• © 2004 by the Association of Clinical Scientists, Inc.

Abstracts of Presentations at the Annual Meeting of the Association of Clinical Scientists in Houston, Texas, on 12 to 16 May 2004

[1] The future of physicians in laboratory medicine. Michael Laposata, Massachusetts General Hospital, Harvard Medical School, Boston, MA.

The test menu for the clinical laboratory continues to grow in size and complexity. With this rapid and substantial growth, there is often added value to the ordering clinician for expert physicians to interpret complex test results. This is analogous to what occurs in anatomic pathology and radiology. The development of narrative interpretations of complex clinical laboratory evaluations that are patient-specific and consider clinical information and all relevant laboratory tests improve the speed and accuracy of the diagnosis and thereby reduce medical error, while minimizing the cost of care to the institution. In addition to providing ordering physicians with an interpretation of the clinical significance of the test results, there is also great need for assistance in the selection of tests. This presentation will present strategies for institutionally derived reflex testing that minimize the challenge for ordering physicians to select the right tests, and a description of the logistics for providing narrative interpretations of complex clinical laboratory evaluations by experts in the field.

[2] Molecular diagnostics of infectious diseases. The Here and Now. James Versalovic, The University of Texas/ M. D. Anderson Cancer Center, Houston, TX.

Diagnostic molecular microbiology has rapidly emerged as an important area of molecular diagnostics. Advances in real-time PCR have resulted in a renaissance of test development for microbial detection. Real-time PCR strategies have resulted in increased versatility of diagnostic laboratories and test menu expansions for qualitative assays. Viral load testing is undergoing a major transformation with homogeneous DNA technologies including real-time PCR. New methods for DNA sequencing and genotyping are enabling laboratories to assimilate these strategies for culture confirmation. The fusion of biochemical testing and DNA sequencing has augmented the abilities of diagnostic laboratories to identify fastidious pathogens. Finally, PCR-based DNA typing methods coupled with informatics have culminated in rapid molecular epidemiologic studies for unusual disease and outbreak investigations. In summary, advances in molecular diagnostics are changing the practice of clinical microbiology and resulting in significant improvements in laboratory services.

[3] When SARS hits home. Jeffrey Tarrand, The University of Texas/M. D. Anderson Cancer Center, Houston, TX.

The historical background and clinical significance of SARS will be briefly discussed. SARS-CoV is a novel coronavirus that is responsible for a highly contagious viral pneumonia. The agent of Severe Adult Respiratory Distress Syndrome derives from a deletion in an animal coronavirus. An unprecedented degree of global cooperation in epidemiology, virology, and public health measures has resulted in the abrupt termination of the initial outbreak. Nonetheless, SARS or similar variants present major diagnostic and organizational challenges to our public health system and to individual hospitals. The diagnosis and management of this novel corona virus syndrome are delineated and particular emphasis is placed on the individual hospital infection control plan.

[4] Is the civet cat the source of the SARS-associated coronavirus? M. Kent Froberg, Department of Pathology. University of Minnesota–Duluth, Duluth, MN.

Severe acute respiratory syndrome (SARS) is a recently recognized infectious disease first reported from the Guangdong Province of southern China. Between November 2002 and July 2003 over 8,000 cases of SARS were reported worldwide from 29 countries with a case fatality ratio of 9.6%. Researchers quickly identified the etiologic agent as a previously unrecognized SARS-associated coronavirus (SARS-CoV). Because its sequence data differ from those of previously known human coronaviruses, it was suspected to have crossed the species barrier between an animal host and humans. A coronavirus with a similar sequence was isolated from feces and secretions of Himalayan palm civet cats from a live-animal market in Guangdong, China. The human SARS-CoV has 29 fewer nucleotides in a region encoding the N-protein, but is otherwise identical to the corona-virus isolated from civet cats. More recent epidemiologic studies in this same area of China found 13.0% of animal traders had IgG antibodies against the SARS-CoV, while 2.9% of hospital workers, 1.6% of CDC workers, and 1.2% of healthy adults at a local clinical were seropositive. If classified by primary animal traded, 72.7% of civet cat animal traders tested positive for IgG SARS-CoV antibodies with a lower seroprevalence rate for animal traders dealing largely with boars, deer, hare, pheasants, cats, other fowl, or snakes. Subsequent PCR studies detected in bats, monkeys, and snakes coronoviruses that were identical or similar to the human SARS-CoV isolates. Thus while the civet cat may be a source of the modified coronavirus responsible for SARS, research findings to date are insufficient to identify either the natural reservoir for SARS-CoV or the animal(s) responsible for crossover to humans.

[5] Pulmonary toxicity of carbon nanotubes: a scientific opportunity and area of concern. Robert L. Hunter and Chiu-Wing Lam, University of Texas Medical School and Wyle Laboratories, Houston, TX

Single-wall carbon nanotubes structurally resemble rolled-up graphite sheets, usually with one end capped; they (individually ~1 nm in diameter and several m long) generally pack tightly together to form rods or ropes. Carbon nanotubes possess unique electrical, mechanical, and thermal properties and have many potential applications in the electronics, computer, and aerospace industries. Because unprocessed nanotubes are very light and could become airborne and potentially reach the lungs, their pulmonary toxicity was investigated. The 3 products studied were made by different methods, and contained different types and amounts of residual catalytic metals. Mice were each intratracheally instilled with 0, 0.1, or 0.5 mg of carbon nanotubes, a carbon black negative control, or a quartz positive control, and euthanized 7 or 90 days after the single treatment for lung histopathological study. All nanotube products induced dose-dependent epithelioid granulomas at 7 days. These lesions persisted and intensified by 90 days. The lungs of mice treated with carbon black were normal except for the presence of carbon in alveolar macrophages. Since nanotubes are a form of pure carbon, it was assumed by some that they would be biologically inert, as is graphite. However, it seems that the differences in electrical and mechanical properties can be accompanied by marked changes in biologic activity. This should be a concern for the health of exposed people, but might also offer opportunities for beneficial products.

[6] Laboratory diagnosis ofBorrelia burgdorferi infection. Raymond W Ryan and Fil Dias, University of Connecticut Health Center, Farmington, CT.

Lyme disease is a multisystemic illness caused by the tick-borne spirochete Borrelia burgdorferi. Early manifestations of the infection include an expanding, erythematous rash at the site of the tick bite called erythema migrans (EM) and/or a nonspecific flu-like illness. Antibody detection methods are the most widely used assays for the laboratory diagnosis of Lyme disease. Some reports have concluded that serologic testing is not useful early in the course of Lyme disease because of the low sensitivity of tests in early disease. Several investigators have used one or more borrelia-specific recombinant proteins in an ELISA format in an attempt to increase the sensitivity and specificity of antibody detection in early disease. In the present study, we compared the detection of B. burgdorferi-specific IgM antibodies in patients with culture-positive EM by an “in house” whole cell ELISA and Western immunoblot. Serum samples were obtained at specific intervals following the appearance of EM. Forty seven percent of patients whose blood was drawn 1–7 days after EM were positive by WC (ELISA), while 80% of those whose blood was drawn 8–19 days post EM were positive by WC (ELISA). The percent positive by IgM Western immunoblot for these groups was 58 and 85, respectively. In conclusion, patients suspected of early Lyme disease whose initial serological test is negative should be retested in 14–21 days

[7] West Nile Virus: What we learned from testing blood donors during the 2003 season. Kathleen Sazama, University of Texas/M.D. Anderson Cancer Center, Houston, TX.

This presentation describes the testing for West Nile Virus (WNV) in US blood donors from mid-yr through the end of the season in 2003. WNV entered the United States via New York in 1999. Initially, there was little concern regarding the potential epidemic that subsequently evolved. In August 2002, reports of transfusion (and transplantation) transmission sparked tremendous interest in developing a method to detect and avert subsequent transmissions. Beginning in mid-June 2003, all blood donors were screened for WNV, using one of two experimental test systems developed by GenProbe in collaboration with Chiron Corporation and Roche. The majority of blood donations were tested using a minipool format of between 8 and 16 samples/pool. Only a few facilities (including ours) used a single sample testing format throughout 2003. In areas of high frequency of pool positivity, blood centers used a combination of pooled and individual sample testing. At a minimum, 1,000 transfusions were interdicted that might have resulted in illness or death of a recipient from a WNV infected donation. The natural history of the epidemic is continuing to unfold, through a combination of animal surveillance, vaccination, and testing and by using human donor testing.

[8] Pathology of surfactant protein abnormalities in infancy. Megan K. Dishop and Claire Langston, Texas Children‘s Hospital, Houston, TX.

This case series describes the spectrum of histopathologic features of surfactant protein abnormalities in infants. Abnormalities of surfactant protein production are rare inherited disorders causing respiratory distress in full term infants. There are 2 major histopathologic patterns: classic alveolar proteinosis and chronic pneumonitis of infancy, which are associated with surfactant protein B (SP-B) and surfactant protein C (SP-C) abnormalities, respectively. We identified 25 cases (from 24 patients) with histologic features of surfactant protein abnormalities, including 3 cases from Texas Children‘s Hospital and 22 cases received in consultation from other institutions. There were 7 patients with a pulmonary alveolar proteinosis pattern (age range, 3 wk to 3 mo) and 17 patients with a chronic pneumonitis of infancy or other atypical pattern (age range, 3 wk to 9 mo). Correlation with known biochemical and molecular data on these patients supports the distinct histopathology of surfactant protein B and protein C abnormalities.

[9] Test consolidation with pre-analytical automation in a small university hospital laboratory. Sidney M. Hopfer and Gregory S. Makowski, University of Connecticut School of Medicine, Farmington, CT.

This presentation reviews the systems integration process involved with ongoing efforts to reorganize and automate high volume tests, regardless of scientific discipline, performed in the clinical laboratories at John Dempsey Hospital of the University of Connecticut Health Center. The Health Center complex contains a small hospital (220 beds), and schools of medicine, dentistry, and public health. In 1995–1996, the clinical laboratory processed 860,000 specimens/yr with 87 full-time employees. In 2002, the laboratory processed approximately 1.5 million specimens/year with 71 staff members. Objectives of the reorganization included: increased staff efficiency, decreased footprint with minimal financial expenditure, workstation consolidation and automation, complete core-lab cross-training, improved workflow, and decreased time from physician order to result report. Definitive actions included movement of the specimen processing area closer to the outpatient phlebotomy areas, conversion to primary closed-tube sampling, utilization of pre-analytical automation for most specimens, reorganization of testing that does not require processing near laboratory entrance, placement of fully automated instrumentation to output station of the pre-analytical workstation, automation of as many tests as possible, implementation of an auto-verification strategy (chemistry/immunochemistry), movement to a paperless environment, and a comprehensive cross training program. Financial benefits included: reduction in cost/test due to increased test volume and standardization of reagents, improved utilization of scientific/clinical directors/technologists/ technicians, decreased instrument footprints, improvements in testing frequency (most testing performed in parallel in real time), menu improvements (decreasing the number of tests sent to reference laboratories), enhanced outreach opportunities, and cost avoidance for instrument and laboratory information systems.

[10] Neurodegenerative diseases: the next frontier. Donald J. Cannon, GeneLink, Teteroboro, NJ.

New approaches to the diagnosis and prognosis of neurodegenerative diseases will be the next frontier in clinical laboratory medicine. Analysis of disease processes affecting nerve cells is becoming a reality. Exploration of diagnostic markers for Alzheimer’s disease is intense. Immunochemistry and molecular diagnostic-based testing is available for Parkinsonism, neuropathies, muscular dystrophies, and other neurogenetic disorders. Immune responses play a role in paraneoplastic neurological disorders and the presence of specific antibodies can indicate the most likely tumor site. Autoimmune and genetic tests assist in the diagnosis of peripheral neuropathies. Risk analysis through nucleotide polymorphism testing appears promising for select disorders. Proteomics of dementia is another area of recent advance. The genetics of schizophrenia has implicated certain biochemical processes in this disease. Knowledge of these areas is progressing rapidly and clinical scientists should be aware of progress and problems in the development of tools for the diagnosis of these conditions.

[11] Prion encephalopathy: CJD and new variants. Peter M. Farmer, North Shore University Hospital, Manhasset, NY.

Prion diseases in humans and other animals are characterized by spongy degeneration of the grey matter of the brain and proliferation of a pathogenetic abnormal or scrapie isoform of the prion protein. In sporatic Creutzfeldt-Jakob Disease (CJD), the scrapie and normal isoforms have identical amino acid sequences but differ in their 3-dimensional conformations. The scrapie isoform is extremely stable, protease resistant, and polymerizes into an amyloid-like compound. In vivo, the scrapie agent acts as a template that converts the normal prion protein to the abnormal isoform. The resulting cascade of transformed protein molecules facilitates the spread of the disease throughout the brain without the need for the nuclear protein biosynthetic mechanisms. In sporatic CJD, conversion to the scrapie isoform may occur spontaneously or possibly through “infection” by an environmental source of prions. Transmission of bovine spongiform encephalopathy (“Mad Cow Disease”) to humans is recognized as “new variant” spongy degeneration with prominent amyloid deposition. The public health risk of prion diseases in the food supply and in wild animals is yet to be determined. The risk of transmission of prion disease to laboratory workers exposed to brain biopsy and autopsy material is small and can be minimized by appropriate precautions.

[12] The role of metal ions as initiators of neuro-degeneration. John Savory and Othman Ghribi, University of Virginia, Charlottesville, VA.

Deposition of A in plaques and the formation of intraneuronal neurofibrillary tangles represent specific features of Alzheimer’s disease and other neurodegenerative disorders. However, whether these neuropatholgical features represent key pathogenic factors in these disorders, or are a response to an upstream process, is unclear. Three metal ions have received the greatest attention as being involved in this process: aluminum (Al), copper (Cu), and zinc (Zn). Cu and Zn are known to induce A flocculation in vitro, and treatment of mice transgenic to APP 2567 with clioquinol, a Cu/Zn chelator, significantly improves general health and body weight parameters in comparison to sham-treated animals. However, it remains to be shown that Cu and Zn cause tau hyperphosphorylation (p-tau), a process integral to the formation of neurofibrillary tangles in Alzheimer’s disease. In contrast, we have demonstrated that administration of Al maltolate intracisternally to New Zealand white rabbits induces tau hyperphosphorylation. Al compounds have been shown by other investigators to increase brain amyloidosis in transgenic mice that accumulate A. These results clearly suggest that Al has the ability to produce p-tau in rabbit brain and to induce A deposition. Although other metal ions have the ability to interact with A, they do not produce p-tau. The endoplasmic reticulum plays a key role in responding to this Al-induced neurotoxic injury. Caspase-12 is activated and there is decrease in Bcl-2 and increase in Bax and caspase-3 activation; the activation of caspase-3 is primarily in the endoplasmic reticulum. Apoptosis is present since, in addition to caspase-3 activation, there is TUNEL positivity, a technique used to detect DNA fragmentation.(Supported by a grant from U. S. EPA.)

[13] Flow cytometric measurement of aluminum chloride, citrate, and maltolate uptake by leukocytes. Roger L. Bertholf and Y. Monique Huggins, University of Florida College of Medicine, Jacksonville, FL.

Aluminum uptake by leukocytes was demonstrated by flow cytometric measurement of a fluorescent aluminum complex, and verified by electrothermal atomic absorption spectrophotemetry. Erythrocytes in whole blood specimens were disrupted and the remaining cells were washed and incubated with phosphate buffer containing aluminum chloride, citrate, or maltolate. After a second washing, aluminum-exposed cells were permeated with a fluorescent stain for aluminum. The fluorescent aluminum complex was measured by flow cytometry. The rate of aluminum uptake was affected by the incubation time and the specific aluminum compound to which the cells were exposed. Aluminum chloride did not enter the cells in appreciable amounts, whereas intracellular aluminum was clearly demonstrated after exposure of leukocytes to aluminum citrate and aluminum maltol. Analysis of flow cytometric data indicated that aluminum uptake occurred most dramatically in lymphocytes. These results are consistent with previous reports of intralymphocytic aluminum accumulation, suggesting a relationship between the solubility of aluminum complexes and their access to the leukocyte cytosol. Flow cytometric measurement of intraleukocytic aluminum may be useful for assessing aluminum exposure and toxicity.

[14] Bio-morphological rationale for laser stimulation of myofascial trigger points and acupuncture points. Alex Tatevian and Artem Grush, Foundation for Research in Acupuncture & Integrative Medicine; Department of Anesthesia, Harvard School of Medicine, Boston, MA.

Routine techniques of myofascial trigger points (MTrP) treatment include needling procedure with an injection of pharmacological substances or without (dry needle technique). Dry needle technique, not much different from acupuncture needling, adds to similarities between MTrP and acupuncture points (AP), including their location and distribution, pain and referred pain patterns, etc. The scientific basis for either MTrP injection or acupuncture is still unclear, although there is a body of evidence relating response in both cases to neuro-transmitter and neuro-hormone release, notably that of the endogenous opioids. With various means of point stimulation granting comparable effect, the least invasive techniques appear most attractive. We investigated the effect of Low Energy Lasers (LEL) in MTrP and AP stimulation. LEL’s are laser devices in which power densities and energy densities of laser beam are lowered to a point where no photo-thermal effects occur, but the photo-osmotic, photo-ionic, and photo-enzymatic effects are still operative. Penetration of a laser beam into tissues falls off at an exponential fashion. Thus, increase of laser power does not result in a linear increase in the penetration depth and in a linear increase of biological effect. The prime determinant of tissue penetration is the wavelength (color) and pulse regime, which makes pulsed Infrared Laser the most suitable laser device. LEL has distinct advantages over needling of MTrP and AP: it is aseptic, non-invasive, and painless; if used properly, it has no reported side effects; it is ideal for children and patients with needle-phobia. Infrared Laser is the only tool for a double-blind, randomized, placebo-controlled study to investigate the effectiveness of acupuncture.

[15] Molecular pathology of alterations in thermoregulation: role of mitochondrial uncoupling proteins. Egil Fosslien, Dept. of Pathology, College of Medicine, University of Illinois at Chicago, Chicago, IL.

The objective of this review is to analyze recent findings of alterations in the function of mitochondrial uncoupling proteins (UCPs) in the etiology of fever compared to other disorders of thermoregulation. Reports on the structure and function of UCPs and studies of in vitro and in vivo signaling pathways that regulate their synthesis and function were compared and analyzed. Mitochondrial oxidative phosphorylation couples inner mitochondrial potential energy derived from transmembrane proton pumping powered by energy from metabolites to generation of adenosine triphosphate (ATP). Uncoupling occurs through proton pump slippage (oxygen consumption without proton pumping), passive membrane leakage, or active protonophoric conductance by mammalian uncoupling proteins (UCPs), which protect against membrane hyperpolarization and radical oxygen species (ROS) formation, regulate ATP synthesis, and control mitochondrial thermogenesis. Body core temperature is regulated by hypothalamic prostaglandin (PG) E2, which signals through its receptors, particularly EP3, via b-adrenergic pathways and stimulates brown adipose tissue (BAT) thermogenesis by inducing UCP-1. Additionally, hepatic heat production contributes to adult thermal homeostasis. Fever hyperthermia caused by infectious agents is centrally regulated and involves signaling by pyrogens such as lipopolysaccharide (LPS), interleukin (IL)-1, and IL-6; LPS derived from infectious agents induce tumor necrosis factor (TNF)-a. Cytomeg-alovirus induces transforming growth factor (TGF)-β1 transcription and upregulates UCP-2 expression via cyclooxygenase (COX)-2, PGE2, and leptin. UCP-3 thermogenesis is regulated by thyroid hormone, β-adrenergic signals, and leptin. Cytokines released by small tumors can cause tumor fever, which often precedes other tumor symptoms. Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit COX-2, reduce PGE2 synthesis, and ameliorate fever. In conclusion, thermogenic mitochondrial uncoupling proteins maintain body core temperature and cause fever induced by infectious agents or cytokines released by tumors.

[16] Development of a mouse model for studies of glucagon secretion. Arun S. Rajan, Min Hu, and Nina Tatevian, Baylor College of Medicine, Houston, TX.

While much is known about the pathophysiology of insulin secretion, the processes underlying the physiology and pathology of glucagon release in diabetes remains poorly understood. This is in part due to the lack of good experimental models to study alpha cell glucagon secretion. In the present study, we have taken advantage of novel genetic engineering approaches to generate a mouse model that may be useful in studies of alpha cell pathophysiology. Using DNA coding for the glucagon gene promoter to drive expression of Cre recombinase, we have generated a Glucagon-CRE mouse (GLU-Cre) which expresses Cre recombinase in alpha cells. Cre-lox recombination will then allow expression or excision of target genes of interest specifically in alpha cells. To verify that Cre recombinase is expressed in alpha cells, we crossed the GLU-Cre mice with a ROSA26 reporter mouse and demonstrated alpha cell-specific expression of β-galactosidase (by frozen section histochemistry and X-gal staining) in the progeny. We observed no β-galactosidase expression in other tissues including liver, lung, and colon. Furthermore, the GLU-Cre mice exhibited glucose tolerance and glucagon secretory profiles comparable to wild-type controls, indicating alpha cell Cre recombinase expression did not interfere with normal metabolism. Thus, the GLU-Cre mice we have generated may serve as a useful model to examine the role of specific proteins in alpha cell function and allow experimental investigation of the cellular mechanisms of glucagon secretion in health and disease.

[17] Drug-induced neutropenia. Abdus Saleem, Varsha M. Bhatt, Baylor College of Medicine, Houston, TX.

Drug therapy plays a significant role in causing neutropenia. The neutropenia may be immune mediated or due to direct inhibition of bone marrow precursors. Recently, due to wide use of chemotherapy, febrile neutropenia has become a common devastating problem. Neutropenia predisposes to many bacterial and fungal infections with organisms including Gram-negative bacilli such as E. coli, Klebsiella, and Pseudomonas, Gram positive organisms such as Staphylococcus, Streptococcus viridans, and Enterococci species and fungi like Candida and Aspergillus. In addition to providing supportive care to the neutropenic patients, recombinant human granulocyte colony-stimulating factor (rG-CSF) has been found rewarding. Though filgrastim was a significant advance in the management of drug induced neutropenia in the last decade, pegfilgrastim (a pegylated form of filgrastim) ushers in the next decade. Pegfilgrastim (Neulasta), is a long acting form of filgrastim, that is administered as a single subcutaneous injection once per chemotherapy cycle. This results in fewer injections, fewer office visits, and better compliance with therapy.

[18] Laboratory methods for hemoglobinopathy screening and detection. Jonathan S. Krauss, Medical College of Georgia, Augusta, GA.

In 1949, Pauling used electrophoresis to identify Hemoglobin (Hb) S by its slower mobility compared to HbA. Itano developed the solubility test for confirmation of HbS (1953). Since that time, laboratory methods for hemoglobinopathy identification have proliferated. The International Committee for Standardization in Hematology (ICSH) recommended a complete blood count (CBC), alkaline electrophoresis, solubility testing for sickling, and quantification of both HbF and HbA2 for hemoglobinopathy investigation (1975). Other useful tests include acid agar electrophoresis, isoelectric focusing (IEF), and globin chain separation, as well as heat- and isopropanol-stability tests. Gradually, cation-exchange high performance liquid chromatography (HPLC) has replaced alkaline (cellulose acetate) electrophoresis as the primary screening step for hemoglobin variant identification. HPLC is also a mainstay for HbA₂ and HbF quantification. A decreased mean cell volume (MCV) with an increased red blood cell count (RBC) and normal red cell distribution width (RDW) are commonly associated with thalassemia minor, whereas a low RBC and elevated RDW with a low MCV are more compatible with iron deficiency. The MCV and RBC have been used specifically to discriminate iron lack from thalassemia minor. Blood smear review and stains for HbH inclusions can also be helpful hemoglobinopathy studies. Immunologic identification has been used for some of the more common Hb variants. Recently, the polymerase chain reaction (PCR) identified alpha and beta chain haplotypes. Electrospray mass spectrometry (ESMS) has been used to identify Hb Kenya. Furthermore, the LightCycler™ real time PCR analyzer has been utilized for rapid beta-globin gene mutation identification. Finally, infrared spectroscopy has been employed in beta-thalassemia diagnosis. Hemoglobinopathy screening and identification is a mature hematologic discipline that is evolving due to technological change. Precise algorithms for hemoglobinopathy investigation may vary due to location, budgetary constraints, and available technology.

[19] Blood transfusion associated fatalities for 1999. Byron Myhre, Harbor-UCLA Med Center, Torrance, CA.

Since 1975, the U S Food and Drug Administration‘s Center for Biological Evaluation and Research (FDA-CBER) has required that all fatalities due to blood transfusion or donation be reported. Several communications have reviewed these statistics for prior years. Employing the Freedom of Information Act (FOIA), the reported fatalities due to blood transfusion for 1999 were requested from the FDA-CBER. These were studied, categorized, and totaled. There were 50 reported fatalities for the year that could be related to blood transfusion. The 3 major causes were: administration of incompatible blood (32%), Transfusion Related Acute Lung Injury (TRALI) (22%), and septicemia due to contaminated blood products (22%). The incidence of fatalities is about 1.2/100,000 patients transfused. Human error still seems to be a significant cause, while the diagnosis of TRALI, poorly understood in 1985, has gained considerable significance. An increase in fatalities due to bacterial contamination, especially with platelets, has occurred, possibly due to longer storage times at higher temperatures. Continual surveillance of transfusion fatalities is needed to determine if these changes reflect a true shift in incidence, or only a periodic fluctuation.

[20] Application of real-time PCR and melting curve analysis in rapid detection of A_el and B_el phenotypes in Taiwan. Chien-Feng Sun, Ding-Ping Chen, Ching-Ping Tseng, Hung-Tse Lin, Wei-Ting Wang, Chang Gung Memorial Hospital and Chang Gung University, Taoyuan, Taiwan.

The ABO blood group is the most important blood group system in transfusion medicine. However, in the A_el and B_el phenotypes, it’s difficult to clearly determine the ABO blood group by conventional serologic methods. Through genetic analysis, we have identified the A_el phenotype to possess an A allele with IVS6+5G→A mutation (Transfusion 2003;43:1138–1144MedlineGoogle Scholar) and the B_el phenotype to possess a B gene with 502C→T mutation. (Vox Sanguinis 2003;85:216–220MedlineGoogle Scholar). We report here the development of a LightCycler (LC) real-time PCR assay and melting curve analysis (Tm) to facilitate the genotype detection of A_el and B_el blood types. The region of mutations was PCR amplified and detected using LC Red640-labeled hybridization probe. For A_el genotype, the melting curve of normal A phenotype appears as one peak at 59 ± 0.5°C, and of A_el appears as 2 peaks at 59 ± 0.5°C and 67 ± 0.5°C, respectively. For B_el genotype, the melting curve of normal B phenotype appears as one peak at 68 ± 0.5°C, and of B_el appears as two peaks at 60 ± 0.5°C and 68 ± 0.5°C, respectively. This genotyping method is 100% accurate as confirmed by automated sequencing of the PCR amplified products. Besides, it takes only 90 min for the completion of this genotyping test. Together, detection of A_el and B_el blood type by

combined LC-PCR and melting-curve analysis is a rapid, reliable, and easy method.

[21] Atypical presentation of anti-Rga. Eiad Kahwash, Sameer S. Talwalkar, Jill Leonard, William Lockwood, Dept of Pathology and Laboratory Medicine, University of Louisville Medical School, Louisville, KY.

The Rodgers (Rga) antigen is strictly a plasma protein, which binds to cell membranes. About 2–3% of the recipient population lacks this antigen and can produce anti-Rga antibody. Rodgers is an allotype of the C4 component of plasma complement and serum or plasma C4 inhibits anti-Rga. This is a case report of a 70-yr-old African-American male with a medical history of hairy cell leukemia and profound pancytopenia that required RBC and platelet transfusions. The patient had been previously transfused with 2 units of RBCs and 4 platelet pools. The patient typed O Rh-negative (rr). The 3-cell antibody screen by routine polyethylene glycol PEG (anti-IgG) was strongly reactive (2+ to 4+) with all 3 cells and the direct antiglobulin test (DAT) was negative. Further serological testing, consisting of 2 manufacturer’s antibody identification panels was non-reactive, including the autologous control. Testing by solid phase technique was found to be non-reactive both in the Capture-R^® Ready Screen and Capture-R^® Ready ID. The antibody screen was repeated using washed cells and was found reactive (1+ to 2+) by PEG (anti-IgG) with all 3-antibody screen cells. The strongly positive reactivity in the antibody screen prompted further investigation. Serological investigation showed reactivity with only 5 of 30 red cells in the antibody identification panels. Anti-Rga was confirmed after the serum was serially diluted, each dilution neutralized with pooled plasma and testing performed by the gel method. The dilutions that were neutralized reacted to a titer of 8. Each control dilution that did not contain pooled plasma reacted to a titer of 32. Undiluted serum was also non-reactive with enzyme treated cells. The patient’s cells were non-reactive with 2 sources of anti-Rga. These findings are consistent with anti-Rga, even though there was limited reactivity in the antibody identification panels. Due to the low percentage of reactive red cells in the antibody identification panels, crossmatch compatible (by PEG) units of RBCs were transfused with no discernible immediate or delayed transfusion reactions. In conclusion, it is important to be aware that the panel cells that are usually reliable for antibody identification purposes may not have the antigens that the screening cells have. (Supported in part by the University of Louisville and by the Norton Healthcare Community Trust Fund # 22-07).

[22] Changing patterns of antibiotic resistance in cancer patients. Issam Raad, The University of Texas/M.D. Anderson Cancer Center, Houston, TX.

Immunocompromised cancer patients, particularly those with neutropenia and fever, require broad-spectrum empiric antimicrobial therapy. In most of these patients, a microbial etiology of the fever is not readily detected and, hence, broad-spectrum antibiotics and antifungal agents are maintained. Over the last 2 decades, there has been a changing pattern in the microepidemiology of organisms causing infections in cancer patients. There has been a shift towards the predominance of resistant Gram-positive bacterial infections, often attributed to the widespread use of central venous catheters and anti-Gram-negative prophylactic antibiotics in this patient population. Leading organisms among the Gram-positive bacteria are the methicillin-resistant staphylococci (S. epidermidis and S. aureus). This has led to excessive use of vancomycin, which in turn resulted in increasing rates of vancomycin-resistant enterococci in the cancer patient population. Novel anti-Gram-positive antibiotic agents have been developed, such as quinupristin/dalfopristin, linezolid, and daptomycin, with concurrent emergence of resistant organisms to these agents. In addition, the excessive use of quinolones and carbapenems in the treatment of Gram-negative bacilli, particularly Pseudomonas aeruginosa, has been associated with emergence of multi-drug resistant P. aeruginosa in cancer and critically ill patients. Cancer patients who are receive broad-spectrum antibacterial and antifungal agents are prone to develop antibiotic-resistant organisms. Study of the epidemiology of these resistant organisms with alternative and appropriate therapy is of paramount importance.

[23] The neuroendocrine tumor concept. Joseph C. Parker, Jr., University of Louisville School of Medicine, Louisville, KY.

Neuroendocrine tumors found throughout the body include discrete neuroendocrine organ systems like the adenohypophysis and adrenal medulla and a dispersed neuroendocrine cell system, found as single or small cell clusters in neural and non-neural sites. The gastrointestinal tract, for example, has more than 20 neuro-endocrine cell types with enterochromaffin cell nerve fiber complexes. Phylogenetically, an integrated neuro-endocrine function is evident with photoreceptors in fish and amphibians transducing light into action potentials received by pineal neurons, while in reptiles and birds, a photoendocrine (pineal) transducer converts light into hormones. Finally, in mammals a neuroendocrine (pineal) transducer converts sympathetic impulses into circulating hormones. The intracellular membrane-bound secretory granules that characterize neuroendocrine tumors can be secreted into vessels as hormones, into synaptic clefts as a neurotransmitter, or into local intercellular spaces. A neural crest origin for neuroendocrine cells is evident in the thyroid, pancreas, adrenals, paraganglia, and clear cells in the respiratory and gastrointestinal systems. On the other hand, paraneuronal cells from neuroectoderm as in the pineal gland and hypothalamus produce chemicals with both neurotransmitter and hormonal functions. Neurosecretory granules in various neuroendocrine tumors have morphologic similarities and may be the same chemical whether of neural crest or neuroectodermal origin. These cells have also been found in non-endocrine neoplasms throughout the body. Characterizing such tumors requires selective neuroendocrine cell markers and an understanding of their benign and malignant behavior, their functional characteristics, their grading, and their sites of origins. Cytokeratin and neurofilament triple protein expressions may help separate neural neuro-endocrine tumors from epithelial neuroendocrine tumors. Their behavior, which varies from just local invasion to aggressive widespread metastases, may be difficult to predict from their morphology alone.

[24] Protein levels of p53 correlate with Velcade-induced inhibitory effects on breast carcinoma cell lines. Robert E. Brown, Mingyue Lun, Nava Siegelmann-Danieli, Thomas M. Blasick and Ping L. Zhang, Geisinger Medical Center, Danville, PA.

To further understand the mechanisms of Velcade-related cell inhibition, we examined its effects on p53-associated inhibition of proliferation and apoptotic pathways. Three human breast carcinoma cell lines (SKBR-3, MDA-175, and MDA-231), negative for hormonal receptors, were studied. The cell lines were incubated with and without 90 nM of Velcade for 2 days and then a tetrazolium-related proliferation assay was performed. Western blot analysis on either whole cell lysates or nuclear components following exposure of the cell lines to 90 nM Velcade for 18 hr was carried out to assess the impact on a series of protein markers. Velcade induced inhibitory rates on the proliferation of the breast carcinoma cells at 62.5 ± 1.0%, 51.9 ± 1.6%,* and 83.0 ± 0.9%*# in SKBR-3, MDA-175, and MDA-231, respectively. (p values, *<0.05 vs SKBR-3 and #<0.05 vs MDA-175). The magnitude of the inhibition paralleled the baseline levels of nuclear p53 protein expression in the 3 cell lines. Subsequent to Velcade treatment, the most striking upregulation of both p53 and p21 waf1 was found in MDA-231 cells, followed by SKBR-3 and MDA-175. In addition, caspase-3, a terminal apoptotic signal, was only cleaved in MDA-231 cells, indicating enhanced apoptosis in this cell line. Interestingly, following Velcade treatment, I-kB in whole cell lysates was reduced, and nuclear I-kB was increased, but p-NF-kB was increased in both whole cell lysates and nuclear components. Conclusions: Our data show that upregulated p53 and p21waf1, after Velcade treatment, were associated with inhibition of cell proliferation and at the same time promoted the formation of an apoptotic protein (cleaved caspase 3) in MDA-231, despite the induction of higher levels of nuclear p-NF-kB in breast cancer cells. In summary, the pretreatment level of nuclear p53, in addition to baseline p-NF-kappaB expression, appears to be a predictive marker for the therapeutic response to Velcade in hormone-receptor-negative breast cancer cells and provides insight into the mechanisms of action of this proteasome inhibitor.

[25] Hoechst 33342-induced apoptosis in promyelocytic leukemia HL-60 cells alters both phospholipid and glycosphingolipid composition. Xinbo Zhang, Frederick L. Kiechle, Dept. of Clinical Pathology, William Beaumont Hospital, Royal Oak, MI.

Our previous results demonstrate that Hoechst 33342-induced apoptosis is associated with alterations in glycosphingolipid composition (Kiechle, Zhang, Ann Clin Lab Sci 2003;33:361–362Google Scholar). To elucidate the potential alterations in intracellular lipid profile, we have determined the effect of Hoechst 33342 on the composition of phospholipids in promyelocytic leukemia HL-60 cells using thin layer chromatography. The present results indicate that Hoechst 33342-induced apoptosis in HL-60 cells is related to a significant decrease of both phosphatidylcholine and sphingomyelin in a dose-dependent and a time-dependent manner. These data suggest that phospholipids and glycosphingolipids play important roles in Hoechst 33342-induced apoptosis.

[26] Chronic lymphocytic leukemia (CLL) conventional karyotypes: comparison of male to female populations. Armand B. Glassman, Edwardo Cantu, The University of Texas/M. D. Anderson Cancer Center, Houston, TX.

Chronic lymphocytic leukemia is a neoplasm of monomorphic small round B lymphocytes found in the peripheral blood, bone marrow, and/or lymph nodes. There is a male to female ratio of approximately 2:1. To determine whether the disproportional sex ratio is characterized by conventional chromosomal karyotypic abnormalities, the following study was performed. 301 CLL patients’ karyotypes from UTMDACC during 2002 were reviewed and analyzed after IRB approval. Standard cytogenetic techniques were used to evaluate karyotypic abnormalities. The sex ratio of the CLL database was 2.4 males to 1 female (212 males and 89 females). Chromosomal abnormalities detected by conventional cytogenetics were 26% (23/89) in females and 31% (66/ 212) in males. Loss of the respective sex chromosomes occurred in 4/23 (17%) females and 10/66 (15%) males. Prominent numerical abnormalities found in females included an additional marker in 8/23 (34%) or trisomy 12 (+12) in 7 of 23 (30%). Structural abnormalities noted in women were deletion of 11q in 8/23 (34%) and deletion of 4q or deletion of 14q in 4/23 (17%). Numerical abnormalities in males included +12 in 18/ 66 (27%); an additional marker in 9/36 (15%), and loss of the Y chromosome in approximately 15%. Structural abnormalities in males included the deletion of the 11q in 10/66 (15%), deletion of 14q in 7/66 (11%), and deletion of 6q in 5/66 (8%). Cytogenetic findings other than markers are not significant predictors for the male to female CLL occurrence ratio.

[27] A decoy androgen responsive element DNA can inhibit androgen receptor transactivation of PSA gene. Pengju Zhang, Jianye Zhang, Charles YF. Young, Pai Chi Kao, Weiwen Chen, Anli Jiang, Lianying Zhang, and Qiang Guo. Shandong University, Jinan, China, and Mayo Clinic, Rochester, MN.

Over-active androgen receptor (AR) may increase the risk for prostate cancers and also play an active role in androgen-refractory prostate cancer. The AR after androgen activation binds a specific genomic DNA sequence called androgen responsive element (ARE) to regulate the gene transcription of prostate-specific antigen (PSA). PSA is the most sensitive prostate cancer biomarker and the most widely used marker for androgen function, because PSA gene is the direct target of AR via its binding to the ARE of the PSA promoter. This study tested the use of a short double-stranded oligo-DNA containing PSA ARE sequence as a decoy that would inhibit the function of AR. A 23-mer phosphorothioated PSA ARE decoy DNA was synthesized. A human androgen receptor (hAR) expression plasmid, a plasmid vector containing the PSA promoter linked to the luciferase gene as a reporter, and the ARE decoy DNA were co-transfected into a human prostate cancer cell line PC3-M by lipofectaminTM2000. After 24 hr of exposure to androgens, cell extracts were prepared to measure luciferase activity in monitoring the effect of the ARE decoy on AR function. The activities of luciferase were significantly reduced in the ARE decoy transfected cells, but not in the cells with the control decoy (inactive decoy). The results demonstrated that ARE decoy DNA can effectively suppress the transactivation of AR in prostate cancer cells and indicated its potential utility as a novel therapeutic tool for prostate cancers.

[28] Development of a multiplex PCR-melting curve mutation detection system to rapidly detect the SHV-and CTX-M-type extended-spectrum β-lactamase-producing Enterobacteriaceae in Taiwan.

Tsu-Lan Wu, Ju-Hsin Chia, Chishih Chu, An-Jing Kuo, Lin-Hui Su, and Cheng-Hsun Chiu, Chang Gung Memorial Hospital and Chang Gung Children’s Hospital, Taoyuan, Taiwan Extended-spectrum β-lactamases (ESBLs) are enzymes produced in some Gram-negative bacilli. These enzymes are capable of hydrolyzing and causing resistance to oxyimino-cephalosporins and aztreonam. In immunocompromised patients, such as neutropenic or neonatal patients, bacteremia caused by ESBL-producing Enterobacteriaceae may lead to a high mortality. Therefore prevention of the spread of ESBL-producing isolates is very important. The prevalence of ESBLs in Klebsiella pneumoniae and Escherichia coli in Taiwan was reported as 8.5% and 30%, respectively. Some ESBL phenotypes, including SHV-2, SHV-5, SHV-12, CTX-M-3, and CTX-M-14, are prevalent all over Taiwan. Direct sequencing of the amplified genes was time-consuming and labor-intensive for the complex population. The aim of this study was to develop a rapid system, which consists of a multiplex PCR to screen for blaSHV, blaCTX-M-3 and blaCTX-M-14 genes and a real time PCR - melting curve method to rapidly discriminate the blaSHV genes prevalent in Taiwan. Using the phenotypic confirmatory method recommended by the NCCLS, a total of 65 clinical ESBL-producing isolates, including 1 Enterobacter aerogenes, 5 Enterobacter cloacae, 18 E. coli, and 41 K. pneumoniae, were identified and subjected for further molecular analysis. A novel multiplex PCR-melting curve mutation detection system was developed to investigate the ESBL genotypes among these isolates and the results were compared with those obtained by a conventional nucleotide sequencing method. CTX-M-3 (36 isolates) was the most prevalent type of ESBLs identified, followed in the order of frequency by SHV-12 (25), SHV-2a (7), CTX-M-14 (6), SHV-5 (5), and SHV-2 (3). Seventeen isolates harbored both SHV- and CTX-M-type ESBLs. Identical results were obtained by both the novel multiplex PCR-melting curve mutation detection system and the conventional nucleotide sequencing method. In conclusion, the accuracy of the novel multiplex PCR-melting curve mutation detection system was confirmed by the conventional nucleotide sequencing method. The system provides an efficient way in the differentiation of ESBLs among the Enterobacteriaceae. Moreover, the design of the system can be easily adapted for similar purposes in areas where different ESBLs are prevalent.

[29] Infections and other inflammatory complications of transplantation. Dani S. Zander, Univiersity of Texas Health Science Center–Houston Medical School, Houston, TX.

In patients undergoing solid organ or bone marrow transplantation, the lungs are a common site of pathology. There are a limited number of histologic reaction patterns that provide clues to the etiology of respiratory decline in these patients, and therapeutic decisions are highly influenced by pathologists’ judgments concerning etiology. The decision-making process is made more complex by the pathogenetic overlap of infections with other immunologic sequelae of allotransplantation. This presentation will address some of the challenging questions that arise in lung biopsies from transplant recipients, and present strategies for the evaluation of these difficult cases.

[30] Air leak phenomenon in a term infant. Drifa Freysdottir, Oluyinka Olutoya, Caraciolo J Fernandes, Nina Tatevian, Baylor College of Medicine, Houston TX.

We present a unique case of the development of an air leak phenomenon in a term, unventilated infant. Pulmonary interstitial emphysema is a form of air block usually seen in ventilated preterm infants with severe lung disease, but perviously unreported in spontaneously breathing term infants. The infant, who had previously been diagnosed as having laryngomalacia, cardiac dysfunction, and evidence of antenatal stroke, presented at 6 wk-of-age with spontaneous pneumothorax and cystic changes in the lungs, despite no history of mechanical ventilation. Repeated chest X-rays previously had shown no evidence of cystic changes. He subsequently underwent resection of the affected area. Pathology revealed persistent interstitial emphysema with cyst formation, pleural blebs, and reactive pleuritis, as well as subpleural air space enlargement. The patient did well postoperatively and was discharged without further problems. This case demonstrates the necessity of a high index of suspicion for pulmonary interstitial emphysema, even in a spontaneously breathing term neonates. Pulmonary interstitial emphysemia needs to be included in the differential diagnosis of cystic lesions of the lungs in this age group.

[31] Stomatococcus mucilaginosus: a rare cause of meningitis in immune suppressed patients. Meena Bhattacharjee, Sara Szabo, James Versalovic, Robert Krance, and John Hicks, Texas Children’s Hospital, Houston, TX.

This report describes a rare cause of bacterial meningitis in a 13-yr-old male, a bone marrow transplant recipient, and emphasizes the importance of molecular characterization in identification of emerging opportunistic organisms, which may elude precise identification by standard cultures. The patient received a second bone marrow transplant for relapsed acute myeloid leukemia. Two months post-transplantation, CT imaging showed multiple hepatic nodules suggestive of abscesses. A needle biopsy of the liver showed small organisms suggestive of fungi. However, cultures revealed coagulase-negative cocci; pyro-sequencing identified these organisms as Stomatococcus mucilaginosus. The boy subsequently developed multiple pulmonary nodules and right lower lobe pneumonia. Head CT was performed due to increasing lethargy. It showed cerebral atrophy without a focal parenchymal lesion; the findings were attributed to steroid and immunosuppressive therapy. Follow-up neuroimaging showed progression with further volume loss, ventricular dilatation, and expansion of extra-axial CSF spaces, again without focal lesions. MR imaging showed generalized meningeal enhancement suggestive of infection, inflammation, or leukemic infiltration. However, before a spinal tap could be obtained, he developed seizures and died. A complete autopsy was performed. Brain examination showed a florid mucoid and gelatinous exudate in the subarachnoid space and filling the ventricles; microscopy showed abundant mucinous material with masses of Gram-positive coccoid organisms with virtually no host reaction. The meningeal reaction consisted solely of macrophages and multinucleate giant cells, many with engulfed bacteria. Neutrophils and lymphocytes were absent Although, there are reports of bacteremia and pneumonia due to Stomatococcus mucilaginosus in neutropenic patients, our patient is the second reported case of bacterial meningitis due to this organism.

[32] Interstitial lung diseases with a familial incidence. Juliana Szakacs, Univ. of Utah Medical School, Salt Lake City, UT.

The pathologic diagnosis of diffuse lung disease is based on the recognition of specific patterns closely correlated with the clinical history and the radiologic findings. The current pathologic criteria for the classification of idiopathic interstitial pneumonias are becoming well defined and guidelines have allowed more specific identification of disease entities. With improved classification, cases of specific familial disorders are becoming evident. Kindreds of desquamative interstitial pneumonia (DIP) and usual interstitial pneumonia (UIP) are now described with inheritance patterns. Genetic abnormalities are being sought and may assist in the future understanding of these debilitating diseases. Patients in suspected kindreds may now present for early screening and the pathologist will be called upon to diagnose interstitial pulmonary fibrosis in pre-symptomatic patients or in pediatric patients in which criteria are not as well defined. The presentation of early disease when suspected in a familial cluster may not fulfill all of the radiologic or pathologic criteria for diagnosis of full blown and symptomatic disease and exposure history may be absent.

[33] Advancing automated technology for the histology laboratory: key design elements. Derek P Lehane and Alan Walton, Thermo-Electron Anatomical Pathology, Pittsburgh, PA, and Runcorn, UK.

Tissue processing is a laborious and time-consuming activity that requires histology laboratory personnel to handle large quantities of hazardous volatile reagents. Histopathologists are demanding systems that not only provide automation and consistency, but shield their staff from exposure to toxic chemicals, provide ease of use, and yield significant savings in total cost of ownership. A structured process for user involvement in design, based in the UK and US, has led to a fundamental rethinking of the design of a modern tissue processor. It is in reagent replenishment that most of the exposure and cost arise. Deploying automatic monitoring of reagent specific gravity allows the replenishment process to be largely automated, and to take place in an enclosed environment. It also has the effect of using reagents optimally. The use of advanced polymers facilitates a design that provides fume extraction around the chamber, minimising exposure. Use of a reagent selection valve integrated into the chamber allows large, individual ports for each reagent, eliminating a source of cross-contamination and allowing faster fluid transfers at low pressure. Energy-efficient design allows battery back-up to preserve specimen quality in the event of power outage. Comprehensive system information can be displayed, printed, accessed by modem, or saved to disc to help in managing the process. The user interface includes training aids and is graphically based in order to provide quick assimilation of information. Extensive system testing in histopathology laboratories has shown that this new approach to tissue processing has met all of its design objectives. More than 300 laboratories across the globe now process specimens in a safer manner, with substantial savings in reagent costs (approx. 65%) and in technologist time.

[34] Anatomic pathology slide seminar: inflammatory and infectious lung diseases. Dani S. Zander, Univ, of Texas Health Science Center at Houston Medical School, Houston, TX; Philip T. Cagle, Baylor College of Medicine, Houston, TX

This slide seminar offers participants a chance to review an unusual and challenging mixture of inflammatory and infectious lung biopsy and cytology cases, discussed by a panel of expert pulmonary pathologists. Prior to the seminar, participants will have opportunity to review representative photographs of the cases and brief histories on a website created for this purpose. Participants are encouraged to submit their diagnoses electronically before the meeting, so that they are eligible to win a prize for the most accurate list of diagnoses. At the seminar, cases will be presented and discussed by the faculty, with attention to other similar-appearing entities in the differential diagnoses.

[35] Introduction of free light chain assays into an outpatient laboratory. Clive R. Hamlin, Case Western Reserve University School of Medicine, Cleveland, OH.

Free light chains (FLC) (ie, peptide chains not associated with intact immunoglobulins), have been difficult to measure in blood or urine, owing to their low levels and the poor sensitivity of reagents. Light chain diseases, including amyloidosis and light chain myeloma, are presently diagnosed by bone biopsy well into the disease course. In 2001, a pair of free light chain assays were introduced, one to quantify free kappa and the second to quantify free lambda. In addition, a kappa/ lambda FLC ratio is calculated. As these assays became more widely utilized, I became interested in having them available on-site. However, patient management strategies are not clear in the absence of large clinical studies. Consequently, it was decided to include FLC assays with each request for protein electrophoresis, once for each patient without charge. Since introduction (October 15, 2003) there have been 307 FLC assays with 222 showing abnormalities. This included 17 IgM-kappa, 9 IgG-lambda, 5 IgG-kappa, 3 each IgA-lambda and free lambda, 2 IgA-kappa, and 1 each IgM-lambda and free kappa multiple myelona patients, of whom 32 showed abnormalities and 9 had completely normal FLC parameters. Additionally, 33 patients showed elevated FLC associated with polyclonal increased immunoglobulins. The remaining 157 patients with FLC abnormalities present a diagnostic dilemma.

[36] Current trends and issues in allergen-specific IgE testing. Thomas M. Li and Debra Hovanec-Burns, Diagnostic Products Corporation, Los Angeles, CA.

The objectives of this presentation are to survey current trends and issues in allergy blood testing, and to describe the development and potential applications of an automated, third-generation, allergen-specific, IgE (sIgE) assay. Earlier generation sIgE assays are associated with problems of precision and accuracy. Due to their use of cellulose discs and cyanogen bromide chemistry, these assays suffer from high NSB and have a tendency to yield false positives. IgE to cross-reactive carbohydrate determinants also reportedly contributes to false positive results. Here, we describe the development of a third-generation immunoassay that allows sIgE to be determined at low concentration (down to 0.1 kU/L, without extrapolation) in a precise and accurate manner, with faster turnaround and no dedicated instrumentation. The assay is run on the same automated platform as most typical immunoassays. It is thus expected to accelerate the ongoing laboratory trend towards consolidation of multiple workstations. We will explain how the assay’s lower detection limit, faster turnaround, and automation on a continuous random access analyzer are achieved through combined use of several technologies, including liquid allergens, universal streptavidin capture, bar-coding, spin wash (to minimize NSB), and enzyme-enhanced chemiluminescent detection. Availability of the third-generation sIgE assay provides clinicians and laboratories with a better tool for allergy blood testing; it has additional potential as well. Lately, there has been interest in the detection of offending allergens in young children, which would make it possible to institute allergen avoidance and treatment as early as possible. The third-generation sIgE assay’s ability to measure at low concentration facilitates exploring the clinical significance of lower cutoffs and earlier trend detection.

[37] A systematic approach to reducing specimen misidentification errors in the clinical laboratory. Roger L. Bertholf and Olga Thompson, University of Florida College of Medicine, Jacksonville, FL.

The integrity of laboratory results, and the safety of patients to whom they apply, are seriously compromised when specimens submitted for laboratory analysis are incorrectly identified. The exact frequency of mislabeled specimens in the laboratory is unknown, but surveys of hospital laboratories in the USA and elsewhere suggest a median frequency of about 1 in 2000 processed specimens. Three factors that contribute to specimen mislabeling are inadequate identification of the patient when the specimen is collected, confusion resulting from delayed labeling, and the need to re-label the specimen in the laboratory. An emphasis on proper patient identification, labeling the specimen at the bedside, and elimination of the need to re-label specimens will improve efficiency in the laboratory and reduce opportunities for errors in specimen labeling. We describe the elements of Project SURE LAB (Stop Unnecessary Re-labeling Errors: Label At the Bedside), which was designed to improve performance in all 3 areas by a system-wide effort to promote rigorous specimen collection procedures, deployment of bar-coded label printers in all areas where patient specimens are collected, and re-engineering laboratory workflow and practice to eliminate, insofar as possible, re-labeling of specimens after receipt. The goal of Project SURE LAB is to improve the safety of Shands Jacksonville patients by minimizing the risk of erroneous laboratory results due to a mislabeled specimen.

[38] Evaluating the linearity of quantitative analytical methods. Joseph P. Laurino, The University of Tampa, Tampa, FL.

While many laboratorians believe that the linearity of a quantitative assay can be assessed visually, most agree that objective measurement criteria are required in most, if not all, situations. For 22 yr, the clinical laboratory community has sought to find an acceptable and practical method to measure straightness. In 1986, the NCCLS Subcommittee on Linearity published a proposed standard, EP6-P, in which a statistical test for nonlinearity was used to determine the significance of a nonlinear observation. This method employed a conventional F test to determine“significant non-linearity.” Due in large part to its reliance on assay precision, this proposed standard failed to adequately detect only those errors that could impact test interpretation. In April 2003, the NCCLS published EP6-A, an improved guideline for the determination of assay linearity. This procedure, by using a polynomial method to identify non-linear conditions, determines the magnitude of non-linearity at each standard concentration, controls for differences in assay precision, utilizes a testable statistical model, and is relatively easy to implement. While there are still differences between the College of American Pathologists’ linearity assessment protocol and the NCCLS EP6-A in the interpretation of the measures of non-linearity, both have adopted this polynomial method as a standard means to determine linearity. Specific examples utilizing this guideline will be provided.

[39]What’s wrong with these results? Effects of complementary and alternative medicines on clinical laboratory testing. Amitava Dasgupta, University of Texas–Houston Medical School, Houston, TX.

Contrary to the popular belief that anything natural is safe, use of complementary and alternative medicines can cause significant toxicity and even death. Death due to use of Kava-Kava, Chan Su, and ephedra-containing weight-loss products have been reported. Unexpected test results in healthy individuals can be correlated to the use of complementary and alternative medicine. Elevated serum liver enzymes along with serum bilirubin have been observed in individuals taking Kava-Kava, Chapparel, Germander, or Comfrey. Unexpected high digoxin levels may be due to interference of certain Chinese medicines such as Chan Su, DanShen, or Ginseng. St. John’s wort, a popular herbal antidepressant, is an inducer of cytochrome P 450 and can cause significant interactions with many Western drugs by reducing their serum levels due to increased clearance by liver. Treatment failure from protease inhibitors in patients with AIDS has been reported due to use of St. John’s wort. Moreover, reduced cyclosporine levels due to use of St. John’s wort led to transplant rejection and eventual death in 2 patients. Contamination by metals in many Chinese medicines is problematic because ingestion of these medicines may cause toxicity from lead, mercury, or arsenic.

[41] Outreach program improves revenue cycle. Joseph E. Skrisson and Frederick L. Kiechle, Department of Clinical Pathology and Beaumont Reference Laboratory, William Beaumont Hospital, Royal Oak, MI.

Since June 2002, Beaumont Reference Laboratory has been developing strategies to improve the revenue cycle including re-engineering the Patient Accounting Department. This effort has produced the following revenue cycle improvements by the end of 2003. In one yr, the accounts receivable days were reduced from 146 to 71. The aged trial balance (ATB) over 180 days was reduced from $4.8 million to $130,000 (3% of total ATB). Over $1.2 million in cash was generated by aggressively working ATB over 180 days. Over $400,000 in cash receipts were collected by involving an outside collection agency (25% collection rate). Over $1.5 million in 3rd party underpayments retrospectively and $300,000 in revenue enhancements prospectively were identified using payment validation reports. The percentage of first time clear claims was improved by creation of 3rd party rejection reports by payor class and by specific payor rejection codes. Eligibility rejections were decreased by 90% with aggressive front end eligibility checking. In conclusion, revenue enhancement in our outreach program has been achieved through systematic evaluation of the entire revenue cycle process.

[41] Web based pathology curriculum: new methods for medical school education. Juliana Szakacs, University of Utah School of Medicine, Salt Lake City, UT.

Medical students are eager to use new modalities for learning, having grown up with computers and virtual reality; however, the use of these new techniques must provide a basic fund of knowledge required to perform in the clinical practice of medicine. We have developed a comprehensive web-based teaching curriculum with components utilizing self-study and assessment tools, lectures, syllabi, and group-interactive case-based laboratories. CasePath was developed in 2002 for the purpose of integrating didactic information from lectures and self-study into a problem-based learning format to be used in small-group teaching. Facilitators assisted the students in working through complex medical issues and problems that required the students to become proficient in the use of computerized records and web data, and the interpretation, integration, and analysis of medical data. The cases are based on real patients; the students review laboratory, clinical, and treatment data during the first laboratory session. On the following week, the students are required to present the case in a concise format integrating all of the knowledge they have acquired about the disease, including searches into the current literature, to their peers. Over the course of the yr, the students are required to progress from a simple oral ward rounds format to a comprehensive grand rounds format with power point presentations, analysis of literature, and outcomes. The program has resulted in improvement of student knowledge base, problem-solving skills on the wards, the ability to extract and interpret pertinent data from the medical record and to present cases formally to their colleagues in the third-yr rotations. CasePath is now in its second version and available on the web at www.path.utah.edu/casepath. The cases have been approved by the hospital HIPPA and conform to strict patient confidentiality requirements.

[42] Consultations in pathology in laboratory medicine via the Internet. Robert L. Hunter Jr., University of Texas–Houston Medical School, Houston, TX.

A system will be demonstrated for the real-time consultation among pathologists via the Internet. The system consists of a microscope with a video camera transmitting images over the Internet and a computer with a web-browser accessing a database. In practice, a person requesting a consultation opens the database and enters the patient’s name and clinical problem, etc. A microscope slide is then viewed over the Internet by both parties simultaneously. The remote consultant is able to capture images and enter an interpretation. These are incorporated into a report that can be electronically signed and distributed over the Internet, by e-mail, or printed on paper. The essential elements of the system are an Axis 2120 network camera, FileMaker Pro Database with a web interface, and active server pages for gathering images from the camera and loading them in the database. Since the entire operation is conducted over the web, the consultant can be located anywhere there is an Internet connection.

[43] Digital imaging library for pathology and laboratory medicine. Luciano B. Lemos, University of Texas–Houston Medical School, Houston, TX.

Presently there are several libraries of images available on the Internet. Most lack a powerful and comprehensive search engine to retrieve images of specific topics. In other libraries, the quality of the digital images is not very good. Our objective was to build a large library of good quality digital images that has a search system to retrieve the images according to specific topographies and anatomical diagnoses. Some of the digital images were obtained through the scanning of 35 mm transparencies and some were obtained directly through digital cameras. The original files are in the TIFF format and range from 0.9 to 10 KB. These digital files are stored on compact disks. The images on the Internet are compressed JPEG versions of the original files. The database behind the search engine was built using Microsoft Access. It includes basic demographic data for the patients. Any data pertaining to patient identification was deleted. It is possible to search images by sex and age range. As for organs and topography, the database includes the general system (ie, heart and large vessels, liver and biliary duct; respiratory system, etc). Additionally, there are 4 levels for organs. This way the visitor to the web page can search for a system (ie, female genital tract), then for an organ (ie, uterus), and more specifically for part of that organ (ie, endometrium). The topography is then combined with the anatomic diagnoses in a similar way. There is a general choice for diseases (ie, parasitic and infectious diseases) and 4 subsequent levels of diagnoses (ie, bacterial diseases to tuberculosis). It is also possible to search images by etiologic agents. It is possible to retrieve all the images belonging to the same patient. An arbitrarily assigned patient ID number links together the images in the database. Another important feature of the database is the random retrieval of images. The images pop up randomly without the diagnosis, which can subsequently be disclosed. This feature can be used as a pop quiz for students and residents.

[44] Computer-assisted interpretations in hemostasis disorders. Nghia D. Nguyen, University of Texas–Houston Medical School, Houston, TX.

We describe the design and implementation of WEB COAG, an interactive program for computer-assisted interpretations of hemostasis disorders on the Internet. Three modules are developed in this program: (a) coagulation profile to display typical results of coagulation screening tests for each disorder; (b) differential diagnosis to generate a list of diagnoses that fit the test results in a given case; and (c) synopsis of coagulopathy and therapy to provide essential information on disorders and therapeutic options. Forty-one coagulation disorders are included in the knowledge base. A web-based course for teaching residents on the coagulation rotation in our pathology department has also been developed to be used in conjunction with WEB COAG. The materials in this course supplement other teaching activities in the rotation. Several modules are included in this course: (a) lessons to cover all aspects of coagulation (quizzes in the form of board-exam type questions and practice cases are used to assess the resident’s competency); (b) algorithms for interpretation of coagulation tests in the form of flowcharts to assist the resident in clinical interpretation; (c) WEB COAG, a decision-support module to introduce the resident to the concept of online consulting; and (d) a discussion forum to allow for communication and exchange of ideas among residents and faculty. Our experience with these programs in teaching pathology residents at our institution has been very encouraging. The programs can be found at: http://dpalm.uth.tmc.edu/faculty/bios/nguyen/nguyen.html. As the web provides residents with convenient tools for self-study and clinical decision-support, web-based education programs may eventually form the core materials for life-long learning by physicians, especially at the point-of-service. Our project aims to spearhead pathology teaching effort in that direction.

[45] On-line dissection manual for surgical pathology. Zhenhong Qu, University of Texas Medical School–Houston, Houston, TX.

The gross examination (encompassing description, dissection, and sampling) is a complex task and an essential component of surgical pathology, being the prerequisite to a correct pathology diagnosis. Due to the complexity of the task, standardized protocols to guide the gross examination often become a bulky manual that is difficult to use. This problem is compounded by the high specimen volume in current practice settings. As a result, the manual is often underutilized, leading to irreparable errors and potentially harmful repercussions. To combat this chronic problem that affects many pathology laboratories, we have developed a simple method to organize and access the complex information of a typical procedure manual. The method employs the Object-Linking-and-Embedding function of Microsoft Word to establish hyperlinks among different contents, and then uses the Touch Screen technology to facilitate navigation through the manual on a computer screen installed at the cutting bench, with no need for a physical keyboard or a mouse. The method allows incorporation of complex text and graphic information but takes <10 sec to reach any intended information in the manual by 3–4 touches on the screen with a cordless disposable stylus. A manual that features comprehensive information, flexible organization, easy access, quick navigation, and minimization of contamination is likely to be utilized more often than a bulky paper manual, resulting in fewer errors at the grossing bench.

[46] Live teleconference imaging of gross and microscopic pathology specimens. Ron Fattor and Biju Philip, Univ. of Texas–Houston Medical School, Houston, TX.

A teleconferencing system was designed specifically for pathology to enable use of live gross and microscopic images. The system has been in use for 2 yr between two large city hospitals. Each hospital has a similar set-up in a conference room, consisting of a microscope with a camera, a document camera, and a camera that looks over the room. The cameras are Axis network cameras that include built-in computers and web servers. A simple program was written to link them all together. In operation, people select 4 live moving images from a list on an web browser page. The 4 chosen are transmitted to the web browser simultaneously as 1 large and 3 small images. These can be viewed on the monitor or projected to a screen. Voice conversation is via a speakerphone greatly reducing problems with HIPPA. The ability to share gross and microscopic images in real time over the internet has proven highly successful for both working and teaching conferences. Currently, the residents in our program utilize this system for weekly clinical pathology conferences. Interesting and challenging cases can thus be shared between two teaching hospitals. The system has also been utilized on occasion by staff pathologists for intradepartmental consultation between hospitals.

[47] Seeing pathogens in three-dimensions–new opportunities offered by electron cryomicroscopy and tomography. Z. Hong Zhou, University of Texas Medical School–Houston, Houston, TX.

Electron microscopy has been widely used in basic cell and molecular biology research as well as clinical diagnostics. In this well-established procedure, thin sections of cells, tissues, or pathogens are fixed and stained to obtain two-dimensional projection images of the specimen, which allow investigators to suggest or infer the structural organization of the samples. Recent advances in electron microscopy have led to the much more powerful electron cryomicroscopy (cryoEM) and electron tomography (ET), which provide 3-dimensional (3D) reconstructions of native, unstained samples at molecular to near-atomic resolutions. In cryoEM, purified macromolecular complexes or viruses are flash-frozen to liquid nitrogen temperature so that they are embedded in a thin layer of vitreous ice and imaged in a transmission electron microscope at low dose condition. Hundreds to thousands of images of the complex, representing different views of the complex in solution, can then be combined to reconstruct the 3D structure of the complex. The 3D structures of complexes at different functional states serve as the structural basis for unraveling their assembly mechanisms and for understanding their biological functions. We have determined the structures of a number of complexes to sub-nanometer resolutions by cryoEM, revealing their folds of secondary structure elements. These structures are further used as constraints for sequence-based molecular modeling to build atomic models. In ET, individual cells or pathogens, often in their unpurified forms, are imaged multiple times by axial tilting to obtain multiple views of the same sample. A 3D reconstruction at ~5 nm resolution is then obtained by combining these views, using an algorithm similar to that of computerized tomography (CT). As will be illustrated using viruses, cryoEM andET offer exciting new opportunities for clinical research and diagnostics.

[48] Middleware: new computer applications in clinical chemistry. Charles A. Bradley, Texas Tech University Health Sciences Center, Lubbock, TX.

The demand for laboratory testing continues to escalate while the personnel and resources required to meet these demands decline. Although an efficient clinical laboratory service has a significant impact on patient care, hospitals continue to limit the technologist workforce due to financial constraints. Therefore, the clinical laboratory has been challenged to do more with less. Laboratories have met this challenge by utilizing automated instruments and laboratory computer systems. These techniques are at the point of diminishing returns as most instruments and computers have not significantly expanded our efficiency in recent years, which brings us to a new concept of computer application that will help to streamline some of the operational bottlenecks within our laboratories. Middleware is simply software that is located between laboratory instrumentation and the laboratory information systems (LIS), that is user programmable, and that enhances productivity of the entire testing and reporting process. It does not interfere with the LIS and does not require reprogramming of the LIS. Some advantages of middleware include consistent review of results, automatic verification of unremarkable results allowing staff to focus on unusual results, consistent response to problems, reduction of training time for new technologists, faster turnaround time for all results, assuring that results in the LIS are the same as reported by the instrument, and automatically commenting on interference with specific tests due to hemoglobin, icterus, or lipemia. Other advantages of middleware are a significant reduction of errors, labor savings, changes in the system that are easy to make, and minimal capital expense. Middleware offers to the clinical laboratorian more options to control laboratory operations without as much dependence on the hospital information technology department.

[49] Development of a modular relational database for pathology research. Myra Wilkerson, Adam Brown, Jeff Prichard, and Greg Strevig, Geisinger Medical Center, Danville, PA.

Geisinger Medical Laboratories is currently developing a web-based research project management software system. This will provide our department with the informatics infrastructural tools to:(a) register patients and specimens; (b) maintain and manage a catalogue of paraffin-embedded tissue, frozen tissue, and DNA specimens; (c) manage common data element sets (cde) for general and organ-specific clinical information; (d) manage cde for tissue microarray (TMA) construction and studies; (e) deidentify data, allowing multiple users access for collaboration; and (f) provide tools for data mining. The software is being developed using Macromedia’s Coldfusion and Flash technologies, including Actionscript. Data storage will be implemented using a Sybase relational database on a UNIX platform. Modules that have been developed include project management, clinical information, tissue microarray, and security. The project management module is a utility that allows an investigator to assign access to other users and track information related to a project, such as Institutional Review Board data, grant numbers, and sponsor information. The clinical information module allows entry and management of general and organ-specific cde including patient demographics, family and social history, tumor staging, pathology, comorbidity, therapeutic interventions, consent, disease progression, and outcomes. The TMA module facilitates finding suitable cases for a project, entry of data related to tissue blocks and cores, virtual construction of a recipient block, viewing digital images of TMA spots online, and entry of immunohisto-chemistry or in situ hybridization scoring data. The security module registers user, assigns passwords and levels of access, prevents bookmarking, and controls session timeout. Modules currently being developed include histology and specimen inventory. A cDNA module may be developed in the near future. This pathology research management system will allow our department to collect, manage, and integrate multiple expanding datasets in a standardized format for easier data analysis.

[50] Informatics: applications in pathology undergraduate medical education. Alex A. Pappas, Michele L. Whitworth, Robin M. Smith. University of Arkansas for Medical Sciences, Little Rock, AR.

Pathology is a visual art and science. With the development of new, easy to use, and easily integrable software, hardware, and high speed Internet connections, it is possible to develop highly complex yet easy to comprehend lectures or teaching modules for medical students. A motivated instructor can use readily available standard personal computer software for development of text documents or PowerPoint™ lectures. Microscopic, gross, radiographic, or patient images are readily available for educators using the Internet. Using imaging software, raw images can be composed or juxtaposed into seamless annotated composites that illustrate salient points from the macroscopic to the molecular level. Using property web-based electron reserve (Docutek ERes™) to manage copyright-protected materials; students can access lectures or teaching modules anytime, anywhere. A different, more comprehensive interactive web-based course management system (WebCT™) is used to integrate lecture notes and images, self-paced quizzes, tests, course information, assignments, and even bulletin boards. This novel integration of concepts and principles with physically observable findings has been well received by students who have rated the lecture series as the best in pathology in our institution, as documented by student surveys and performance on step 1 of the United States Medical Licensure Examination (USMLE™) this past year. Such systems allow the motivated instructor to update and integrate new information in a timely, efficient manner.