduction in 1994, only 4 (11.4%) cases occurred in the parotid gland. There was a 71.4% female predilection. In all reported cases, tumors showed monotonous clear cell nests surrounded by hyalinized stroma. Tumor cells in all cases were positive for epithelial markers and negative for myoepithelial markers. Cervical lymph node metastatsis occurred in 9 patients (25.7%). Two cases (5.7%) developed distant metastasis. A 58-yr-old woman with the synchronous occurrence of hyalinizing clear cell carcinoma of the submandibular gland and adenocarcinoma ex-mixed tumor of the parotid gland is reported. Tumor cells in submandibular gland were positive for cytokeratins and P63, and negative for S-100, SMA, and CD43. This case provides further evidence that HCCC lacks myoepithelial differentiation. A summary of the demographic and clinical features of the reported cases is presented.
[32] Cutaneous angiosarcoma: a case series with prognostic correlation. MB Morgan, S Somach, W Eng, B Smoller, University of South Florida College of Medicine, Tampa, FL.
Cutaneous angiosarcoma (CA) is a rare and aggressive endothelial-derived sarcoma. Few large studies have examined the clinicopathologic and prognostic attributes of CA. We sought to discern the potential prognostic significance of a variety of demographic features (ie, age, sex, location), histologic attributes (ie, depth of invasion, tumor necrosis, tumor cell morphology, margin status, mitoses), and follow-up data (ie, tumor recurrence, metastases) in CA. The statistical influence of age, sex, anatomic location, tumor depth of invasion, tumor cell morphology, presence or absence of necrosis, number of mitoses, and margin status on time to tumor recurrence and metastases was examined in 47 patients with CA. Angiosarcoma arising within the breast, in a previously irradiated anatomic site, and a pre-existing vascular malformation or one associated with a lymphedematous extremity were excluded from study. Most of the patients were men (76%), with an average age of 75.1 yr (range: 59–92 yr). The most common location was the head and neck region (96%). The most common presentation was of a rapidly expanding erythematous patch, and the most common clinical impression was angiosarcoma. The average external diameter of the tumor was 5.3 cm (range: 1.1–8.9 cm). The most common histologic pattern was characterized by anastomosing dissecting sinusoids lined by atypical endothelial cells (64%) with 15% of cases showing a diffuse epithelioid or spindle cell proliferation and 21% showing a mixture of the 2 histologic patterns. The average depth of tumor invasion was 2.86 mm (range: 1.8->6.0 mm). Of the tumors, 78% had a mitotic rate that exceeded 3 per mm2. Follow-up was available in 37 of the patients and ranged from 6 to 65 months. The 5-year local recurrence rate was 84% and the overall 5-year survival was 34%. Most patients died as a result of their disease with widespread pulmonary, cardiac, and/or brain metastases. In conclusion, the gross and histologic features, external diameter (>5 cm), depth of invasion (>3 mm), mitotic rate (>3 HPF), positive surgical margins, tumor recurrence, and metastases correlated with adverse outcome by univariate analysis and, with the exception of mitotic rate, by multivariate analysis. Of the foregoing, tumor diameter, depth of invasion, positive margins, metastases, and tumor recurrence were the most robust predictors of outcome. None of the demographic factors was associated with outcome. This study confirms the poor prognosis of patients with CA. Among all demographic and histologic patterns examined for prognostic significance, tumor diameter, tumor depth of invasion, margin status, tumor recurrence, and metastases emerged as the most important determinants of outcome.
[33] How M. tuberculosis turns the immune response against us. RL Hunter Jr, M Olsen, J Actor, C Jagannath, University of Texas–Houston Medical School, Houston, TX
Tuberculosis is an obligate human parasite whose survival depends upon transmission from person to person. It is one of the most studied diseases in history and has been declared a public health emergency by the WHO. Nevertheless, several fundamental questions remain. How does tuberculosis turn our immune response against us to produce caseating granulomas and cavities? Why do individual lesions in the lung develop independently of one another and why don’t they heal? We propose that three essential clues to these questions have been largely ignored for decades. First, textbook descriptions of developing pulmonary tuberculosis are based on the rabbit in which macrophage activation, driven by cell mediated immunity, is followed by destruction of macrophages that accumulate as slowly expanding caseous necrosis. As reported by Osler in 1892 and subsequently by many others, human pulmonary tuberculosis begins differently as a tuberculous pneumonia that smolders before rapidly undergoing necrosis to form caseous masses and cavities. Second, early tuberculous pneumonia is characterized by progressive accumulation of host lipids and mycobacterial antigens within foamy alveolar macrophages. Finally, the most abundant lipid produced by virulent MTB, trehalose 6,6'-dimycolate (TDM or cord factor), has the ability to switch between two different sets of biologic activities depending on its environment. When located on organisms, TDM inhibits phagosome-lysosome fusion, acidification of phagosomes, and protects MTB from killing by macrophages. If TDM comes off the organism and attaches to the surface of sufficiently large lipid droplets, it becomes highly toxic for macrophages and induces immune granulomas and caseation necrosis. We propose that the two sets of biologic activities of TDM are central to the pathogenesis of tuberculosis. TDM on organisms facilitates their survival in alveolar macrophages in otherwise immune hosts. The infection quietly stimulates accumulation of host lipid, TDM, and other mycobacterial products in these cells until conditions develop for the activation of the toxicity of TDM. This rapidly kills macrophages and immune cells producing caseating lesions and cavities that are impervious to immune responses and facilitate transmission of infection to new hosts.
[34] Human papillomaviruses (HPV): challenges in the 21st Century. DC Halstead, North Florida Pathology/Baptist Health, Jacksonville, FL
Data from several studies collected over the past two decades have provided a better understanding of the pathogenesis of HPV and its role in the development of cervical cancer, one of the most common cancers in the world. In 2000, the Food and Drug Administration approved triage to high-risk HPV DNA testing for atypical squamous cells of undetermined significance (ASC-US) Pap smears. In addition, revised screening guidelines have been published for the use of HPV testing as an adjunct to cervical cytology in women 30 years of age and older. According to the guidelines, if both tests are negative, there is no need to repeat either test for 3 years. Although screening programs have dramatically reduced the number of cancer-related deaths, HPV vaccinations will undoubtedly change our approach to current screening and therapy. One of the most important contributions in developing a prophylactic vaccine is the identification of capsid-specific virus-like particles (VLPs) that are antigenic and induce neutralizing antibodies but are non-pathogenic. In contrast, therapeutic vaccines have been developed using HPV oncogenes to stimulate cell-mediate immunity. Currently there are several ongoing phase III studies to evaluate prophylactic and therapeutic vaccines that have the potential to prevent a major sexually transmitted disease and eradicate cervical cancer.
[35] Avian infuenza: not just for the birds. A Glassman, Medical University of South Carolina, Charleston, SC.
Millions of domestic birds have been killed in attempts to stop the spread of the avian influenza (AI) strain H5N1 to humans. The present outbreak (dating from the late 1990’s) of the disease has caused approximately 100 deaths in humans. Only persons in direct contact with fowl have been infected. This study examines the role of AI in human pandemics of the 20th century, its pathogenicity for people, and the potential for an early 21st century epidemic. Recent genetic analyses of material from the 1917–1918 pandemic that killed an estimated 20 – 40 million people have revealed the causative agent to have been AI. Other human epidemics of AI have occurred in 1957 and 1968, resulting in hundreds of thousands of deaths in the USA. The natural barrier to human infection is the fact that AI viruses have generally not been able to attach to human respiratory epithelial cells. Adaptation to human receptors has been attributed to (i) reasortment or (ii) genetic mutation/evolution. Human to human transmission does not occur unless changes in the H5N1 that result in sharing of characteristics with other human influenza (HI) types. Most often these changes occur by reassortment as the virus passes through other species, eg, porcine, and acquires genome segments that permit person to person transmission. The AI of 1918 has hemmaglutinin and nucleoprotein segments related to HI. In general, people have little immunity to AI so if a sustainable transmissible strain of AI arises the risk of a pandemic would be high. No useful vaccine or therapy is available. AI is resistant to amantadine and rimantadine. Oseltamivar and zanamivir are of questionable effectiveness. The concern is that if the present AI adapts to humans a pandemic will result and no effective measures for prevention or treatment exist.
[36] The exponential amplification of new PCR technologies. CA Holland, William Beaumont Hospital, Royal Oak, MI.
The polymerase chain reaction (PCR) is well known for preferential amplification of small fragments of DNA for subsequent use in downstream applications. Since its advent in the mid-1980’s several adaptations of the classical PCR method have been described. These methods include the more common reverse transcriptase-PCR (RT-PCR), nested-PCR, multiplex-PCR, and real time-PCR, as well as some lesser known methods such as arbitrarily primed-PCR (AP-PCR), random amplified polymorphic DNA-PCR (RAPD-PCR), rapid amplification of cDNA ends (RACE), differential display-PCR (DD-PCR), inverse PCR, and asymmetric-PCR. RT-PCR is often used in the clinical laboratory to identify infectious agents such as viruses or to follow minimal residual disease in leukemias. Nested-PCR has been employed to increase sensitivity and minimize false priming, while multiplex-PCR can identify more than one target in a single reaction. Recently, the advent of new fluorescent reactions has allowed PCR to be viewed in realtime, thus greatly reducing subsequent detection manipulations and possible addition of contaminating amplicons. Other PCR reactions can identify unknown sequences (inverse, AP, RAPD, RACE, DD), generate ssDNA (asymmetric-PCR), or be performed in situ (in situ-PCR). Additional methods have been designed to produce cleaner PCR products (Hot Start-PCR) or to bypass complicated optimization steps (Touchdown-PCR). This review will cover the differences among these methods and give examples of their appropriate use in the clinical molecular pathology laboratory.
[37] Validation of a real-time MRSA-PCR assay for the detection of MRSA colonization in nasal swabs. M Pieretti, N Christopher, Morton Plant Hospital, St. Joseph Hospital, BayCare Laboratories, Clearwater, FL.
Rapid detection of colonization with methicillin-resistant Staphylococcus aureus is an increasingly important infection control measure. The FDA-approved IDI-MRSA real time PCR qualitative assay has been developed to rapidly identify MRSA colonization and thus help patient isolation procedures in a timely fashion. We have evaluated the performance of this molecular assay in a microbiology laboratory setting by performing a side-by-side comparison of this assay to that of direct plating followed by plating on ChromAgar plates to identify MRSA. Nasal swabs (n = 1480) received in the laboratory for MRSA screening were evaluated in this study. The same nasal swab was first used to plate a blood agar plate and then utilized for the IDI-MRSA assay according to the manufacturer’s recommendations. PCR of individual samples was repeated if the internal control was invalid. Colonies identified as Staphylococcus aureus on blood agar plates were subcultured on ChromAgar plates. The PCR assay identified 386 specimens as positive and 1020 as negative for MRSA; 74 samples were invalid in the first PCR because of internal control failure. Of the latter, 81% gave a valid result after a freeze-thaw cycle of the sample lysate. Of the 386 PCR positive samples, 364 were also MRSA positive by culture. MRSA screening by culture identified 8 samples that yielded a negative result by PCR. PCR results were available within 8–10 hours of sample collection. The IDI-MRSA real-time PCR assay allowed rapid and accurate identification of MRSA colonization. The assay showed high positive (94.3) and high negative (99.2) predictive values compared to the culture method used.
[38] A multiplex PCR assay to distinguish Aspergillus and species of Candida from fixed tissue. J Ernst, N Laposky, MK Froberg, University of Minnesota School of Medicine, Duluth, MN
Serious nosocomial infections have increased in the United States with the advent of increased bone marrow and solid organ transplantation, immunosuppressive therapy, cancer chemotherapy, AIDS, and the use of high-risk medical devices. Fungi are now the fourth most common bloodstream pathogens in hospital settings with a mortality rate greater than 50%. Fungal resistance to many antimicriobial agents has been reported, making early diagnosis and treatment vital. Proper diagnosis of fungal infections requires clinical suspicion and culture of appropriate tissue specimens, which may take weeks. Rapid and accurate diagnosis of the specific fungus would aid in proper drug and dose selection, as well as provide invaluable epidemiological data on trends in fungal infections. Species diagnosis from fixed tissues may not be possible histologically; therefore, we developed a rapid, multiplex PCR assay in order to identify the most common nosocomial fungi at the species level from formalin-fixed paraffin embedded tissue specimens. Our goal was to distinguish Aspergillus fumigatus, Candida albicans, Candida glabrata, Candida parapsilosis, and Candida tropicalis using a single PCR reaction. The multiplex primer mixture consisted of two sets of universal fungal primers targeting the 5.8 and 28S rRNA (CTSF and CTSR), one specific primer for each of the four Candida species, and two specific primers for Aspergillus. This technique allows rapid identification of the most common species of fungi involved in serious nosocomial infections in a multiplex design, thereby minimizing the use of reagents and time required for species identification.
[39] Hepatitis C virus genotyping: a comparison of two methodologies. RW Ryan, LA Avery, I Ratkiewicz, J Aslanzadeh, University of Connecticut School of Medicine, Farmington, CT, and Hartford Hospital, Hartford, CT.
Hepatitis C virus (HCV) infects over 170 million people worldwide. Chronic infection occurs in 50–80% of the cases and eventually leads to cirrhosis and hepatocellular carcinoma. HCV consists of a family of highly related but distinct genotypes numbered 1 through 6, with various subtypes, and differing geographical distribution. HCV genotype is often utilized to determine the duration of therapy with pegylated interferon and ribavirin. Invader HCV genotyping assay (Third Wave Technology, Madison, WI) was compared to the VERSANT HCV LiPA test (Bayer Diagnostics, Tarrytown, NY) using 40 HCV+ plasma samples. Specimens were subjected to RT-PCR using both the COBAS Amplicor HCV monitor for the LiPA test and the Roche COBAS TaqMan48 analyte-specific reagent (ASR) for the Invader test. The generated amplicons were used to ascertain the genotype for each sample with the corresponding test. Overall, there was 95% correlation between the two tests. Both methods genotyped 18 samples as genotype 1,2 samples as genotype 2,11 samples were genotyped 1b by the LiPA and 1 by the Invader assay; 2 samples were genotyped 1a by the LiPA and 1 by the Invader test. The genotypes on 2 samples were 2b by the LiPA and 2 by the Invader assay and the genotypes on 4 samples were 3a by the LiPA test and 3 by the Invader test. Two samples with viral loads of 2000 and 2900 IU/ml were genotyped indeterminate by the LiPA test but were genotype 1 by the Invader assay. The Invader HCV genotyping assay was more sensitive, predicting the genotype for all 40 samples. In addition, the invader system with its automated reader was easier to interpret and required no more than 30 min hands-on-time to complete the test. The LiPA assay was superior to the Invader HCV system in predicting the HCV subtypes within each genotype.
[40] Total quality management of medical device applications. KJ Aziz, Kensington, MD.
The principles and concepts of total quality management (TQM) comprise quality management, assurance, and control. These principles and concepts can provide the essential foundation for healthcare laboratories in regard to their routine management and operations, based on the needs of the users and customers and their understanding of quality and cost. Quality management functions determine quality policy, objectives, responsibilities for continuous quality monitoring, and improvements. As more technologies transfer from research and development to actual clinical use, laboratory managers must integrate these new systems into existing healthcare services. Careful adherence to manufacturers’ labeling instructions, good laboratory practice, and availability of reliable new medical device systems can contribute to the desired goal of quality patient care. Manufacturing QA covers all stages in the total product life cycle (TPLC), including approaches to a clinical system’s design, manufacturing, clinical testing, and final availability of finished product. It is expected that information about validating QC procedures may help the user’s understanding of a device’s overall QA requirements so that users can benefit from the results of such device systems. Prior to marketing, design controls require a risk analysis and validation of the design; they incorporate user requirements and the intended use of the device. Additionally, manufacturers are encouraged to design medical devices with the ability to detect potential failures and alert the users to take corrective actions. Such built-in control systems potentially reduce or eliminate the need for users to run quality control samples to monitor the performance of these devices. In evaluating a device’s market readiness and final approval, the FDA must consider the product’s benefits against its known or potential risks. Based on the premarket application studies and approval process, the manufacturer determines the quality control validation requirements and makes them available in the product’s labeling. This presentation presents an overview of FDA’s Quality System Regulations (QSR) as they relate to TPLC approaches to new medical systems, in respect to premarket review and evaluation, manufacturing process, and post-marketing commercial use.
[41] Mortality in patients with heart failure: telehealth versus standard medical care. P Foulis, B Neugaard, G Barbier, N Chumbler, R Schofield, University of South Florida, Tampa, FL, James A Haley Veterans Administration Hospital, Tampa, FL, University of Florida, Gainesville, FL, North Florida/South Georgia Veterans Health Care System, Gainesville, FL
This study explored mortality differences between heart failure (HF) patients receiving a telehealth intervention to similar individuals who received care in either a primary care setting or in an ambulatory HF clinic. Heart failure poses an important public health problem and is increasing in both incidence and prevalence. New HF treatment options need to be explored that reduce utilization while maintaining quality. The treatment group consisted of 162 patients enrolled in a HF telehealth intervention at a VA Medical Center. These patients were monitored remotely in their homes through an in-home messaging device. A nurse practitioner monitored the patients’ vital signs including blood pressure and weight on a daily basis and adjusted medications as necessary. The three comparison groups consisted of 149 HF patients who were referred to the telehealth program but did not enroll, 141 HF patients who were treated at a specialized ambulatory HF clinic, and 532 HF patients receiving primary care. The random sample of primary care patients was selected by searching electronic medical records using the ICD-9 codes for HF. Patients who had no ejection fraction data or who had ejection fractions above 40% were excluded. Using Cox proportional hazards regression models, mortality rates were compared between the treatment and comparison groups. The risk of dying during the 12 month follow-up period was significantly higher in patients receiving standard medical care. Intensive monitoring through a HF telehealth disease management model may lead to a reduced risk of mortality by providing increased access to health care resources.
[42] Cryoglobulins: an important but neglected test. Z Shihabi, M Hinsdale, Wake Forest University School of Medicine, Winston-Salem, NC.
Cryoglobulins (CRY) analysis is important for patient care with different kinds of vasculitis–those with purpura, and arthritis. However, it does not receive much attention in routine analysis. This is due to several problems. The CRY in >80% of the cases exists in low concentrations of ~100–300 mg/L among normal serum proteins (~60,000 mg/L). It is difficult to measure accurately because of contamination from normal serum proteins. There is lack of appreciation that the low concentrations can be associated with severe symptoms. The analytical aspects of CRY have not kept up with advances in technology. For example, sample volumes are too large, and the methods of detection remain crude (mainly visual). Here we describe a simple and sensitive method for CG analysis based on measuring the cold precipitate as total urine protein, but correcting for the contamination from normal serum proteins present in the precipitate based on albumin measured as microalbumin. After a few wash steps, albumin remains high and variable in the cryoprecipitate, which, without corrections, can lead to false results. In healthy individuals, cryoglobulin is present in the cold precipitate at an average of 51 mg/L (range 2–150 mg/L, n = 32), while the total proteins in the precipitate average 141 mg/L (range 10–300 mg/L). However, the mean for the corrected cryoglobulins based on 32 samples from out-patients is 36 mg/L with a reference range of 0–120 mg/L. The test is close to linearity (r = 0.992) between 0– 1000 mg/L. It correlates well with capillary electrophoresis (r = 0.7). We measured cryoglobulins in 190 positive samples that were phenotyped by electrophoresis. Nine samples were type I with an average CRY of 10,222 mg/L; 109 samples were type II with an average of 372 mg/L; and 76 samples were type III with an average of 216 mg/L. In conclusion, we present a method for measuring CRY based on measuring the total proteins but with correction for normal serum proteins by measuring albumin as microalbumin.
[43] An unusual cause of elevated serum total ß-hCG: a case study. C Buckner, L Wilson, C Papadea, Medical University of South Carolina, Charleston, SC.
Beta-human chorionic gonadotropin (ß-hCG), a hormone synthesized by the placenta, is measured in women for many reasons including the detection of normal and abnormal (ectopic and molar) pregnancy, screening for congenital defects, and diagnosing and following choriocarcinoma. A small amount of the nicked form of ß-hCG is also secreted by the pituitary under normal conditions. For rare cases in which the most common causes of elevated serum ß-hCG are ruled out, the result is considered either false-positive or true for an unusual reason. Laboratory identification of unexpected results is important since these can have serious clinical consequences for the patient. Ways to investigate possible causes of an unexplained elevated serum ß-hCG include several laboratory testing options: ß-hCG in urine; serum heterophile antibodies; ß-hCG in serial serum dilutions; and ß-hCG by different commercial immunoassays. This paper will discuss the case of a nonpregnant patient with chronic renal failure who had a preoperative negative urine pregnancy test with elevated serum total ß-hCG twice (ie, 290 mIU/ml; 285 mIU/ml), several days apart. She underwent a dilation/curettage and bilateral salpingectomy. The immunoassays were repeated several times postoperation and showed seven similarly elevated results by two immunoassays but a third immunoassay measured ß-hCG <5 mIU/mL. The conflicting results were explained by the form of ß-hCG measured by each assay and the patient’s low renal clearance of the hormone. This case illustrates a rare cause of an elevated serum ß-hCG and the importance of detecting the true nature of such a result.
[44] Concomitant use of benzodiazepines and other drugs and risk of injury in community-dwelling veterans. AM Spehar, D French, R Campbell, H Means, T Chirikos, T Bulat. VISN-8 Patient Safety Center; University of South Florid,; VISN-8 Measurement and Evaluation Team; VISN-8 Pharmacy Benefit Management; and Moffitt Cancer Center; Tampa, FL
Benzodiazepines are medications that are acknowledged to independently increase injury risk by increasing the likelihood of falls, fall-related injuries, adverse drug events, and car accidents. Benzodiazepines used concomitantly with other high-risk medications can further increase this risk. This study quantifies the increased risk of healthcare treatment for injuries that occurred while taking benzodiazepines and other medications, adjusted for other risk factors. We linked outpatient prescription data and inpatient/outpatient medical utilization records for 13,745 veterans receiving outpatient benzodiazepine and >1.5 million non-benzodiazepine prescriptions over three years. Injuries and adverse events were identified using ICD-9-CM coding for injuries and poisonings (800–999), and associated into episodes of care. Software was used to identify major severity combinations of benzodiazepines and other drugs. Analysis was limited to drug combinations within 30 days prior to an injury-related healthcare episode. A multivariate model was used to adjust this risk for impact of co-morbidities, hospital discharges, marital status, age, benzodiazepine dose and duration, etc. Of 1110 unique individuals with an injury, 790 (71%) had used benzodiazepines and another high-risk drug, increasing the odds of an injury more than two-fold. Dose and duration of benzodiazepines and certain co-morbidities were associated with increased injury risk; being married reduced the risk. This study quantified the impact of concomitant use of benzodiazepines and other drugs on the risk of injury. Better patient and provider education is needed when prescribing high-risk medications. Future injury risk models must incorporate these complex aspects of medication usage linked to adverse outcomes.
[45] Case studies in forensic toxicology. DJ Cannon, University of Tampa, Tampa, FL.
The use of chemical experiments and advanced instrumentation to study the science of poisons and toxic substances is an integral part of criminal investigations. These studies involve the use of postmortem tissue, human performance effects, and forensic drug testing. The history, methodologies utilized, and the various substances subjected to forensic toxicology analyses will be described. Modern and ancient poisoners and victims produce fascinating patterns of toxicological investigation. Crime scene actions, autopsy results, and other steps in the process of proving criminal poisoning are absolute keys to successful court outcomes. Case studies highlight this observation and emphasize the need for continued advanced, high quality work in forensic chemistry that deals with toxic compounds. Case examples will be discussed to illustrate the historical evolution of poisonings and to describe recent homicidal poisonings, ranging from the "Playboy Poisoner" to the "Eccentric Genius."
[46] Celiac disease: the modern-day impersonator. M Salwen, SUNY Downstate Medical Center, Brooklyn, NY.
Celiac disease (CD) is common but under-recognized, with about a 1% prevalence in those of European origin, and is seen worldwide, even in developing nations. Most cases are undiagnosed. In others, diagnosis is often delayed because of the heterogeneity of symptoms, which cause diverse presentations. Fewer than one-half have enteric symptoms. Patients consult many different medical specialties. This genetically determined T cell-mediated autoimmune enteropathy of the proximal small intestine is triggered by gluten. The classic symptoms of either abdominal pain or chronic diarrhea occur in about one-third of the patients. Fecal occult blood is present in one-half. Symptoms are often extraintestinal involving many organs, and include anemias, osteoporosis, arthritis, short stature, neuropathies, and infertility. Patients are also examined because of the diagnosis of a family member (10% of siblings are at risk), endoscopy for dyspepsia or reflux, other autoimmune diseases, or pruritic papulovesicular skin lesions. Lymphomas, other cancers, Type 1 diabetes and other endocrine autoimmune and genetic disorders are more frequent in association with CD. Only a small percentage of those at risk, who ingest wheat, develop CD. Gluten in infant formula during the first three months of life, gastrointestinal infections, or a lack of breastfeeding increase the risk of CD in infancy. Serological testing for IgA endomysial or transglutaminase antibody identifies those to have a small bowel biopsy, which is the diagnostic standard. IgA deficiency is common in CD, necessitating testing sometimes for IgG antibodies. HLA-DQ2 and/or DQ8 are present in nearly all cases, and if absent, virtually exclude CD. The typical duodenal mucosal inflammatory lesions demonstrate villous atrophy, crypt hyperplasia, and intraepithelial lymphocytosis, which cause impaired nutrient absorption (B12, iron, cholesterol, or calcium deficiencies) and other biochemical changes. This multisystem disorder requires laboratory testing for diagnosis.
[47] Utility of flow cytometry in the diagnosis of cutaneous lymphomas. S Oshtory, N Apisarnthanarax, AC Gilliam, KD Cooper, HJ Meyerson, University Hospitals of Cleveland/Case Western Reserve University, Cleveland, OH.
To determine the usefulness of flow cytometry for the diagnosis of cutaneous lymphomas, particularly cutaneous T cell lymphoma (CTCL), we analyzed skin biopsies from 25 patients with clinical signs or suspicion of cutaneous lymphoma. Of 14 patients with histopathology results suggesting CTCL, 10 had positive molecular studies and 11 had detectable T cell abnormalities by flow cytometry. Three of the 14 patients had negative molecular studies and no abnormality by flow cytometry. Seven of the 25 patients had cutaneous B cell lymphoma (CBCL); all 7 patients had detectable flow cytometric abnormalities, but only 3 of 6 patients had positive molecular studies. In the remaining 4 cases, which were suspicious for reactive lymphoid infiltrates, no abnormality was identified by flow cytometry. Flow cytometry of skin biopsy specimens is a sensitive method for detecting abnormalities in CTCL, documenting clonality, and sub-classifying B cell lymphomas and should be considered part of the routine work-up of patients presenting with a clinical suspicion of cutaneous lymphoma.
[48] Urinary bladder augmentation: comparison of bladder acellular matrix versus a commercial xenograft. M Wilkerson, M Parekh, A Cavanaugh, L Rothblum, Geisinger Medical Center and Weis Center for Research, Danville, PA.
Bladder reconstruction or augmentation is one recommended treatment for voiding disorders. Currently, a segment of small bowel is grafted in situ for bladder construction. Complications include electrolyte imbalance, mucus production, stone formation, abscess, peritoneal adhesions, and malignant potential. An alternative that has been tried in animal models is the preparation of an acellular matrix from a donor bladder of the same species. This study compares bladder augmentation in a rabbit model using allograft and xenograft acellular matrices. One group of 15 rabbits had bladder augmentation with a donor allograft acellular matrix prepared from rabbit bladder mucosa. A second group of 15 had augmentation using a commercially available xenograft prepared from porcine small bowel mucosa. The bladder from 5 animals in each group was harvested after 1, 2, and 3 mo. All bladders had epithelialization of the luminal aspect of the grafts and muscle had grown into the grafts. These findings suggest that bladder augmentation using a donor acellular matrix may some day be an alternative to the small bowel grafting that is presently offered for treatment of a non-compliant human bladder.
[49] Synergistic effects of acetaminophen and deferoxamine against iron-induced cardiac damage in gerbils. EM Walker Jr, ER Blough, RG Morrison, KM Rice, S Cansino, GL Wright, P Wehner, Marshall University, School of Medicine, Huntington, WV.
The objective of this study was to test possible additive or synergistic effectiveness of acetaminophen and deferoxamine combinations in the gerbil iron-overload model. Male gerbils were divided into 5 groups: Controls: iron-overloaded (120 mg/kg ferric hydroxide complex, ip); iron-overloaded treated with acetaminophen (200 mg/kg, ip); iron-overloaded treated with deferoxamine (83 mg/kg, ip); and iron-overloaded treated with a combination of acetaminophen and deferoxamine (half-doses = 100 mg/kg acetaminophen and 42 mg/kg deferoxamine, ip). Iron dextran was injected every 5 days for 8 weeks. Acetaminophen and deferoxamaine treatments (Mondays, Wednesdays, Fridays) followed the same schedule for 8 weeks and were continued for 4 weeks after completion of iron overloading. Echocardiograms were performed on all gerbils 12 weeks after completion of treatments. Liver and cardiac iron contents were determined using inductively coupled plasma atomic emission spectrometry. Iron overloading resulted in 52.0- and 6.4- fold increases in liver and cardiac iron content, respectively. Echocardiographic evaluation indicated cardiac hypertrophy, right and left ventricular distension, and declines in systolic function. Injections of acetaminophen and deferoxamine, alone, or in combination, were equally effective in preventing cardiac structural and functional changes. Half-dose combinations of acetaminophen and deferoxamine reduced cardiac iron content (56%) compared to full doses of either agent, alone (acetaminophen, 47 %; deferoxamine, 43 %) (p <0.05). Acetaminophen or deferoxamine or half-dose combinations were equally effective in reducing liver iron content (acetaminophen, 32%, deferoxamine, 31%, acetaminophen-deferoxamine, 36%, p >0.05). In conclusion, there are possible additive or synergistic effects of acetaminophen and deferoxamine combinations in reducing excess cardiac iron content and in preventing or reversing iron-induced cardiac pathology.
[50] NNK stimulated lung cancer cell proliferation and growth via the induction of nuclear factor-kappa B. GG Chen, MY Li, Y Johnson, WL Tak, SKM Tong, APC Yim. The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong.
Smoking is the single most extensively documented risk factor for all histological types of lung cancer. Among the numerous toxic and carcinogenic agents contained in tobacco products, the nicotine-derived 4-(N-methyl-N-nitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) is the most potent carcinogen. However, the mechanism responsible is not completely known. In the present study, we examined whether the NF-
B activation could be induced by NNK in lung cancer cells (NCI-H23 & CRL-2066). Experiments showed that NNK significantly up-regulated the expression of NF-B p65 and the translocation of p65 into the nucleus. The activation of p65 was also in agreement with the result of the time-course study of NF-B transcriptional activity. Consistent with above data, the phosphorylated I B was increased in the cytosol, resulting in the translocation of NF-B into the nucleus. Data obtained by electrophoretic mobility shift assay (EMSA) also supported that the activity of NF-B was enhanced in the cells after NNK treatment. Having established the promoting-effect of NNK on the survival molecule, NF-B, we continued to demonstrate that such an effect of NNK could significantly stimulate the cell proliferation and growth. NNK significantly increased cell viability and proliferation of the cells in a time-dependent manner as determined by MTT assay. After pre-treatment with NNK, lung cancer cells showed a significant resistance to the cell death induced by staurosporine, a strong cell death stimulus. SN50, a specific inhibitor to block NF-B translocation, markedly suppressed the translocation of NF-B p65 into the nucleus of the cell treated by NNK, and thus inhibited the activity of NF-B and the proliferation of the cell. In conclusion, our data clearly indicated that the activity of NF-B was obviously increased by NNK, which subsequently led to increased proliferation and growth of lung cancer cells.
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