Overutilization of Test for Hemoglobinopathy Evaluation: Experience from a Tertiary Care Academic Medical Center.

BACKGROUND: Hemoglobin electrophoresis is a common clinical laboratory test for the identification of hemoglobinopathies in clinical practice. We investigated the utilization of this test in our academic teaching hospital and hypothesized that hemoglobin electrophoresis evaluation is overutilized at this institution. METHODS: 128 consecutive cases were analyzed and their medical records were studied to determine the clinical indication for the hemoglobin electrophoresis. RESULTS: Of the 128 cases studied, only 44% of cases had a justifiable reason for obtaining the hemoglobin electrophoresis, whereas the remaining 56% of cases did not have an acceptable indication for obtaining the test. CONCLUSIONS: We conclude that hemoglobin electrophoresis is overutilized in our academic medical center and we recommend consultation with clinical pathologists prior to ordering such tests.

Biotin at High Concentration Interferes with the LOCI Digoxin Assay but the PETINIA Phenytoin Assay Is Not Affected.

Many automated immunoassays incorporate biotinylated antibodies and streptavidin-coated magnetic beads in the assay design. Biotin at elevated concentrations may interfere with these immunoassays. We evaluated potential interference of biotin on serum digoxin (LOCI assay utilizing biotinylated antibody) and phenytoin (PETINIA assay; no biotinylated antibody) measurements using the Vista 1500 analyzer. Aliquots of drug-free serum pool were supplemented with various biotin concentrations (range: 1 ng/mL to 250 ng/mL) followed by measuring apparent digoxin and phenytoin levels using appropriate immunoassays. In the second set of experiments, one serum pool was prepared from patients taking digoxin and another from patients taking phenytoin. Then aliquots of these serum pools were further supplemented with biotin followed by measuring digoxin or phenytoin concentrations. We observed apparent digoxin levels at 50 ng/mL biotin concentration or higher and also significant interference of biotin in serum digoxin measurement at a biotin concentration of 250 ng/mL. In contrast, we observed no interference of biotin in serum phenytoin measurement. We conclude that biotin interferes with the LOCI digoxin assay at a high concentration only.

Multimodal Genetic Approach for Molecular Imaging of Vasculature in a Mouse Model of Melanoma.

PURPOSE: In this study, we evaluated a genetic approach for in vivo multimodal molecular imaging of vasculature in a mouse model of melanoma. PROCEDURES: We used a novel transgenic mouse, Ts-Biotag, that genetically biotinylates vascular endothelial cells. After inoculating these mice with B16 melanoma cells, we selectively targeted endothelial cells with (strept)avidinated contrast agents to achieve multimodal contrast enhancement of Tie2-expressing blood vessels during tumor progression. RESULTS: This genetic targeting system provided selective labeling of tumor vasculature and showed in vivo binding of avidinated probes with high specificity and sensitivity using microscopy, near infrared, ultrasound, and magnetic resonance imaging. We further demonstrated the feasibility of conducting longitudinal three-dimensional (3D) targeted imaging studies to dynamically assess changes in vascular Tie2 from early to advanced tumor stages. CONCLUSIONS: Our results validated the Ts-Biotag mouse as a multimodal targeted imaging system with the potential to provide spatio-temporal information about dynamic changes in vasculature during tumor progression.

Engineering an effective Mn-binding MRI reporter protein by subcellular targeting.

PURPOSE: Manganese (Mn) is an effective contrast agent and biologically active metal, which has been widely used for Mn-enhanced MRI (MEMRI). The purpose of this study was to develop and test a Mn binding protein for use as a genetic reporter for MEMRI. METHODS: The bacterial Mn-binding protein, MntR was identified as a candidate reporter protein. MntR was engineered for expression in mammalian cells, and targeted to different subcellular organelles, including the Golgi Apparatus where cellular Mn is enriched. Transfected HEK293 cells and B16 melanoma cells were tested in vitro and in vivo, using immunocytochemistry, MR imaging and relaxometry. RESULTS: Subcellular targeting of MntR to the cytosol, endoplasmic reticulum and Golgi apparatus was verified with immunocytochemistry. After targeting to the Golgi, MntR expression produced robust R1 changes and T1 contrast in cells, in vitro and in vivo. Co-expression with the divalent metal transporter DMT1, a previously described Mn-based reporter, further enhanced contrast in B16 cells in culture, but in the in vivo B16 tumor model tested was not significantly better than MntR alone. CONCLUSION: This second-generation reporter system both expands the capabilities of genetically encoded reporters for imaging with MEMRI and provides important insights into the mechanisms of Mn biology which create endogenous MEMRI contrast.

Divalent metal transporter, DMT1: a novel MRI reporter protein.

Manganese (Mn)-enhanced MRI (MEMRI) has found a growing number of applications in anatomical and functional imaging in small animals, based on the cellular uptake of Mn ions in the brain, heart, and other organs. Previous studies have relied on endogenous mechanisms of paramagnetic Mn ion uptake and enhancement. To genetically control MEMRI signals, we reverse engineered a major component of the molecular machinery involved in Mn uptake, the divalent metal transporter, DMT1. DMT1 provides positive cellular enhancement in a manner that is highly sensitive and dynamic, allowing greater spatial and temporal resolution for MRI compared to previously proposed MRI reporters such as ferritin. We characterized the MEMRI signal enhancement properties of DMT1-expressing cells, both in vitro and in vivo in mouse models of cancer and brain development. Our results show that DMT1 provides an effective genetic MRI reporter for a wide range of biological and preclinical imaging applications.

Novel genetic approach for in vivo vascular imaging in mice.

RATIONALE: The formation and maintenance of a functional vasculature is essential for normal embryonic development, and genetic changes that affect the vasculature underlie pathogenesis in many human diseases. In vivo imaging in mouse models is required to understand the full complexity of mammalian vascular formation, which is a dynamic and 3-dimensional process. Optical microscopy of genetically expressed fluorescent reporter proteins offers high resolution but limited depth of penetration in vivo. Conversely, there are a plethora of molecular probes for alternative in vivo vascular imaging modalities, but few options for genetic control of contrast enhancement. OBJECTIVE: To develop a reporter system for multimodal imaging of genetic processes involved in mammalian vascular biology. METHODS AND RESULTS: To approach this problem, we developed an optimal tagging system based on Biotag-BirA technology. In the resulting Biotag reporter system, coexpression of 2 interacting proteins results in biotin labeling of cell membranes, thus enabling multimodal imaging with "avidinated" probes. To assess this approach for in vivo imaging, we generated transgenic mice that expressed the Biotag-BirA transgene from a minimal Tie2 promoter. A variety of imaging methods were used to show the utility of this approach for quantitative analysis in embryonic and adult models of vascular development, using intravascular injection of avidinated probes for near infrared, ultrasound, and magnetic resonance imaging. CONCLUSIONS: The present results demonstrate the versatility of the Biotag system for studies of vascular biology in genetically engineered mice, providing a robust approach for multimodal in vivo imaging of genetic processes in the vasculature.

In vivo MRI of neural cell migration dynamics in the mouse brain.

Multipotent neuroblasts (NBs) are produced throughout life by neural stem cells in the forebrain subventricular zone (SVZ), and are able to travel long distances to the olfactory bulb. On arrival in the bulb, migrating NBs normally replace olfactory neurons, raising interest in their potential for novel cell replacement therapies in various disease conditions. An understanding of the migratory capabilities of NBs is therefore important, but as yet quantitative in vivo measurement of cell migration has not been possible. In this study, targeted intracerebral injections of iron-oxide particles to the mouse SVZ were used to label resident NBs in situ, and their migration was tracked noninvasively over time with magnetic resonance imaging (MRI). Quantitative intensity metrics were employed to identify labeled cells and to show that cells are able to travel at speeds up to 100 microm/h en route to the olfactory bulb, but that distribution through the olfactory bulb occurs at a much slower rate. In addition, comparison of histological and MRI measures of iron-oxide particle distribution were in excellent agreement. Immunohistochemistry analysis 1-3 weeks after labeling revealed that the majority of labeled cells in the olfactory bulb were immature neurons, although iron-oxide particles were also found in astrocytes and microglia. This work indicates that dynamic measurements of endogenous cell migration can be made with MRI and represents the first in vivo measurement of NB migration rates. The use of MRI in future studies tracking endogenous NB cells will permit a more complete evaluation of their role during homeostasis at various developmental stages and during disease progression.