2011 Travel Award: Jeffrey H. Bock. Pfizer Global Research and Development, Groton, CT
“Comparison of Sysmex Flow Cytometric and Microscopic Bone Marrow Differentials in Wistar Rats" [K. A. Criswell, J. H. Bock, R. P. Giovanelli]
Background: Preclinical drug trials frequently require evaluation of animal bone marrow to evaluate hematopoietic safety. Microscopic bone marrow evaluation is time-consuming and requires highly trained individuals. Flow cytometric determination of peroxidase activity, cell size, and lymphocyte immunophenotyping has previously been used for rodent bone marrows.
Methods: The Sysmex XT-2000iV hematology analyzer contains gates that may be manually set to capture individual populations. Therefore, it has the potential to further automate bone marrow analysis. This study was conducted to validate the cellular populations observed in Sysmex cytograms from rat bone marrow. Rats were treated with 0, 50, or 100 U/kg erythropoietin (EPO) or with 0, 5, 20, or 40 mg/ kg cyclophosphamide (CP). Pharmacologic modulation of hematopoietic populations with these compounds was evaluated using flow cytometric, microscopic, and Sysmex technologies.
Results: Erythropoietin produced a dose-dependent decrease in M:E (myeloid to erythroid ratio). Sysmex and manual mean M:E values were 1.3, 0.9, 0.8 vs. 1.3, 0.8, 0.7, respectively for the EPO-treated groups. Linear regression of M:E ratio was 0.8090 and 0.8996 for Sysmex and flow cytometric differentials and Sysmex and microscopic differentials, respectively. CP produced a decrease in bone marrow cellularity. Total nucleated cell count (TNC) decreased from 59.4 in controls to 51.5, 33.7, and 16.1 x 103 cells in CP-treated groups. Sysmex and manual mean M:E values were 1.0, 1.1, 2.9, 9.0 and 1.1, 1.1, 2.1, 5.0, respectively in the CP cohort, demonstrating the instrument’s ability to measure progressive changes in M:E. The linear regression between Sysmex and flow cytometry in CP-treated rats for M:E ratio reached 0.7819. Outliers in the flow method were due to extremely hypocellular bone marrow, resulting in flow results that failed to match microscopic differentials. In contrast, the linear regression for M:E between Sysmex and microscopic differentials was 0.9158 in CP-treated animals. This demonstrates that Sysmex technology can more accurately delineate bone marrow M:E ratio in rats under extreme hypocellularity conditions than flow cytometry. Lymphoid and erythroid populations overlapped in the Sysmex cytogram. Subsequent experiments depleted lymphocytes using a magnetic cell sorting (MACS) method (B cells with anti-rat CD45RA and T cells with anti-rat OX52). The positively selected populations were reanalyzed to demonstrate the specificity of each component cell type within the Sysmex cytogram. MACS effectively removed all lymphocytes from the samples and the recovered aliquots definitively linked Sysmex cytogram location for recovered lymphocytes. The average standard deviation from six bone marrows that were analyzed four times each for lymphocyte population was 0.37. An anti-myeloid MACS method was also used to verify the location of the myeloid population within the Sysmex histogram.
Conclusion: These studies demonstrate that the bone marrow populations achieved good separation when analyzed by the Sysmex XT-2000iV coupled with antilymphocyte MAC. Sysmex gating into mature and immature populations of myeloid and erythroid cells, and of lymphocytes was comparable to those achieved by flow cytometry or microscopic evaluations. This suggests that the Sysmex technology holds significant promise in demonstrating increased reproducibility while reducing analysis time and cellular expertise required to evaluate rodent bone marrow.
2010 Travel Award: David F. Adams, GlaxoSmithKline, King of Prussia, PA
“Evaluation of Species Specific Calibrations for Measuring Albumin in Urine of Dogs and Monkeys using an Automated Human Immunoturbidometric Assay" [D. F. Adams, K. M. Lynch, T. S. Sellers, and D. Ennulat.]
The Siemens human immunoturbidometric microalbumin assay for the ADVIA® 1650 Chemistry System is an immunological method used to assess and/or monitor renal disease in clinical medicine. Application of this method for nonclinical species could provide a reliable and automated method for assessing kidney injury in nonclinical safety studies. However, the antihuman antibody used in this assay demonstrated only partial cross-reactivity to dog and cynomolgus monkey albumin. Here we describe the use of commercially available dog and cynomolgus monkey albumin to create species-specific calibrations for accurately measuring albumin in urine from beagle dogs and cynomolgus monkeys using the Siemens assay on the ADVIA 1650 Chemistry System. Stock solutions of dog and cynomolgus monkey albumin diluted in diH2O to concentrations of 500 mg/L were used to prepare multi-point calibration curves. Intra- and interassay precision was acceptable (CVs < 5%) using commercially available human controls (albumin concentrations of 30-40 mg/L and 150-200 mg/L) and dog and monkey urine. Linearity was demonstrated in both species across the calibration range of 5 to 500 mg/L (dog: R2= 0.9999, monkey: R2 = 0.9997). Additionally, the stock 500 mg/L calibrators were stable at -70oCfor 2 months. Spike/recovery evaluations demonstrated acceptable recovery (87-104%) in both species. Mean (min-max) urinary albumin (normalized to creatinine) values in clinically healthy beagle dogs and cynomolgus monkeys were 0.43 (0.03-2.80) mg/mmol and 1.47 (0.41-8.69) mg/mmol, respectively (n = 24/species). Additionally, urinary albumin concentrations for 20 clinically healthy beagle dogs correlated well (y = 2.87x + 12.93, R2 = 0.9488) with albumin concentrations measured using a dog specific microalbumin ELISA method. Species-specific calibration of the Siemens human immunoturbidometric microalbumin assay provides an easily adaptable automated method for measurement of urinary albumin concentrations in dogs and monkeys
2010 Travel Award: Cynthia I. Starks, Huntingdon Life Sciences, East Millstone, NJ
"The Determination of Enzygnost Thrombin Anti-Thrombin III Complex in Pig Citrated Plasma to Support Pre-Clinical Toxicology Studies" [C. Starks, B. Litzenberger, M. Genato, J. Dharmadhikari, C. McDonough].
Objective: The purpose of this project was to determine if the performance of the Siemens Diagnostics EIA test kit Enzygnost Thrombin Anti-Thrombin III Complex (TAT) (product #OWMG-15 or equivalent) was fit-for-purpose when analyzing pig citrated plasma to support pre-clinical safety assessment toxicology studies. Since the method is specific for human, matrix effect interference was evaluated.
Methods and Materials: The Siemens Diagnostics EIA test kit Enzygnost Thrombin Anti-Thrombin III Complex (TAT) (product #OWMG-15 or equivalent) is a sandwich enzyme immunoassay. TAT concentrations are quantified by measuring the test sample solution absorbance at a specified wavelength and comparing the values with those from a standard curve. The color intensity is proportional to the concentration of TAT. The calibration range was 2 to 60 ug/L. The Spectra Max 340 PC 384 Microplate Reader version 1.0 was used for testing. There were no design modification made to this assay, however a second control was made from the two highest standards provided in the kit. in order to demonstrate adequate analytical quality control over the calibration range (kit only offers 1 QC material).
Results: The Intra-assay precision and accuracy was conducted by analysing QCs and a pooled pig sodium citrated plasma in duplicate replicates of 5 within one run. The Intraassay precision measurements ranged from 2.6% to 4.2 % RSD for QC samples and 8.4% RSD for Pig sodium citrated plasma pool. The recovery of the QCs ranged from 84.1%to 86.9 %. The Inter-assay precision and accuracy was performed by analysing QCs and pool pig sodium citrated plasma in separate runs against at least three independent standard curves. The Inter-assay precision measurements ranged from 7.2 to 8.1 % relative standard deviation (RSD) for QC samples and 10.7 % RSD for the pig sodium citrated plasma pool. Matrix Effect was performed by preparing spiked samples, which consist of mixing each QC level with the pooled pig sodium citrated plasma at a 1:20 dilution. The mean recovery of the spikes samples ranged from 95.6% to 99.2 %. The assay was used on a study to evaluate treatment effects of a test device applied to a coagulopathic swine model. The group mean values. for pretest and two post treatment timepoints were 25.42 ug/L, 30.67 ug/L and 41.79 ug/L respectively.
Conclusion: The quantitative method for the measurement of Thrombin Anti-Thrombin in pig sodium citrated plasma using the Siemens Diagnostics EIA test kit Enzygnost Thrombin Anti- Thrombin III Complex (TAT) (product #OWMG-15 or equivalent) is fit-for-purpose when analyzing pig citrated plasma. The pre-clinical safety trial results demonstrates that the assay translates from human to pig and can serve as a biomarker for hemostasis evaluations.
2009 Travel Award: Janet DiPiero. Bristol Myers Squibb, Lawrenceville, NJ
A Comparison Study of the Hemoglobin A1c Assay in Rats and Monkeys using Siemens Advia® 1800 Chemistry System and Hemoglobin Electrophoresis
This comparison study was designed to determine if the Hemoglobin A1c (HbA1c) assay using Siemens Advia® 1800 (Advia 1800) automated chemistry system was applicable for veterinary samples and would produce accurate results based on species. A study was designed to compare the automated assay to Hemoglobin Electrophoresis (electrophoresis), which is the universally accepted method for evaluating Hemoglobin A1c (HbA1c). Electrophoresis is a process that causes movement of particles in an electric field, resulting in formation of "bands" that separate toward one end or the other in the field. The components then move away from each other at different rates, and when separated, form a series of distinctly pigmented bands. The bands are then compared with those of a normal sample with the concentration of HbA1c reported out as a percentage of total hemoglobin. Siemens Advia HbA1c automated assay is based on a two part methodology. First a total hemoglobin (tHb) concentration is determined by converting all different forms of hemoglobin into a single form to be measured spectrophotometrically, then a latex agglutination inhibition method is used for the measurement of specific HbA1c. The concentration of HbA1c and the concentration of tHb are measured and the ratio is reported as percent HbA1c.
Whole blood samples were obtained in tubes containing the anticoagulant potassium ethylenediaminetetraacetate (K2-EDTA) from 11 non-diabetic and 3 diabetic cynomologus monkeys via the femoral vein and 12 Zucker Diabetic Fatty lean (ZDF lean) and 2 Zucker Diabetic Fatty obese (ZDF obese) rats bled by cardiac puncture. Samples were aliquoted and stored at -80°C for approximately 1 month. One set of samples was sent to a reference laboratory for analysis of HbA1c by the electrophoresis method, and the other set of aliquots was analyzed on the Advia 1800. The results for HbA1c by electrophoresis were compared to the automated assay for cross reactivity in each species and correlation between methods.Results showed that the Advia 1800 automated assay did exhibit cross reactivity in each species and correlated to the standard electrophoresis method. For each method tested, an approximate 2-fold increase in HbA1c results were observed in the diabetic rat and monkey populations when compared to their respective non-diabetic populations.These findings indicate that selection of the automated Siemens Advia HbA1c assay can be used for veterinary testing in both rats and monkeys.
2009 Travel Award: Karen M. Lynch, GLAXOSMITHKLINE, Dept of Safety Assessment, King of Prussia, PA
Development of an electrochemiluminescent immunoassay for the measurement of regucalcin in serum and urine of rats.
Regucalcin is a calcium-binding protein involved in regulation of intracellular homeostasis. The abundance of regucalcin in liver and kidney suggests that measurable amounts of regucalcin in serum or urine can reflect liver or kidney injury. A rat-specific assay for measurement of regucalcin in rat serum or urine was developed using a sandwich immunoassay format and Meso Scale Discovery® (MSD) platform. Recombinant rat regucalcin was expressed by baculovirus in SF9 cells with a flag/hexahis tag engineered on the N-terminus. The flag/hexahis/regucalcin was solubilized from cell lysate pellet, purified by Ni-NTA, refolded, and reprocessed under non-denaturing conditions on Ni-NTA followed by size exclusion chromatography. Purified rat regucalcin was used to immunize rabbits for polyclonal antibody production. Reactivity of the antibodies with the recombinant regucalcin protein was confirmed by demonstration of acceptable linearity (0.1-1000 ng/mL). All standards demonstrated acceptable precision (CV<15%) and accuracy (Relative Error + 10%) compared to nominal values when analyzed in duplicate over 8 analytical runs. Specificity of the assay for native rat regucalcin was demonstrated with dilutional linearity (y=142.47x +263.97; r2=0.997) using freshly prepared rat liver homogenate (0.98-22.1 ng/mL). Acceptable intra- and inter-assay precision (CVs <15%; 0.58-71.6 ng/mL) was demonstrated for rat serum, plasma, urine and liver homogenates. Acceptable recoveries (88%-119%; 0.186-18.6 ng/mL) were observed for rat liver homogenates, serum and plasma. Linearity (y=133.62x + 484.07; r2=0.999) and recovery (115-130%) of regucalcin in rat urine (10-100 ng/mL) were also demonstrated. Regucalcin is not detectable in the serum of naïve, clinically healthy Sprague-Dawley (SD) rats however preliminary investigations have shown that regucalcin increases during liver or renal injury in the rat. In a 4-day study with methapyriline (150mg/kg), regucalcin was detected (0.4-0.5 ng/mL) in Day 5 serum samples from 2 of 5 high dose rats with periportal necrosis and maximum ALT increases (11X and 35X control). In a 7-day toxicity study with a compound that caused hepatobiliary degeneration in male SD rats, serum regucalcin was increased in 2 of 10 treated rats on Day 8 (up to 9 ng/mL). In contrast, low levels of regucalcin are normally present in urine of control rats (0.19-0.78 ng/μmol creatinine), and regucalcin has demonstrated potential for use as a marker of nephrotoxicity. In male Wistar rats given single doses of the proximal tubular toxicants hexachlorobutadiene (HCBD; 100mg/kg ip) or potassium dichromate fixanal (25mg/kg sc), mean urinary regucalcin (normalized to creatinine) values were increased at 6, 12, 24, 48, 72, and 96 hours postdose (up to 65-fold control) in both studies, and maximum regucalcin changes preceded maximum urinary albumin increases by 12 to 36 hours in both studies, suggesting potential use as an early marker of kidney injury in the rat. In conclusion, a sensitive and precise rat-specific immunoassay has been developed for measurement of regucalcin in serum, plasma, tissue homogenates and urine using the Meso Scale Discovery® platform. Further investigations to assess the potential use of regucalcin as a biomarker of drug-induced tissue injury in rats are warranted.
2008 Travel Award: Qing H. Meng MD, PhD, Assistant Professor
Department of Pathology
Royal University Hospital, College of Medicine
University of Saskatchewan
Beneficial Effects of Soy Isoflavones on Insulin Secretion, Glucose Control, and Cataract Prevention in Streptozotocin-induced Diabetic Rats
Objectives: Isoflavones, mainly derived from soybean, are a group of biologically active substances with a chemical structure similar to that of estrogen. Although studies have shown the beneficial effects of soy protein consumption on diabetes, the effective components and the underlying mechanisms of soy protein have not been fully understood. In this study, we investigated the beneficial effects and mechanisms of soy isoflavones using streptozotocin (STZ)-induced diabetic rats.
Materials and methods: Diabetes (type 1) was induced in male Sprague-Dawley rats by intraperitoneal injection of 100 mg/kg STZ. Diabetic rats were then randomly divided into 3 groups (n=9 each group) and received a special diet supplemented with 20% casein (control), 20% low-isoflavone soy protein (LIS), and 20% high-isoflavone soy protein (HIS) for 8 weeks, respectively. Serum lipid and glucose levels were measured on a Beckman Synchron LX20. Serum insulin was assayed using a commercial immunoassay kit by ELISA (Mercodia). Methylglyoxal, a glucose metabolite and strongly associated with diabetes and diabetic complications, was quantitated by HPLC.
Results: The body weights of diabetic rats in the HIS group were significantly increased compared to those in the LIS or control group (303.11 ± 15.65 vs. 257.14 ± 8.81 or 255.43 ± 11.9 g, p < 0.05). Compared with the LIS or control rats, serum glucose levels were significantly reduced (19.50 ± 1.85 vs 28.24 ± 3.56 or 26.94 ± 1.82 mmol/L, p < 0.05, respectively) and serum insulin levels were significantly increased (2.46 ± 0.35 vs 0.55 ± 0.04 or 0.39 ± 0.04 μg/L, p < 0.01, respectively) in the HIS rats. Serum methylglyoxal concentrations were lower in HIS rats than that in the LIS or control rats (0.77 ± 0.09 vs 1.57 ± 0.17 or 1.91 ± 0.28 nmol/L, p < 0.05). Serum glutathione levels were significantly increased in the HIS rats compared to the LIS or control rats. (13.26 ± 1.61 vs 7.35 ± 1.55 or 5.54 ± 1.10 mmol/L, p < 0.01). Serum HDL cholesterol levels were significantly increased in the HIS rats compared to that of the LIS or control rats but no significant changes in total cholesterol and triglycerides were observed. Immunohistostaining demonstrated increased insulin production in the pancreatic islets of Langerhans in diabetic rats treated with the HIS compared to the LIS or control group. Moreover, the death rate of diabetic rats was reduced. The development of cataracts was significantly delayed and the incidence of cataracts of diabetic rats was markedly decreased in the HIS group compared to that in the LIS or control group (p<0.05). There were no differences in all the above parameters between the LIS and control rats.
Conclusions: Ingestion of high soy isoflavones not only lowers glucose levels but also reduces the death rate and the incidence of cataracts in diabetic rats, which is demonstrated for the time. The beneficial effects of soy isoflavones are attributed to increased insulin secretion and antioxidant protection.
2008 Travel Award: M. M. Kuo. PhD, Amgen, Inc., Seattle, WA
Short-term and Long-term Stability of Sorbitol Dehydrogenase in Cynomolgus Monkey Serum
Objective: Sorbitol dehydrogenase (SDH) along with other liver enzymes such as aspartate aminotransferase (AST) and alanine aminotransferase (ALT) is used to assess hepatotoxicity in preclinical studies. The purpose of this study was to determine short-term and long-term stability of SDH under various storage conditions.
Methods: Serum samples from 10 different cynomolgus monkeys were spiked with extract of cynomolgus liver to achieve final SDH activities of approximately 100, 50, 25 and 12.5 U/L. To assess long-term storage stability, multiple serum aliquots for each level were frozen at -200C and -800C for 24 hrs, 48 hrs, 7 days, 14 days, 1 month, and 3 months. The frozen aliquots were thawed at room temperature (RT) and analyzed on the Olympus AU400 chemistry analyzer for AST, ALT and SDH at the above time points. To assess short-term storage stability, duplicate aliquots from each level were placed at RT and 40C for 1 hr, 2 hrs, 4 hrs, 6 hrs, 8 hrs and 24 hrs and analyzed for AST, ALT and SDH. In addition, to assess the effects of preanalytical and processing conditions neat (unspiked) aliquots of blood from one cynomolgus monkey were centrifuged 30 or 90 minutes after collection. Samples were analyzed for AST, ALT and SDH immediately after centrifugation and again after the following incubation conditions: 1 hr at 40C, 3 hrs at 40C, 3hrs at 40C + 2.5 hrs at RT.
Short-Term and Long-Term Stability (%CV)
- Storage Conditions AST ALT SDH
- RT for 24 hrs 1.6 1.3 3.5
- 40C for 24 hrs 1.1 1.0 2.2
- -200C for 3 months 2.8 50.8 6.9
- -800C for 3 months 1.5 1.6 5.8
Preanalytical and Processing Conditions (%CV)
- Processing Conditions AST ALT SDH
- 30 min Post Collection 1.1 0.0 0.9
- 90 min Post Collection 0.9 1.2 2.5
Conclusion: AST, ALT and SDH were stable up to 24 hours at room temperature and 40C, and up to 3 months at -800C. AST and SDH were stable up to 3 months at -200C, but ALT became unstable by 7 days. Variations in preanalytical and processing conditions had no effect on ALT, AST, and SDH stability up to 5.5 hours.
2007 Travel Award: Octavia M. Peck-Palmer, Washington University School of Medicine, St. Louis, MO
Comparison of Analytical Performance of Three Murine Insulin Enzyme-Linked Immunoassays
Background: As research continues to grow exponentially in the fields of diabetes, obesity and metabolic disease, the analysis of insulin concentration in rodent disease models is paramount. Commercially available enzyme-linked immunoassays (ELISA) can conveniently be performed by using a small amount of sample for the quantification of insulin in serum or plasma. Pre-analytical variables such as hemolysis need to be controlled especially in fasting animals since insulin concentrations in the fasting state is usually close to the limit of detection of the assays. During hemolysis, an insulin-degrading enzyme present in the red blood cells is responsible for the loss of insulin. Previous studies have shown that diamide, a thiol-oxidizing agent, prevents the hemolysis-induced loss of insulin without interfering with the assays studied.
Objective: To determine the analytical performance of 3 different commercially available ELISA kits and to determine if diamide is needed to prevent hemolysis-induced insulin loss.
Methods: Murine insulin ELISA kits were purchased from Alpco Diagnostics, Linco Research Inc. or Crystal Chem, Inc. to investigate precision, accuracy, and diamide effect. Testing was performed following manufacturer’s instructions. Optical density was measured using the ELx800TM ELISA plate reader. Data was converted to ng/mL using a quadratic parametric equation using Gen5TM software. Statistical analysis was performed using GraphPad Prism 4 (GraphPad Software Inc.). Precision and accuracy were expressed as the percentage of the coefficient of variation (%CV) and the percentage of recovery from the expected value. A commercial quality control material was used (Millipore Corporation, Billerica, MA) at different dilutions ranging from 0.2, to 6.4 ng/mL. Diamide was tested at a final concentration of 5 mM. Percent recovery from the expected insulin concentration or percent difference between mice insulin samples treated with and without diamide was calculated to measure the effect of diamide in the assays. Heparinized plasma from 18 hours fasting male and female CD-1 mice (n=20) or 18 hours fasting or fed GSK-3 female wild type (n=16) was obtained and stored at -20° C until assayed.
Results: Precision ranged from 31% to 2.6%, 12% to 0.7%, and 18% to 0.9% for the Alpco, Linco or Crystal Chem kits at concentrations of 0.2, 3.5, and 6.4 ng/mL, respectively. Accuracy averaged 120%, 101% and 109% for the Alpco, Linco or Crystal Chem assay kits for the range of concentrations tested. Percent recovery of insulin in a commercial material without diamide averaged 129%, 105%, and 111%; and 119%, 108% and 116% with diamide for the Alpco, Linco or Crystal Chem kits respectively. Percent difference between insulin concentrations in fasting and fed stages in the same GSK-3 wild type animal without diamide averaged an increase of 20%, 2.3% and 23%; and a decrease of 7%, 8% and 5% for diamide treated samples for the Alpco, Linco or Crystal Chem kits, respectively.
Conclusion: The murine ELISA Linco kit has the highest precision and accuracy at fasting insulin concentrations. When hemolysis is avoided during the pre-analytical process, diamide may not be needed to prevent hemolysis-induced insulin loss in the fasting stage.