Raquel M. Walton

ASVCP reference interval guidelines: determination of de novo reference intervals in veterinary species and other related topics

Reference intervals (RI) are an integral component of laboratory diagnostic testing and clinical decision-making and represent estimated distributions of reference values (RV) from healthy populations of comparable individuals. Because decisions to pursue diagnoses or initiate treatment are often based on values falling outside RI, the collection and analysis of RV should be approached with diligence. This report is a condensation of the ASVCP 2011 consensus guidelines for determination of de novo RI in veterinary species, which mirror the 2008 Clinical Laboratory and Standards Institute (CLSI) recommendations, but with language and examples specific to veterinary species. Newer topics include robust methods for calculating RI from small sample sizes and procedures for outlier detection adapted to data quality. Because collecting sufficient reference samples is challenging, this document also provides recommendations for determining multicenter RI and for transference and validation of RI from other sources (eg, manufacturers). Advice for use and interpretation of subject-based RI is included, as these RI are an alternative to population-based RI when sample size or inter-individual variation is high. Finally, generation of decision limits, which distinguish between populations according to a predefined query (eg, diseased or non-diseased), is described. Adoption of these guidelines by the entire veterinary community will improve communication and dissemination of expected clinical laboratory values in a variety of animal species and will provide a template for publications on RI. This and other reports from the Quality Assurance and Laboratory Standards (QALS) committee are intended to promote quality laboratory practices in laboratories serving both clinical and research veterinarians.

In vivo and ex vivo MRI detection of localized and disseminated neural stem cell grafts in the mouse brain

The application of stem cells as delivery vehicles opens up the opportunity for targeting therapeutic proteins to the damaged or degenerating central nervous system. Neural stem cell (NSC) lines have been shown to engraft, differentiate and correct certain central nervous system diseases. The present study was performed to test the ability of magnetic resonance imaging (MRI) in detecting transplanted NSCs under conditions of limited migration in the normal adult mouse brain versus widespread migration when the cells are transplanted neonatally. The C17.2 NSC line was labeled in vitro with superparamagnetic iron oxide (SPIO) particles and the labeled cells were implanted intracranially. Serial in vivo gradient echo MR imaging was performed using a 4.7 T horizontal bore magnet. High resolution ex vivo images of the isolated brains were performed at 9.4 T, and the presence of iron was correlated with Prussian blue staining in histological sections. Adult animals injected with SPIO-labeled stem cells exhibited hypointense regions near the injection site that were observed up to 32 days after injection. In neonatally transplanted animals, MR signal intensity from transplanted NSCs was not apparent in in vivo imaging but ex vivo MR images revealed small hypointense regions throughout the brain including the olfactory bulbs, cortex and the cerebellum, reflecting the wide distribution of the engrafted cells. These regions were correlated with Prussian blue staining, which confirmed the presence of SPIO particles inside the engrafted cells. We have shown that MRI is capable of differentiating localized and widespread engraftment of C17.2 stem cells in the central nervous system.

Subject-based reference values: biological variation, individuality, and reference change values.

Subject-based reference values have been largely overlooked in veterinary medicine. These values represent longitudinal data rather than the cross-sectional data represented by standard population-based reference values. As such they provide information about biological and analytical variation. Inherent random variation of analytes around a homeostatic set point is referred to as biological variation; data on biological variation are underutilized in veterinary medicine and have multiple applications that include setting analytical goals, predicting the utility of population-based reference intervals (RIs), assessing the value of partitioning reference values, and evaluating the significance of changes in serial results. To generate these data, relatively few individuals are sampled for a short period of time. Given the difficulty of obtaining specimens from large number of healthy individuals to establish a cross-sectional RI for many veterinary species, especially exotic species, use of subject-based RIs is a practical alternative approach for the veterinary diagnostician. Furthermore, for the majority of biochemical analytes and even many hemostatic variables, population-based reference values are less sensitive than subject-based reference values for detecting pathologic changes in an individual. The focus of this review is the clinical usefulness of subject-based reference values and diagnostic implications for their use. Implementation of the concepts of biological variation, individuality, and reference change value (RCV) may allow large diagnostic laboratories to offer more sensitive reference values to assess health and detect disease.

Magnetic Resonance Imaging Detects Differences in Migration Between Primary and Immortalized Neural Stem Cells

RATIONALE AND OBJECTIVES The study was performed to evaluate the effect of magnetic resonance imaging (MRI) contrast agent (super paramagnetic iron oxide [SPIO]) on differentiation and migration of primary murine neural stem cells (NSCs) in comparison to a neural stem cell line (C17.2). Because detection of labeled cells depends on the concentration of SPIO particles per imaging voxel, the study was performed at various concentrations of SPIO particles to determine the concentration that could be used for in vivo detection of small clusters of grafted cells. MATERIALS AND METHODS Murine primary NSCs or C17.2 cells were labeled with different concentrations of SPIO particles (0, 25, 100, and 250 microg Fe/mL) and in vitro assays were performed to assess cell differentiation. In vivo MRI was performed 7 weeks after neonatal transplantation of labeled cells to evaluate the difference in migration capability of the two cell populations. RESULTS Both the primary NSCs and the C17.2 cells differentiated to similar number of neurons (Map2ab-positive cells). Similar patterns of engraftment of C17.2 cells were seen in transplanted mice regardless of the SPIO concentration used. In vivo MRI detection of grafted primary and C17.2 cells was only possible when cells were incubated with 100 microg/mL or higher concentration of SPIO. Extensive migration of C17.2 cells throughout the brain was observed, whereas the migration of the primary NSCs was more restricted. CONCLUSIONS Engraftment of primary NSCs can be detected noninvasively by in vivo MRI, and the presence of SPIO particles do not affect the viability, differentiation, or engraftment pattern of the donor cells.

Biodistribution and pharmacodynamics of recombinant human alpha-l-iduronidase (rhIDU) in mucopolysaccharidosis type I-affected cats following multiple intrathecal administrations

The storage disorder mucopolysaccharidosis type I (MPS I) is caused by a deficiency in lysosomal α-L-iduronidase activity. The inability to degrade glycosaminoglycans (GAG) results in lysosomal accumulation and widespread tissue lesions. Many symptoms of MPS I are amenable to treatment with recombinant human α-L-iduronidase (rhIDU), however, peripherally administered rhIDU does not cross the blood-brain barrier and has no beneficial effects in the central nervous system (CNS). A feline model of MPS I was used to evaluate the CNS effects of rhIDU following repeated intrathecal (IT) administration. Twelve animals were randomized into four groups based on the time of euthanasia and tissue evaluation following three repeat IT administrations of 0.1 mg/kg rhIDU or placebo on Study Days 1, 4 or 5, and 9. Two days after the final IT injection, the mean tissue α-L-iduronidase (IDU) activity in the brains of the two treated animals were approximately 3-times higher (50.1 and 54.9 U/mg protein) than the activity found in normal cat brains (mean of 18.3 U/mg), and remained higher than untreated MPSI brain at 1 month (2.4 and 4.1 U/mg protein) before returning to near-baseline levels after 2 months. This activity corresponded with decreased brain GAG concentrations after 2 days (1.4 and 2.0 μg/mg) and 1 month (0.9 and 1.1 μg/mg) which approached levels observed in normal animals (0.7 μg/mg). Attenuation of GAG, gangliosides GM2 and GM3, and cholesterol reaccumulation was identified at both two days and one month following final IT injection. No adverse effects attributable to IT rhIDU administration were observed. IT rhIDU may be an effective means for providing enzyme replacement therapy for the central manifestations of MPS I.

Suppurative, Nonseptic Polyarthropathy in Dogs

The goals of this study were to determine the historical, physical examination, and clinicopathologic findings in dogs with suppurative, nonseptic polyarthropathy and to identify concurrent disorders associated with this syndrome. Medical records of 52 dogs with cytologic evidence of suppurative inflammation in two or more joints were examined retrospectively. Age of dogs was 4.8 years (median, range: 0.5-12 years). There was no clear breed or sex predilection, but most were large-breed dogs (body weight > or = 20 kg [44.4 lbs] in 40/52). Body temperature was 103.0 degrees F (39.4 degrees C) (median, range: 100.0-105.9 degrees F), with 29 of 52 dogs having a body temperature > or = 103 degrees F (39.4 degrees C). Lameness was identified in 42 of 52 dogs. Erosive changes were found in only 1 of 37 dogs that had radiography performed. A clear underlying disease process was not identified in 34 of 52 dogs. Seven dogs had evidence of infectious or inflammatory processes at extra-articular sites; 4 dogs were diagnosed with systemic lupus erythematosus (SLE); 2 dogs had gastrointestinal disease; 2 dogs had been vaccinated within 1 month before onset of polyarthritis; 1 dog had cancer; 1 dog had polyarthritis and meningitis; and 1 dog had erosive polyarthritis. Of the 44 dogs tested, 25 had antibodies to Borrelia burgdorferi, detected by an ELISA assay, which was significantly greater than the general hospital population (P = .007). Antibodies against Rickettsia rickettsiae and Ehrlichia canis were not definitively identified in the sera of any dog tested in this study (45 and 44 dogs, respectively). We conclude that an underlying disease process is not identified in most cases of suppurative polyarthropathy in dogs and that intestinal disease, neoplasia, and SLE are uncommon causes of polyarthritis. While seropositivity against the causative agent of Lyme disease was common and possibly a cause of polyarthritis in some dogs of our study, evidence of other vector-borne infection was not identified.

In vitro growth and differentiation of canine olfactory bulb-derived neural progenitor cells under variable culture conditions.

The dog serves as a large animal model for multiple neurologic diseases that may potentially benefit from neural progenitor cell (NPC) transplantation. In the adult brain, multipotent NPCs reside in the subventricular zone and its rostral and caudal extensions into the olfactory bulb and hippocampus. The olfactory bulb represents a surgically accessible site for obtaining cells for autologous NPC transplantation. To model conditions that would occur for ex vivo gene therapy in the postnatal brain, NPCs were isolated from the canine olfactory bulb, expanded ex vivo under different culture conditions, and compared quantitatively for growth and immunophenotype. Under standard growth conditions, canine olfactory bulb-derived NPCs (OB-cNPCs) could be expanded nearly 500-fold in the time evaluated. Canine OB-cNPCs grown on poly-d-lysine (PDL) or on PDL-fibronectin had similar growth rates, whereas supplementation with leukemia inhibitory factor (LIF) resulted in significantly slower growth. However, when OB-cNPC cultures were grown on PDL-fibronectin or PDL supplemented with LIF, a greater proportion of cells with neuronal markers were generated upon differentiation.

Magnetic resonance imaging as a tool for monitoring stem cell migration.

Noninvasive monitoring of stem cells is an important step in developing stem-cell-based therapies. Among several imaging techniques available, magnetic resonance imaging (MRI) provides an effective way to detect implanted stem cells in live animals. In this mini-review, we discuss the available MRI contrast agents and different cell-labeling strategies used for detection of stem cell migration in the brain. The potential effects of MRI contrast agents on stem cell viability and differentiation are also discussed.

Hepatic and metabolic changes in surgical colic patients: a pilot study

Objective – To determine: (1) changes in blood ammonia, bile acid (BA), bilirubin, triglyceride, and glucose concentrations and liver enzyme activities in perioperative colic patients and (2) the association between these laboratory findings and short-term survival. Design – Prospective observational clinical study. Animals – Thirty-two adult horses undergoing exploratory celiotomy for colic. Interventions – None. Measurements and Main Results – Blood samples were collected preoperatively and at 24–36 and 72–84 hours postoperatively and analyzed for blood ammonia, BA, bilirubin, triglyceride, and glucose concentrations and sorbitol dehydrogenase (SDH) and gamma glutamyl transferase (GGT) activities. Short-term survival was defined as survival to hospital discharge. Data were analyzed using a Fishers exact test and analysis of variance. Mildly increased blood ammonia concentrations were present in 2 horses at admission. Postoperative blood ammonia concentrations were within reference intervals in all horses. There were increases in liver enzyme activities as well as in BA, triglyceride, and total bilirubin concentrations. Horses with markedly increased admission BA concentrations and SDH activities did not survive. BA concentrations and SDH activities decreased postoperatively. There was no association between GGT activity and survival; GGT activity remained increased postoperatively. Blood triglyceride concentration was increased in almost all horses postoperatively; horses that did not survive had higher triglyceride concentrations at 24–36 hours postoperatively than horses that survived. Conclusion – Alterations in metabolism and hepatobiliary function are common in colic patients. The results of this study provide further prognostic indices for colic patients and highlight areas for improvement in patient management.OBJECTIVE To determine: (1) changes in blood ammonia, bile acid (BA), bilirubin, triglyceride, and glucose concentrations and liver enzyme activities in perioperative colic patients and (2) the association between these laboratory findings and short-term survival. DESIGN Prospective observational clinical study. ANIMALS Thirty-two adult horses undergoing exploratory celiotomy for colic. INTERVENTIONS None. MEASUREMENTS AND MAIN RESULTS Blood samples were collected preoperatively and at 24-36 and 72-84 hours postoperatively and analyzed for blood ammonia, BA, bilirubin, triglyceride, and glucose concentrations and sorbitol dehydrogenase (SDH) and gamma glutamyl transferase (GGT) activities. Short-term survival was defined as survival to hospital discharge. Data were analyzed using a Fishers exact test and analysis of variance. Mildly increased blood ammonia concentrations were present in 2 horses at admission. Postoperative blood ammonia concentrations were within reference intervals in all horses. There were increases in liver enzyme activities as well as in BA, triglyceride, and total bilirubin concentrations. Horses with markedly increased admission BA concentrations and SDH activities did not survive. BA concentrations and SDH activities decreased postoperatively. There was no association between GGT activity and survival; GGT activity remained increased postoperatively. Blood triglyceride concentration was increased in almost all horses postoperatively; horses that did not survive had higher triglyceride concentrations at 24-36 hours postoperatively than horses that survived. CONCLUSION Alterations in metabolism and hepatobiliary function are common in colic patients. The results of this study provide further prognostic indices for colic patients and highlight areas for improvement in patient management.

Abnormalities in neural progenitor cells in a dog model of lysosomal storage disease.

Abstract Lysosomal storage disorders constitute a large group of genetic diseases, many of which are characterized by mental retardation and other neurologic symptoms. The mechanisms of neural dysfunction remain poorly understood. Because neural progenitor cells (NPCs) are fundamentally important to normal brain development and function, we investigated NPC properties in a canine model of mucopolysaccharidosis VII (MPS VII). MPS VII is a lysosomal storage disorder characterized by defects in the catabolism of glycosaminoglycans. NPCs were isolated from the olfactory bulb, cerebellum, and striatal subventricular zone of normal and MPS VII (&bgr;-glucuronidase-deficient) postnatal dog brains. Canine NPCs (cNPCs) from normal and MPS VII brains had similar growth curves, but cerebellar-derived cNPCs grew significantly slower than those derived from other regions. In differentiation assays, MPS VII cNPCs from the striatal subventricular zone and cerebellum generated fewer mature neuronal and/or glial cells than normal, and MPS VII olfactory bulb-derived cNPCs retained significantly more phenotypically immature cells. These differences were only present at the earliest time point after isolation; at later passages, there were no differences attributable to genotype. The data suggest that MPS VII cNPCs respond differently to developmental cues in vivo, probably because of the diseased neural microenvironment rather than intrinsic cellular deficits.