INVESTIGATION OF THE USE OF SERUM BIOMARKERS FOR THE DETECTION OF CARDIAC DISEASE IN MARINE MAMMALS.

Cardiac disease has been extensively documented in marine mammals; however, it remains difficult to diagnose antemortem. Assays measuring cardiac troponin I (cTnI) and N-terminal pro-brain natriuretic peptide (NT-proBNP) are used as sensitive and specific biomarkers of cardiac disease in many species, but have not been widely investigated in marine mammals. This study aimed to provide a set of reference values for cTnI and NT-proBNP in belugas (BW) (Delphinapterus leucas), sea otters (SO) (Enhydra lutris), Steller sea lions (SSL) (Eumetopias jubatus), and California sea lions (CSL) (Zalophus californianus) with and without cardiac disease, and to determine if these biomarkers are useful indicators of cardiac disease in these species. First, existing immunoassays for cTnI and NT-proBNP were successfully validated utilizing species-specific heart lysate spiked serum. Cohorts were defined by histopathology as animals with no evidence of cardiac disease ("control"), with confirmed cardiac disease ("disease"), and with concurrent renal and cardiac disease ("renal") for which serum samples were then analyzed. Serum concentration ranges for cTnI (ng/ml) and NT-proBNP (pmol/L) were determined for control and disease cohorts. There was significantly higher cTnI (P= 0.003) and NT-proBNP (P= 0.004) concentrations in the CSL disease cohort, as well as positive trends in BW, SO, and SSL disease cohorts that did not reach statistical significance. NT-proBNP concentrations were significantly higher in the CSL renal cohort compared with the control (P < 0.001) and disease (P= 0.007) cohorts. These results suggest that cTnI and NT-proBNP may be clinically useful in the antemortem diagnosis of cardiac disease in CSL, and warrant further investigation in BW, SO, and SL.

CIRCULATING CONCENTRATIONS OF THYROID HORMONE IN BELUGA WHALES (DELPHINAPTERUS LEUCAS): INFLUENCE OF AGE, SEX, AND SEASON.

Thyroid hormones play a critical physiologic role in regulating protein synthesis, growth, and metabolism. To date, because no published compilation of baseline values for thyroid hormones in beluga whales (Delphinapterus leucas) exists, assessment of thyroid hormone concentrations in this species has been underused in clinical settings. The purpose of this study was to document the concentrations of total thyroxine (tT4) and total triiodothyronine (tT3) in healthy aquarium-maintained and free-ranging beluga whales and to determine the influence of age, sex, and season on the thyroid hormone concentrations. Archived serum samples were collected from healthy aquarium-maintained (n=43) and free-ranging (n=39) belugas, and serum tT4 and tT3 were measured using chemiluminescence immunoassay. The mean tT4 concentration in aquarium-maintained belugas was 5.67±1.43 µg/dl and the mean tT3 concentration was 70.72±2.37 ng/dl. Sex comparisons showed that aquarium-maintained males had significantly greater tT4 and tT3 (9.70±4.48 µg/dl and 92.65±30.55 ng/dl, respectively) than females (7.18±2.82 µg/dl and 77.95±20.37 ng/dl) (P=0.004 and P=0.013). Age comparisons showed that aquarium-maintained whales aged 1-5 yr had the highest concentrations of tT4 and tT3 (8.17±0.17 µg/dl and 105.46±1.98 ng/dl, respectively) (P=0.002 and P<0.001). tT4 concentrations differed significantly between seasons, with concentrations in winter (4.59±1.09 µg/dl) being significantly decreased compared with spring (P=0.009), summer (P<0.0001), and fall (P<0.0001) concentrations. There was a significant difference in tT4 and tT3 concentrations between aquarium-maintained whales (5.67±1.43 µg/dl and 70.72±15.57 ng/dl, respectively) and free-ranging whales (11.71±3.36 µg/dl and 103.38±26.45 ng/dl) (P<0.0001 and P<0.001). Clinicians should consider biologic and environmental influences (age, sex, and season) for a more accurate interpretation of thyroid hormone concentrations in belugas. The findings of this study provide a baseline for thyroid health monitoring and comprehensive health assessments in both aquarium-maintained and free-ranging beluga whales.

Validation of Sysmex XT-2000iV analyzer-generated quantitative bone marrow differential counts in cynomolgus monkeys, Beagle dogs, and CD-1 mice.

BACKGROUND: In a previous study, the validation of rat bone marrow (BM) collection, processing, and analysis using the Sysmex XT-2000iV (Sysmex Corporation, Kobe, Japan) hematology analyzer showed that the Sysmex hematology analyzer produced BM differential counts that were comparable to those obtained with microscopic differential counts. OBJECTIVE: This study was conducted to expand the validation of the Sysmex TNCC (total nucleated cell count) and 5-part BM differential in cynomolgus monkeys, Beagle dogs, and CD-1 mice, which are alternate species that are also frequently used in preclinical safety studies. METHODS: The Sysmex 5-part BM differential counts were generated with a two-step process, whereby proliferating and maturing erythroid and myeloid cells were determined by preset gating and lymphocytes were determined using species-specific B- and T-lymphocyte antibodies and a magnetic cell-sorting method (MACS). Agreement with microscopic myelograms with 500-cell differential counts was determined from BM suspensions of 62 cynomolgus monkeys, 47 Beagle dogs, and 44 CD-1 mice. RESULTS: The correlation coefficients between methods for myeloid to erythroid (M:E) ratios in all three species was > 0.928. The Bland-Altman differences between methods were approximately ± 0.3 units for the M:E ratio in dogs and mice, and +0.6 and -0.4 in monkeys. The upper limits of agreement for all three species were ≤7% for maturing myeloid cells, ≤6% for maturing erythroid cells, and ≤4% for proliferating myeloid cells, proliferating erythroid cells, and lymphocytes. CONCLUSIONS: The Sysmex XT-2000iV produces an automated M:E ratio and a 5-part differential count equivalent to microscopic differential counts in cynomolgus monkeys, Beagle dogs, and CD-1 mice.

Liver Microvascular Injury and Thrombocytopenia of Antibody-Calicheamicin Conjugates in Cynomolgus Monkeys-Mechanism and Monitoring.

Purpose: Adverse reactions reported in patients treated with antibody-calicheamicin conjugates such as gemtuzumab ozogamicin (Mylotarg) and inotuzumab ozogamicin include thrombocytopenia and sinusoidal obstruction syndrome (SOS). The objective of this experimental work was to investigate the mechanism for thrombocytopenia, characterize the liver injury, and identify potential safety biomarkers.Experimental Design: Cynomolgus monkeys were dosed intravenously at 6 mg/m2/dose once every 3 weeks with a nonbinding antibody-calicheamicin conjugate (PF-0259) containing the same linker-payload as gemtuzumab ozogamicin and inotuzumab ozogamicin. Monkeys were necropsied 48 hours after the first administration (day 3) or 3 weeks after the third administration (day 63).Results: PF-0259 induced acute thrombocytopenia (up to 86% platelet reduction) with nadirs on days 3 to 4. There was no indication of effects on megakaryocytes in bone marrow or activation of platelets in peripheral blood. Microscopic evaluation of liver from animals necropsied on day 3 demonstrated midzonal degeneration and loss of sinusoidal endothelial cells (SECs) associated with marked platelet accumulation in sinusoids. Liver histopathology on day 63 showed variable endothelial recovery and progression to a combination of sinusoidal capillarization and sinusoidal dilation/hepatocellular atrophy, consistent with early SOS. Among biomarkers evaluated, there were early and sustained increases in serum hyaluronic acid (HA) that correlated well with serum aspartate aminotransferase and liver microscopic changes, suggesting that HA may be a sensitive diagnostic marker of the liver microvascular injury.Conclusions: These data support the conclusion that target-independent damage to liver SECs may be responsible for acute thrombocytopenia (through platelet sequestration in liver sinusoids) and development of SOS. Clin Cancer Res; 23(7); 1760-70. ©2016 AACR.

Evaluation of the Relative Performance of Drug-Induced Skeletal Muscle Injury Biomarkers in Rats.

Novel skeletal muscle (SKM) injury biomarkers that have recently been identified may outperform or add value to the conventional SKM injury biomarkers aspartate transaminase (AST) and creatine kinase (CK). The relative performance of these novel biomarkers of SKM injury including skeletal troponin I (sTnI), myosin light chain 3 (Myl3), CK M Isoform (Ckm), and fatty acid binding protein 3 (Fabp3) was assessed in 34 rat studies including both SKM toxicants and compounds with toxicities in tissues other than SKM. sTnI, Myl3, Ckm, and Fabp3 all outperformed CK or AST and/or added value for the diagnosis of drug-induced SKM injury (ie, myocyte degeneration/necrosis). In addition, when used in conjunction with CK and AST, sTnI, Myl3, CKm, and Fabp3 individually and collectively improved diagnostic sensitivity and specificity, as well as diagnostic certainty, for SKM injury and responded in a sensitive manner to low levels of SKM degeneration/necrosis in rats. These findings support the proposal that sTnI, Myl3, Ckm, and Fabp3 are suitable for voluntary use, in conjunction with CK and AST, in regulatory safety studies in rats to monitor drug-induced SKM injury and the potential translational use of these exploratory biomarkers in early clinical trials to ensure patient safety.

Comparison of the Sysmex XT-2000iV and microscopic bone marrow differential counts in Wistar rats treated with cyclophosphamide, erythropoietin, or serial phlebotomy.

BACKGROUND: In a previous study, it was demonstrated that bone marrow analysis using the Sysmex XT-2000iV hematology analyzer produced differential counts in untreated rats that were comparable to microscopic differential counts. OBJECTIVE: The aim of this study was to modulate hematopoiesis in rats in vivo either through pharmacologic treatment or serial phlebotomy, and to determine whether the Sysmex XT-2000iV could accurately analyze bone marrow quantitative changes when compared with results obtained by microscopy. METHODS: Rats were treated once with 0, 5, 20, and 40 mg/kg cyclophosphamide (CP), 0, 50, 100 IU/kg erythropoietin (EPO) on 4 consecutive days, or serial phlebotomy of 1-2 mL of blood for 4 days. Modulation of hematopoietic populations in bone marrow was evaluated using the Sysmex XT-2000iV hematology analyzer, and compared with microscopic differential counts. RESULTS: Correlation coefficients between M:E ratios determined by Sysmex and the microscopic method were 0.94, 0.96, and 0.98 for CP, EPO, or serial phlebotomy treatments, respectively. Mean concordance correlation coefficients for M:E demonstrated method agreement of 0.63, 0.92, and 0.85 for the 3 treatments. Quantitative automated and microscopic bone marrow differential counts were within the expected 95% confidence intervals for CP, EPO or serial phlebotomy. CONCLUSIONS: The Sysmex XT-2000iV provides quantitative bone marrow differential counts of bone marrow cell series in rats with treatment-induced changes which are comparable to microscopic differential counts. Reliable automatic bone marrow differential counting allows increased throughput, sensitivity, reproducibility, and enhanced interpretation of bone marrow evaluation in rodent preclinical studies.

Validation of Sysmex XT-2000iV generated quantitative bone marrow differential counts in untreated Wistar rats.

BACKGROUND: Preclinical drug trials frequently require assessment of bone marrow toxicity in animals to evaluate hematopoietic safety. Since the gold standard, cytologic evaluation, is time consuming and requires highly trained individuals, automated methods remain intriguing. OBJECTIVE: The Sysmex XT-2000iV hematology analyzer allows user-developed customizable gating. This study was conducted to validate the gating of bone marrow cell populations in Sysmex cytograms from untreated rats. METHODS: B- and T-lymphocytes and myeloid cells were experimentally depleted from Charles River Wistar Han IGS (CRL: WI [Han]) rat whole bone marrow suspension using a magnetic cell sorting (MACS) method. The positively and negatively selected populations were used to verify select gates within the Sysmex cytogram. Intra- and inter-animal precision, comparability between right and left femur, as well as agreement with microscopic myelograms based on 500 counted cells, were determined. RESULTS: Intra-sample precision and right-to-left femur comparability confirmed that gating was reproducible and stable. In 50 tested rats, myeloid to erythroid ratios (M:E) were 1.32 ± 0.33 in males and 1.38 ± 0.29 in females by Sysmex compared to 1.36 ± 0.32 in males and 1.42 ± 0.32 in females by microscopic evaluations. Bland-Altman differences between methods was ≤ ± 0.35 units for M:E, ≤ 5.4% for maturing myeloid cells, ≤ 3.4% for proliferating myeloid cells, ≤ 6.0% for maturing myeloid cells, ≤ 3.4% for proliferating myeloid cells, and ≤ 4.1% for lymphocytes. CONCLUSIONS: In untreated control Charles River Wistar Han IGS (CRL: WI [Han]) rats, the Sysmex XT-2000iV produced an automated M:E and 5-part differential count equivalent to microscopic differential counts.