Validation of a Multiparametric, High-Content-Screening Assay for Predictive/Investigative Cytotoxicity: Evidence from Technology Transfer Studies and Literature Review.

A multiparametric, live-cell, high-content-screening (HCS) cytotoxicity assay was first demonstrated in 2006 ( Arch. Toxicol. 2006 , 80 , 580 - 604 ) to be highly concordant with human hepatotoxicity, including idiosyncratic hepatotoxicities and other target organ toxicities in contrast to historical assays. The success of the assay was attributed to its simultaneous measurement of multiple appropriate "cytobiomarkers": use of human cells with xenometabolic competence for toxicities mediated by metabolites, 72 h exposure to enable expression of slower-acting toxicants, exposure to a wide-range of concentrations from 30- to 100-fold the efficacious concentration, and normalizing the in vitro cytotoxic concentration to an estimate of the in vivo concentration of exposure. An overwhelming volume of evidence has accumulated over the last 10 years to support this approach as necessary in predictive toxicology. Equivalent assays have now been successfully applied in ∼50 studies across a wide variety of toxicants, toxicities, cell types, and disciplines. Review herein of the wider literature on cytotoxicity since the first assay was reported 100 years ago supports the selection of key cytobiomarkers along a final common pathway of cell injury, including cell proliferation, mitochondrial activity, apoptosis, lysosomal mass, oxidative stress, and cell membrane permeability. HCS studies without inclusion of such key cytobiomarkers or without testing to sufficiently high concentration have not been as successful. Furthermore, a subset of the original toxicants has been reanalyzed herein using the original HCS assay and has confirmed their high sensitivities and specificities across locations, HCS technologies, staff, laboratories, and time. A protocol is demonstrated for operational validation of the assay within laboratories to demonstrate proficiency and quality management.

High content imaging for the morphometric diagnosis and immunophenotypic prognosis of canine lymphomas.

BACKGROUND: The common, symptomatic form of canine lymphoma (multicentric, medium-to-large cell, advanced) is consistently diagnosed manually and qualitatively by veterinary cytologists mainly based on increased lymphocyte size. The most effective prognostic feature is immunophenotype based on dual-antibody labeling for T versus B cells. High content imaging (HCI) is a novel, semi-automated, fluorescence microscopy and image-analysis technology used in research and predictive toxicology. OBJECTIVE: We tested the hypothesis that HCI could semi-automatize the quantitative diagnosis of canine lymphoma and simultaneously determine immunophenotypic prognosis. METHODS: Lymphocytes were obtained from lymph nodes of six lymphoma-free and five lymphomatous dogs, stained with antibodies against CD21 and CD3 (B- vs. T-cells), Hoechst-33342 and seeded into 96-well plates. Morphological parameters were examined: (a) cell area, (b) nuclear area, (c) nuclear displacement, (d) cytoplasmic area, (e) nucleus to cell area ratio (N/Cell), (f) nucleus to cytoplasm ratio (N/Cyt), and (g) cell roundness. RESULTS: HCI unequivocally discriminated malignant from benign lymphocytes, and provided immunophenotype. Cell and cytoplasmic area, nuclear displacement and roundness showed biggest differences and the means for each of the samples were not overlapping between the two groups. Mean/median/SD for control versus lymphoma samples were: (a) cell area (µm(2) ): 55.5/53.6/7.5 versus 80.3/75.5/8.7, (b) nuclear area (µm(2) ): 33.4/34.8/3.5 versus 40.2/38.5/5, (c) cytoplasm area (µm(2) ): 22.1/21/6.2 versus 40/38.4/4.9, (d) N/cell: 0.62/0.62/0.06 versus 0.52/0.52/0.03, (e) nuclear displacement (arbitrary units): 0.139/0.149/0.055 versus 0.33/0.30/0.056, (f) N/Cyt: 1.84/1.76/0.44 versus 1.19/1.24/0.17, and (g) roundness index: 1.22/1.21/0.03 versus 1.42/ 1.42/0.11 (P < 0.05 for all). CONCLUSION: HCI identified several, novel, morphometric parameters that effectively diagnose the common, symptomatic form of canine lymphoma, and also simultaneously determine prognostic immunophenotype.

"High Content Imaging for the Morphometric Diagnosis and Immunophenotypic Prognosis of Canine Lymphomas"

Background: The common, symptomatic form of canine lymphoma (multicentric, medium-to-large cell, advanced) is consistently diagnosed manually and qualitatively by veterinary cytologists based on increased lymphocyte size. The most effective prognostic feature is immunophenotype based on dual-antibody labeling for T versus B cells. High Content Imaging (HCI) is a novel, semi-automated, fluorescence microscopy and image-analysis technology used in research and predictive toxicology. Objective: We tested the hypothesis that HCI could semi-automatise the quantitative diagnosis of canine lymphoma and simultaneously determine immunophenotypic prognosis. Methods: Lymphocytes were obtained from lymph nodes of 6 lymphoma-free and 5 lymphomatous dogs, stained with antibodies against CD21 and CD3 (B- vs. T- cells), Hoechst-33342 and seeded into 96-well plates. Morphological parameters were examined: a) cell area, b) nuclear area, c) nuclear displacement, d) cytoplasmic area, e) nucleus to cell area ratio (N/Cell), f) nucleus to cytoplasm ratio (N/Cyt), g) cell roundness. Results: HCI unequivocally discriminated malignant from benign lymphocytes, and provided immunophenotype. Cell and cytoplasmic area, nuclear displacement and roundness showed biggest differences without value overlap between groups. Mean/ median/SD for control versus lymphoma samples were: a) cell area (µm2 ): 55.5/53.6/7.5 versus 80.3/75.5/8.7, b) nuclear area (µm2 ): 33.4/34.8/3.5 versus 40.2/38.5/5, c) cytoplasm area (µm2 ): 22.1/216.2 versus 40/38.4/4.9, d) N/Cell: 0.62/0.62/0.06 versus 0.52/0.52/0.03, e) nuclear displacement (arbitrary units): 0.139/0.149/0.055 versus 0.33/0.30//0.056, f) N/Cyt: 1.84/1.76/0.44 versus 1.19/1.24/0.17, g) roundness index: 1.22/1.21/0.03 versus 1.42/ 1.42/0.11 (p<0.05). Conclusion: HCI identified several, novel, morphometric parameters that effectively diagnose the common, symptomatic form of canine lymphoma, and also simultaneously determine prognostic immunophenotype. © 2014 Clinical Cytometry Society.

Serum cardiac troponin I concentrations in dogs with leishmaniasis: correlation with age and clinicopathologic abnormalities.

BACKGROUND: There is anecdotal evidence of myocardial injury in dogs with leishmaniasis due to generalized vasculitis and myocarditis. OBJECTIVE: The aims of this study were to evaluate serum concentration of cardiac troponin I (cTnI) as an indicator of myocardial injury in dogs with leishmaniasis and to assess the relationship between cTnI concentration and age, serum antibody titer, and a variety of blood analytes. METHODS: In this retrospective study, serum cTnI concentration was measured in dogs with leishmaniasis and in age-matched healthy dogs. Diagnosis was based on clinical signs and moderate-to-high seropositivity for Leishmania as measured by ELISA. Correlations between cTnI concentration and ELISA seropositivity, PCV, concentrations of serum creatinine, total protein, albumin, and globulin, albumin:globulin ratio (A/G), and urine protein:creatinine ratio (UPC) were investigated. The Mann-Whitney test was used to compare analytes between dogs with normal and increased (> 0.06 µg/L) cTnI concentration and to compare cTnI concentrations between dogs with and without anemia, azotemia, and proteinuria. RESULTS: In dogs with leishmaniasis (n = 40), median cTnI concentration was higher than in control dogs (n = 11) (P = .011). Sixteen dogs (40%) with leishmaniasis had increased cTnI concentration; cTnI was moderately to weakly correlated with decreased albumin concentration, decreased A/G, increased UPC, decreased PCV, positive Leishmania titer, and increased age. Dogs with leishmaniasis had significantly higher total protein and globulin concentrations and lower PCV, albumin concentration, and A/G than control dogs. Hematologic and biochemical analytes did not differ significantly between dogs with cTnI concentration within the reference interval and those with increased concentrations. Concentration of cTnI was higher in proteinuric dogs compared with nonproteinuric dogs (P = .017). CONCLUSION: A proportion of dogs with leishmaniasis have increased serum cTnI concentration, indicative of some degree of cardiac injury. Additional studies are needed to investigate the relationship between leishmaniasis and possible myocardial injury.

Veterinary and toxicological applications for the detection of cardiac injury using cardiac troponin.

The use of cardiac troponin (cTn), the 'gold-standard' biomarker of myocardial injury in humans, is growing in veterinary medicine and in animal safety studies, although there are differences in its application in animals. In this study six new assays for the marker were assessed in 619 animals of six different species (dog, cat, horse, cattle, rat and rabbit), in clinical and drug-safety studies. Healthy animals and clinical cases without cardiac disease served as controls. Several of the tested assays had poor analytic or diagnostic sensitivity and only one test was effective in all species and in all models of cardiac injury. This assay had the highest sensitivity and widest dynamic range, and identified cardiac injury due to anaemia, pancreatitis, uncontrolled Addison's and Cushing's disease, old age, renal disease, severe colic, lymphoma and neoplasia. Detection of the cTnI and cTnT forms correlated with loss of cardiac function in toxicity studies in rodents and rabbit. Increased serum cTnI was not found to correlate with disease aetiology or pathogenesis, but was effective in detecting, monitoring and quantifying ongoing cardiac injury. Cardiac injury, as demonstrated by elevated cTnI in blood, appears to be a common sequel to a wide variety of both primarily cardiac disease and of other diseases that do not primarily involve the cardiovascular system.

Cardiac troponin is the most effective translational safety biomarker for myocardial injury in cardiotoxicity.

There is an overwhelming weight of evidence that certifies cardiac troponin (cTn) as the preferred, defacto, translational, safety biomarker for myocardial injury in cardiotoxicity. As well as being the gold standard for cardiac injury in man, it has been widely used for clinical assessment and monitoring of cardiac toxicity in humans being treated for cancer. Furthermore, several dozen preclinical published studies have directly confirmed its effectiveness in laboratory animals for assessment of cardiotoxicity. It is gradually being reverse translated from human into animal use as a safety biomarker. Its use is especially merited whenever there is any safety signal indicating potential cardiotoxicity and its required inclusion as a routine biomarker in preclinical safety studies seems on the horizon. There are some considerations that are unique to use of cTn assays in animals. Lack of awareness of these has, historically, significantly inhibited the introduction of cTn as a safety biomarker in preclinical toxicology. Firstly, cross-species reactivity is usually but not always high. Secondly, there is a background of cardiac injury that needs to be controlled for, including spontaneous cardiomyopathy in Sprague Dawley rats, and inappropriate blood collection methods. Also, there are faster kinetics of clearance in rats than for humans. Also, coincident muscle injury is frequent with cardiotoxicity and requires a skeletal muscle biomarker. Because cTn assays were developed for detection of gross cardiac necrosis, such as occurs with myocardial infarct, the more sensitive assays should be used for preclinical studies. However, analytic sensitivity is higher for standard preclinical studies than for clinical diagnostic testing because of use of concurrent controls and use of batch analysis that eliminates interassay variability. No other biomarker of myocardial injury comes close to cTn in effectiveness, including CK-MB, LDH-1 and 2, myoglobin, and FABP3. In addition to the use of cTn for monitoring active myocardial degeneration, there is growing evidence that measurements of brain natriuretic peptide (BNP) may be effective for monitoring drug-induced left ventricular dysfunction.

Blood cardiac troponin in toxic myocardial injury: archetype of a translational safety biomarker.

A translational safety biomarker for toxic myocardial injury is needed in drug discovery and development. This need reflects the increasing recognition of occurrence of cardiotoxicities, prior lack of preclinical blood biomarkers for toxic cardiac injury, introduction of troponin as a biomarker, and regulatory and industry drivers. Cardiac troponin is considered the gold-standard biomarker in humans for cardiac injury due to ischemic injury and drug toxicity. It has been demonstrated to correlate highly with histopathological extent of injury, degree of impairment of cardiac function, and prognosis. Numerous studies have now clearly demonstrated that both cardiac troponin T and cardiac troponin I are sensitive and specific biomarkers of cardiac injury in laboratory animals. Their use is highly recommended for incorporation into preclinical drug-safety studies, especially whenever there is any history of cardiac effect in prior studies with a compound of the same or similar chemical or pharmacological class. The main caveats with respect to cross-species use of specific cardiac troponin assays are the need for species-specific validation, definition of cut-offs based on relevant assessments of imprecision and reference ranges or concurrent controls, and knowledge of the species-dependent kinetics of release into, and clearance from, the blood. Future development of high-sensitivity assays should determine whether minimal increases below a threshold concentration of troponin might reflect reversible myocardial effects.