A review of guidelines for evaluating a minor modification to a validated assay.

Any modification to a validated assay must be evaluated in terms of the impact on the assay's performance characteristics and whether the assay remains fit for the intended purpose. The comparison is referred to as a 'method comparison', 'method comparability', 'method change', or 'comparative validation'. This review presents recommendations and examples of studies found in the current literature as a means of assessing minor modifications. In addition, the authors discuss common statistical approaches used for these comparisons.

Toute modification apportée à un essai validé doit être évaluée afin de mesurer l'impact de cette modification sur les paramètres de performances de l'essai et déterminer si l'aptitude à l'emploi qui lui a été assigné demeure valable suite à la modification en question. Cette comparaison est désignée sous les termes de « comparaison de méthodes d'essai ¼, « comparabilité de méthodes ¼, « changement de méthode d'essai ¼ ou « validation comparative ¼. Les auteurs font part de leurs recommandations et donnent des exemples d'études émanant de la littérature récente concernant l'évaluation de modifications mineures. En outre, ils examinent les approches statistiques couramment utilisées pour ces comparaisons.

Toda modificación que se introduzca en un ensayo validado debe ser objeto de evaluación para determinar la influencia del cambio en las características de funcionamiento del ensayo y saber si este sigue estando adaptado a su función. Para referirse a la comparación, los autores emplean expresiones como 'comparación de métodos', 'comparabilidad de métodos', 'cambio de método' o 'validación comparativa'. Los autores presentan aquí recomendaciones y ejemplos de estudios extraídos de la bibliografía actual como medio de evaluar modificaciones de importancia menor. Además, los autores examinan las lógicas estadísticas comunes utilizadas para estas comparaciones.

Long-term functional outcomes and quality of life in adult survivors of childhood extremity sarcomas: a report from the St. Jude Lifetime Cohort Study.

PURPOSE: This study compared measured physical performance, health-related quality of life (HRQOL), and social role attainment between extremity sarcoma survivors and controls, and evaluated associations between disease and treatment exposures, health conditions, and performance measures. METHODS: Survivors of extremity sarcoma from the St. Jude Lifetime cohort and controls frequency matched by age-, sex-, and race completed physical performance testing and questionnaires. Survivors with Z-scores on outcome measures ≤ -2.0 SD (compared to controls) were categorized with severe impairment/limitation. RESULTS: Among 206 survivors (52.4 % male median age 36 years (range 19-65)), 37 % had low relative lean mass, 9.7 % had an ejection fraction <50 %, 51.5 % had diffusion capacity for carbon monoxide <75 %, 27.7 % had sensory and 25.2 % motor neuropathy, and 78.2 % had musculoskeletal complications. Severe impairments/limitations were present among ≥25 % of survivors on fitness, balance, and physical HRQOL measures, and among ≥15 % on strength and activity of daily living measures. Lower extremity tumor location (OR 8.23, 95 % CI 2.54-26.67, P value 0.0004) and amputation (OR 8.07, 95 % CI 3.06-21.27, P value <0.0001) were associated with poor fitness. Poor fitness was associated with increased odds of scoring <40 on the SF-36 physical component summary (OR 4.83, 95 % CI 1.95-11.99, P value 0.001) and role-physical subscale (OR 3.34, 95 % CI 1.33-8.43, P value 0.01). Survivors and controls had similar rates of marriage, independent living, employment, and college attendance. CONCLUSIONS: Extremity sarcoma survivors experience high rates of physical impairment and report lower than expected physical HRQOL. However, they are as likely as peers to be married, live independently, be employed, and attend college. IMPLICATIONS FOR CANCER SURVIVORS: Follow-up for extremity sarcoma survivors should include assessment of need for further orthopedic care and rehabilitation to address cardiopulmonary and musculoskeletal health.

Vitamin D status among long-term survivors of hematopoietic cell transplantation.

Little is known about serum vitamin D levels following hematopoietic cell transplantation (HCT). Patients are instructed to avoid sun exposure because of an increased risk of skin cancers. Altered gastrointestinal absorptive capacity as a result of GVHD, bile acid or pancreatic enzyme insufficiency or bacterial overgrowth may lead to difficulty in absorbing the fat-soluble vitamin D. This study was undertaken to determine the prevalence of serum 25-hydroxyvitamin D (25(OH)D) deficiency, and factors associated with 25(OH)D deficiency, among children and adults who were at least 1 year following HCT. A total of 95 participants (54 males and 41 females) completed a questionnaire on usual diet and lifestyle, and provided a blood sample for 25(OH)D determinations between November 2008 and July 2009. The majority of participants had serum 25(OH)D levels ≥75 nmol/L (n=62, 65%), 23 had insufficient levels (50-75 nmol/L) and 10 participants were deficient (<50 nmol/L). The majority of participants reported regular use of vitamin D supplements (n=58, 61%). Prednisone use was significantly inversely associated with serum 25(OH)D concentrations. Total vitamin D intake was the strongest single predictor of 25(OH)D concentrations. These findings suggest that 400-600 IU vitamin D per day appears to be required to achieve optimal serum 25(OH)D concentrations following HCT.

Screening, prevention and management of osteoporosis and bone loss in adult and pediatric hematopoietic cell transplant recipients.

Long-term survivors of hematopoietic cell transplantation (HCT) are at risk for loss of bone mineral density (BMD) and subsequent osteoporosis. There is a lack of clear guidelines for the screening, prevention and treatment of bone loss after HCT. We reviewed the prevailing literature and provide guidelines developed by our center for the screening and management of this complication. Bone loss occurs predominantly within the first 6-12 months after autologous and allogeneic HCT. Recovery first occurs in the lumbar spine and is followed by a slower recovery of BMD in the femoral neck. BMD may not return to baseline levels in patients with continuing exposure to corticosteroids and calcineurin inhibitors. All HCT recipients should be advised general interventions to reduce fracture risk including adequate intake of calcium and vitamin D. We recommend screening all adult allogeneic and autologous HCT recipients with dual-energy X-ray absorptiometry 1 year after transplantation. Patients at high risk for bone loss (for example, patients receiving ≥ 5 mg of prednisone equivalent daily for > 3 months) can be screened earlier (for example, 3-6 months after HCT). Where indicated, bisphosphonates or other anti-resorptive agents (for example, calcitonin) can be used for prevention or treatment of osteoporosis in adult HCT recipients. Pediatric HCT recipients should be referred to a pediatric endocrinologist for evaluation and treatment of bone loss. There remain several areas of uncertainty that need further research in adult and pediatric HCT recipients, such as the optimal timing and frequency of screening for loss of bone mineral density, relationship of bone loss with risk of fractures, selection of appropriate patients for pharmacologic therapy, and optimal dosing schedule and duration of therapy with anti-resorptive agents.

Pathology and viral antigen distribution of lethal pneumonia in domestic cats due to pandemic (H1N1) 2009 influenza A virus.

A novel swine-origin H1N1 influenza A virus has been identified as the cause of the 2009 influenza pandemic in humans. Since then, infections with the pandemic (H1N1) 2009 influenza virus have been documented in a number of animal species. The first known cases of lethal respiratory disease associated with pandemic (H1N1) 2009 influenza virus infection in house pets occurred in domestic cats in Oregon. A 10-year-old neutered domestic shorthair and an 8-year-old spayed domestic shorthair died shortly after developing severe respiratory disease. Grossly, lung lobes of both cats were diffusely firm and incompletely collapsed. Histologically, moderate to severe necrotizing to pyonecrotizing bronchointerstitial pneumonia was accompanied by serofibrinous exudation and hyaline membranes in the alveolar spaces. Influenza A virus was isolated from nasal secretions of the male cat and from lung homogenate of the female cat. Both isolates were confirmed as pandemic (H1N1) 2009 influenza virus by real-time reverse transcriptase polymerase chain reaction. With immunohistochemistry, influenza A viral antigen was demonstrated in bronchiolar epithelial cells, pneumocytes, and alveolar macrophages in pneumonic areas. The most likely sources of infection were people in the household with influenza-like illness or confirmed pandemic (H1N1) 2009 influenza. The 2 cases reported here provide, to the best of the authors' knowledge, the first description of the pathology and viral antigen distribution of lethal respiratory disease in domestic cats after natural pandemic (H1N1) 2009 influenza virus infection, probably transmitted from humans.

Late occurrence of chronic immune-mediated axonal polyneuropathy following bone marrow transplant for juvenile-onset alpha-mannosidosis.

A 23-year-old woman with juvenile-onset alpha-mannosidosis developed an axonal polyneuropathy more than a year following successful unrelated donor (URD) BMT complicated by chronic graft-versus-host disease (GVHD). Progressive muscle weakness and paresthesias developed over at least 4 months, and made her nonambulatory. Nerve conduction and EMG studies demonstrated an axonal sensorimotor neuropathy. Cerebral spinal fluid (CSF) IgG was elevated with two peaks not identified in serum. Strength improved after a single course of plasma exchange and continued to improve over 12 months. The response to plasma exchange, elevated CSF IgG production, and evidence of a serum IgM peak suggest an immune-mediated mechanism. Chronic polyneuropathies following BMT are rare and are usually temporally related to GVHD or infection. This patient's disease was unusual because of its late occurrence and chronic onset in the face of resolved GVHD and in the absence of infection.

Hepatic mesenchymal hamartoma in a neonate: a case report and review of the literature.

To describe an unusual presentation of mesenchymal hamartoma in a critically ill neonate necessitating a novel therapeutic embolization before definitive resection. An unusual presentation of a large hepatic mass in a newborn complicated by pulmonary hypertension and vascular "steal" with renal insufficiency is presented. The mass was initially successfully embolized, but then revascularized, necessitating resection in an attempt to improve the clinical status of the critically ill neonate. The resected mass was a mesenchymal hamartoma with a necrotic center and extensive arterial collateralization. The patient began improving immediately after resection. Mesenchymal hamartoma may present in the neonate as a diagnostic dilemma. This is the first case report describing persistent pulmonary hypertension and renal compromise from this tumor. Embolization as a therapeutic modality to address this tumor is described. The cause of the persistent and severe pulmonary hypertension remains unclear,but may be related to the tumor.

Characteristics of a recent isolate of Sarcocystis neurona (SN7) from a horse and loss of pathogenicity of isolates SN6 and SN7 by passages in cell culture.

An isolate of Sarcocystis neurona (SN7) was obtained from the spinal cord of a horse with neurologic signs. The parasite was isolated in cultures of bovine monocytes and equine spleen cells. The organism divided by endopolygeny and completed at least one asexual cycle in cell cultures in 3 days. The parasite was maintained by subpassages in bovine monocytes for 10 months when it was found to be non-pathogenic to gamma interferon knockout (KO) mice. Revival of a low passage (10th passage) of the initial isolate stored in liquid nitrogen for 18 months retained its pathogenicity for KO mice. Merozoites (10(6)) of the late passage (22nd passage) were infective to only one of four KO mice inoculated. Similar results were obtained with SN6 isolate of S. neurona. No differences were found in Western blot patterns using antigens from the low and high passage merozoites of the SN7 and SN6 isolates. These results suggest that prolonged passage in cell culture may affect the pathogenicity of some isolates of S. neurona.

Characterization of a Sarcocystis neurona isolate (SN6) from a naturally infected horse from Oregon.

An isolate of Sarcocystis neurona (SN6) was obtained from the spinal cord of a horse from Oregon with neurologic signs. The parasite was isolated in cultures of bovine monocytes and equine spleen cells. The parasite divided by endopolygeny and completed at least one asexual cycle in cell cultures in three days. Two gamma interferon knockout mice inoculated with cell culture-derived merozoites became ill 35 d later and S. neurona schizonts and merozoites were found in encephalitic lesions. The parasite in tissue sections of mice reacted with S. neurona-specific antibodies and S. neurona was reisolated from the brain of knockout mice.

Isoelectric focusing of soluble proteins in the characterization of species and isolates of Nematodirus (Nematoda: Trichostrongyloidea).

Isoelectric focusing was performed on extracts from Nematodirus spathiger, Nematodirus filicollis, Nematodirus helvetianus, and 3 geographic isolates of Nematodirus battus. Gender-specific differences were noted within species; however, the overall protein profile of each species and isolate was distinct and reproducible and allowed unequivocal differentiation. A coefficient of similarity (Sm) for males of each species and isolate was calculated, and a dendrogram, based on evaluation of Sm by the unweighted pair-group method with arithmetic means, was produced. Although cluster analysis of the 3 isolates of N. battus indicates the North American and Weybridge isolates are similar, interpretation of the relationships and thus the history of introduction based on these data is equivocal. Isoelectric focusing is a robust method for establishing identity and has great utility in diagnostics. However, in the absence of selective histochemical staining, interpretation of identity and homology for specific bands and banding patterns is problematic, thus limiting the utility of this method for phylogenetic inference.

Identification and characterization of cDNA clones encoding plant calreticulin in barley.

Two cDNA clones (CRH1 and CRH2) homologous to animal calreticulin, a major calcium storage protein in the lumen of the endoplasmic reticulum, were isolated from an ovary cDNA library of barley through differential screening. The two clones differ in the 3' untranslated region and the 5' region that encodes a putative N-terminal signal sequence. CRH1 was mapped to the minus arm of chromosome 1. CRH2 was mapped to the minus arm of chromosome 2. The deduced amino acid sequences share 50 to 55% identity with animal calreticulins and exhibit the same three-zone characteristic. Recombinant protein stained blue with Stains-all and bound 45Ca2+ when transferred to nitrocellulose membranes. A native protein of approximately 55 kD was identified in ovary extract. Elevated gene expression was observed in ovaries 1 day after pollination and during early embryogenesis. CRH1 was expressed at a higher level than CRH2. These studies demonstrate the presence of calreticulin in plant cells and its developmental regulation in fertilization.

Mapping genes for resistance to barley stripe rust (Puccinia striiformis f. sp. hordei).

Two genes conferring resistance to the barley stripe rust found in Mexico and South America, previously identified as race 24, were mapped to the M arms of barley chromosomes 7 and 4 in a doubled haploid population using molecular markers and the quantitative trait loci (QTL) mapping approach. The resistance gene on chromosome 7 had a major effect, accounting for 57% of the variation in disease severity. The resistance gene on chromosome 4 had a minor effect, accounting for 10% of the variation in trait expression. Two pairs of restriction fragment length polymorphism markers are being used to introgress the resistance genes to North American spring barley using molecular marker-assisted backcrossing.

Seroprevalence of Toxoplasma gondii in llamas (Lama glama) in the northwest USA.

Serum samples from 283 llamas (Lama glama) from Oregon, Washington State and Idaho were tested for antibodies to Toxoplasma gondii using the modified agglutination test. Antibodies were found in 95 (33.5%) llamas. Percent seropositivity in serum dilutions of 1:25, 1:50, 1:500, and 1:5000 was 9.5%, 18.3%, 4.9%, and 0.7%, respectively.

Isoelectric focusing of soluble proteins from Fasciola hepatica L, 1758 and Fascioloides magna B, 1875.

Isoelectric focusing was performed on the soluble proteins of whole-body and excretory-secretory products (ESP) of Fasciola hepatica and Fascioloides magna. Adult F hepatica flukes were recovered from experimentally infected sheep and ESP obtained from the flukes; portions of liver were cut and frozen at -70 C. Fascioloides magna adults were collected from naturally infected white-tailed deer and ESP obtained; portions of liver were collected from noninfected white-tailed deer. Adult flukes and their host tissues were homogenized and centrifuged; protein concentrations with their ESP were determined and adjusted to less than 2.50 mg/ml. Seven ESP samples from F hepatica and 1 from Fascioloides magna were subjected to isoelectric focusing with the 2 species of fluke and their respective host liver homogenates. After separation, gels were stained with silver and scanned on a laser densitometer. Protein banding patterns of the 2 species of flukes were dissimilar. In the pH range of 3.5 to 9.6, the body protein had approximately 30 peaks and ESP about 23 peaks in both species. Overall banding patterns of the body protein and ESP of both species were distinct from those of respective host tissues. Of the peaks reported as dominant, 3 of the body protein and 2 of ESP were shared between the 2 species. Fascioloides magna had more dominant peaks than F hepatica. This technique of soluble protein isoelectric focusing is simple and reproducible, and the 2 fluke species can easily be differentiated by this technique, as well as by morphologic characteristics.

Efficacy of ivermectin administered via sustained-release bolus against gastrointestinal nematodes in cattle.

Twelve calves (mean weight, 175.5 kg) were used to confirm efficacy of ivermectin delivered from a prototype sustained-release bolus against naturally acquired gastrointestinal nematodes including early fourth-stage (inhibited) larvae of Ostertagia ostertagi. The calves were allocated by restricted randomization on weight to 1 of 2 groups: controls, to which a placebo bolus was given orally, and treated calves, to which a sustained-release bolus designed to deliver 8 mg of ivermectin/day at a steady rate was given orally. After treatment, the 2 groups were housed in separate pens with concrete flooring. Twenty-eight days after treatment, all calves were euthanatized and necropsied. The ivermectin-treated calves had no larval or adult Ostertagia spp and significantly (P less than 0.01) fewer adult Trichostrongylus axei and adult Cooperia (C oncophora, C punctata and C surnabada) than control calves. Efficacy of ivermectin was greater than 99% for Cooperia spp, and 100% for other parasites. Drug-related adverse reactions were not observed.

Clinical reference values for serum protein electrophoresis for the llama (Lama glama).

Serum protein electrophoresis was performed on 71 clinically healthy juvenile and adult llamas (6 juvenile males, 7 juvenile females, 25 adult males, 13 adult females, and 20 pregnant females) to determine normal serum protein concentrations. Values were reported for each of the 5 groups because the groups were not homogeneous in all 8 peaks. Although the values reported here may serve as reference values for adults, they represent only a guideline for the juveniles because of the limited number of animals in each of these groups.