Michael Laposata

Operational Implementation of Prospective Genotyping for Personalized Medicine: The Design of the Vanderbilt PREDICT Project

The promise of “personalized medicine” guided by an understanding of each individuals genome has been fostered by increasingly powerful and economical methods to acquire clinically relevant information. We describe the operational implementation of prospective genotyping linked to an advanced clinical decision‐support system to guide individualized health care in a large academic health center. This approach to personalized medicine entails engagement between patient and health‐care provider, identification of relevant genetic variations for implementation, assay reliability, point‐of‐care decision support, and necessary institutional investments. In one year, approximately 3,000 patients, most of whom were scheduled for cardiac catheterization, were genotyped on a multiplexed platform that included genotyping for CYP2C19 variants that modulate response to the widely used antiplatelet drug clopidogrel. These data are deposited into the electronic medical record (EMR), and point‐of‐care decision support is deployed when clopidogrel is prescribed for those with variant genotypes. The establishment of programs such as this is a first step toward implementing and evaluating strategies for personalized medicine.

Pancreatic injury in rats induced by fatty acid ethyl ester, a nonoxidative metabolite of alcohol

BACKGROUND & AIMS The mechanism by which alcohol injures the pancreas remains unknown. Alcohol-intoxicated humans have high levels of fatty acid ethyl esters (FAEEs), nonoxidative products of ethanol metabolism, in blood, pancreas, and liver. The aims of this study were to determine whether FAEEs are toxic to the pancreas in vivo and, if so, to assess whether this injury is specific to the pancreas and to compare it to the injury observed in acute pancreatitis. METHODS FAEEs were infused into Sprague-Dawley rats. Levels of FAEEs in plasma and pancreas were measured, and pancreatic injury was assessed during a 48-hour period for edema formation and ectopic trypsinogen activation and by light and electron microscopy. RESULTS FAEEs induced highly significant increases in pancreatic edema, pancreatic trypsinogen activation, and vacuolization of acinar cells. These findings were specific to the pancreas and were not found in liver, lung, myocardium, skeletal muscle, or subcutaneous fat. CONCLUSIONS FAEEs at concentrations found in human plasma produce a pancreatitis-like injury in rats, providing direct evidence that FAEEs can produce organ-specific toxicity. Thus, FAEEs may contribute to acute alcohol-induced damage to the pancreas.

Omega-3 fatty acid monotherapy for pediatric bipolar disorder: A prospective open-label trial

BACKGROUND To test the effectiveness and safety of omega-3 fatty acids (Omegabrite(R) brand) in the treatment of pediatric bipolar disorder (BPD). METHOD Subjects (N=20) were outpatients of both sexes, 6 to 17 years of age, with a DSM-IV diagnosis of BPD and Young Mania Rating Scale (YMRS) score of >15 treated over an 8-week period in open-label trial with omega-3 fatty acids 1290 mg-4300 mg combined EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid). RESULTS Subjects experienced a statistically significant but modest 8.9+/-2.9 point reduction in the YMRS scores (baseline YMRS=28.9+/-10.1; endpoint YMRS=19.1+/-2.6, p<0.001). Adverse events were few and mild. Red blood cell membrane levels of EPA and DHA increased in treated subjects. CONCLUSIONS As only 35% of these subjects had a response by the usual accepted criteria of >50% decrease on the YMRS, omega-3 fatty acids treatment was associated with a very modest improvement in manic symptoms in children with BPD.

Adenoviral gene transfer of Caenorhabditis elegans n−3 fatty acid desaturase optimizes fatty acid composition in mammalian cells

Omega−3 polyunsaturated fatty acids (PUFAs) are essential components required for normal cellular function and have been shown to exert many preventive and therapeutic actions. The amount of n−3 PUFAs is insufficient in most Western people, whereas the level of n−6 PUFAs is relatively too high, with an n−6/n−3 ratio of >18. These two classes of PUFAs are metabolically and functionally distinct and often have important opposing physiological functions; their balance is important for homeostasis and normal development. Elevating tissue concentrations of n−3 PUFAs in mammals relies on chronic dietary intake of fat rich in n−3 PUFAs, because mammalian cells lack enzymatic activities necessary either to synthesize the precursor of n−3 PUFAs or to convert n−6 to n−3 PUFAs. Here we report that adenovirus-mediated introduction of the Caenorhabditis elegans fat-1 gene encoding an n−3 fatty acid desaturase into mammalian cells can quickly and effectively elevate the cellular n−3 PUFA contents and dramatically balance the ratio of n−6/n−3 PUFAs. Heterologous expression of the fat-1 gene in rat cardiac myocytes rendered cells capable of converting various n−6 PUFAs to the corresponding n−3 PUFAs, and changed the n−6/n−3 ratio from about 15:1 to 1:1. In addition, an eicosanoid derived from n−6 PUFA (i.e., arachidonic acid) was reduced significantly in the transgenic cells. This study demonstrates an effective approach to modifying fatty acid composition of mammalian cells and also provides a basis for potential applications of this gene transfer in experimental and clinical settings.

Fatty acid ethyl esters decrease human hepatoblastoma cell proliferation and protein synthesis.

BACKGROUND/AIMS Fatty acid ethyl esters (FAEEs) are nonoxidative products of ethanol metabolism. They have been implicated as mediators of ethanol-induced organ damage because FAEE and FAEE synthase have been found specifically in the organs damaged by ethanol abuse. This study showed toxicity specifically related to FAEE or their metabolites for intact human hepatoblastoma-derived cells (HepG2). METHODS The lipid core of human low-density lipoprotein (LDL) was extracted and the LDL particle reconstituted with either ethyl oleate or ethyl arachidonate. Cultured HepG2 cells were incubated with LDL containing FAEE. Cell proliferation was measured by [methyl-3H]thymidine incorporation. Protein synthesis was determined using L-[35S]methionine. RESULTS Incubation of cells with 600 mumol/L ethyl oleate or 800 mumol/L ethyl arachidonate decreased [methyl-3H]thymidine incorporation into HepG2 cells by 31% and 37%, respectively. LDL reconstituted with 400 mumol/L ethyl oleate decreased protein synthesis in intact HepG2 cells by 41%. Electron microscopy revealed significant changes in cell morphology, particularly involving the cell nucleus. FAEE delivered in reconstituted LDL were rapidly hydrolyzed and the fatty acids re-esterified into phospholipids, triglycerides, and cholesterol esters, with preference for triglycerides. CONCLUSIONS These findings provide evidence that FAEE are toxic for intact human hepatoblastoma cells and that they or their metabolites may be an important causative agent in ethanol-induced liver damage.

“Pre-pre” and “post-post” analytical error: high-incidence patient safety hazards involving the clinical laboratory

Abstract Data from recent studies suggest that the highest incidence of laboratory-related errors occurs in the pre-analytical phase of laboratory testing. However, few studies have examined the frequency of errors in laboratory test selection and interpretation. A survey of physicians who use our clinical laboratory demonstrated that the largest number of test ordering errors appear to involve physicians simply ordering the wrong test. Diagnostic algorithms providing guidance for test selection in specific disorders are also used as the basis for the establishment of reflex protocols in the clinical laboratory. The provision of an expert-driven interpretation by laboratory professionals resulted in improvements both in the time to and the accuracy of diagnosis. A survey of our physician staff has shown that in the absence of such an interpretation, for patients being assessed for a coagulation disorder, approximately 75% of the cases would have involved some level of test result misinterpretation. Clin Chem Lab Med 2007;45:712–9.

Fatty acid ethyl esters: Toxic non-oxidative metabolites of ethanol and markers of ethanol intake

This report describes the biochemistry of fatty acid ethyl esters (FAEE), non-oxidative metabolites of ethanol, and their clinical significance. We review information regarding the enzymes responsible for FAEE synthesis and degradation, and the mechanisms involved with the intracellular and extracellular transport of FAEE and FAEE-mediated cytotoxicity. Also a summary of reports on the emerging clinical significance and diagnostic utility of FAEE as short and long-term markers of alcohol consumption, and the methodological aspects of FAEE assessment is included.

Laboratory Diagnosis of Dysfibrinogenemia

Dysfibrinogenemia is a coagulation disorder caused by a variety of structural abnormalities in the fibrinogen molecule that result in abnormal fibrinogen function. It can be inherited or acquired. The inherited form is associated with increased risk of bleeding, thrombosis, or both in the same patient or family. Traditionally, dysfibrinogenemia is diagnosed by abnormal tests of fibrin clot formation; the thrombin time and reptilase time are the screening tests, and the fibrinogen clotting activity-antigen ratio is the confirmatory test. The inherited form is diagnosed by demonstrating similar laboratory test abnormalities in family members, and if necessary by analysis of the fibrinogen protein or fibrinogen genes in the patient. The acquired form is diagnosed by demonstrating abnormal liver function tests and by ruling out dysfibrinogenemia in family members. This article reviews the laboratory testing of dysfibrinogenemia and presents an algorithm for sequential test selection that can be used for diagnosis.

Physician Survey of a Laboratory Medicine Interpretive Service and Evaluation of the Influence of Interpretations on Laboratory Test Ordering

CONTEXT Complex coagulation test panels ordered by clinicians are typically reported to clinicians without a patient-specific interpretive paragraph. OBJECTIVES To survey clinicians regarding pathologist-generated interpretations of complex laboratory testing panels and to assess the ability of the interpretations to educate test orderers. DESIGN Surveys were conducted of physicians ordering complex coagulation laboratory testing that included narrative interpretation. Evaluation of order requisitions was performed to assess the interpretations influence on ordering practices. SETTING Physicians ordering coagulation testing at a large academic medical center hospital in Boston, Mass, and physicians from outside hospitals using the academic medical center as a reference laboratory for coagulation testing. OUTCOME MEASURES Physician surveys and evaluation of laboratory requisition slips. RESULTS In nearly 80% of responses, the ordering clinicians perceived that the interpretive comments saved them time and improved the diagnostic process. Moreover, the interpretations were perceived by ordering clinicians to help prevent a misdiagnosis or otherwise impact the differential diagnosis in approximately 70% of responses. In addition, interpretations appeared to be able to train the ordering clinicians as to the standard ordering practices. CONCLUSIONS The results demonstrate physician satisfaction with an innovative information delivery approach that provides laboratory diagnostic interpretation and test-ordering education to clinicians in the context of their daily workflow.

The Brain-to-Brain Loop Concept for Laboratory Testing 40 Years After Its Introduction

Forty years ago, Lundberg introduced the concept of the brain-to-brain loop for laboratory testing. In this concept, in the brain of the physician caring for the patient, the first step involves the selection of laboratory tests and the final step is the transmission of the test result to the ordering physician. There are many intermediary steps, some of which are preanalytic, ie, before performance of the test; some are analytic and relate to the actual performance of the test; and others are postanalytic and involve transmission of test results into the medical record. The introduction of this concept led to a system to identify and classify errors associated with laboratory test performance. Errors have since been considered as preanalytic, analytic, and postanalytic. During the past 4 decades, changes in medical practice have significantly altered the brain-to-brain loop for laboratory testing. This review describes the changes and their implications for analysis of errors associated with laboratory testing.