Recognizing and Reducing Analytical Errors and Sources of Variation in Clinical Pathology Data in Safety Assessment Studies.

Veterinary clinical pathologists are well positioned via education and training to assist in investigations of unexpected results or increased variation in clinical pathology data. Errors in testing and unexpected variability in clinical pathology data are sometimes referred to as "laboratory errors." These alterations may occur in the preanalytical, analytical, or postanalytical phases of studies. Most of the errors or variability in clinical pathology data occur in the preanalytical or postanalytical phases. True analytical errors occur within the laboratory and are usually the result of operator or instrument error. Analytical errors are often ≤10% of all errors in diagnostic testing, and the frequency of these types of errors has decreased in the last decade. Analytical errors and increased data variability may result from instrument malfunctions, inability to follow proper procedures, undetected failures in quality control, sample misidentification, and/or test interference. This article (1) illustrates several different types of analytical errors and situations within laboratories that may result in increased variability in data, (2) provides recommendations regarding prevention of testing errors and techniques to control variation, and (3) provides a list of references that describe and advise how to deal with increased data variability.

A model for educational enrichment and employment recruitment for clinical laboratory science students.

An educational partnership was initiated between a pharmaceutical company and a university-based clinical laboratory science program to achieve mutually beneficial objectives. This external enrichment site provides a unique educational experience for the students that cannot be duplicated any where else in the community. The framework for the educational experience was established with a full day's schedule of visits and presentations guided by a list of twenty learning objectives. Clinical laboratory science students interact with laboratory professionals who are employed by the pharmaceutical company and assigned to a variety of traditional and non-traditional roles. During the visit, pharmaceutical company employees observe student interactions in small group settings and assess the learners' interest in the work environment and specimen testing process. Employee feedback may be applied to future employment decision making. This article describes how employer outreach goals and initiatives and educational enrichment objectives can be met through cooperative team work.

Gene expression analysis of the acute phase response using a canine microarray.

The safety of pharmaceuticals is typically assessed in the dog and rat prior to investigation in humans. As a result, a greater understanding of adverse effects in these preclinical testing species would improve safety assessment. Despite this need, there is a lack of tools to examine mechanisms and identify biomarkers in the dog. To address this issue, we developed an Affymetrix-based oligonucleotide microarray capable of monitoring the expression of thousands of canine genes in parallel. The custom canine array contains 22,774 probe sets, consisting of 13,729 canine and 9045 human-derived probe sets. To improve cross-species hybridization with human-derived probes, the detection region was moved from the variable 3' UTR to the more homologous coding region. Testing of this strategy was accomplished by comparing hybridization of naive dog liver RNA to the canine array (coding region design) and human U133A array (standard 3' design). Although raw signal intensity was greater with canine-specific probe sets, human-derived probes detected the expression of additional liver transcripts. To assess the ability of this tool to detect differential gene expression, the acute phase response was examined in beagle dogs given lipopolysaccharide (LPS). Hepatic gene expression 4 and 24 h post-LPS administration was compared to gene expression profiles of vehicle-treated dogs (n=3/group). Array data was consistent with an acute inflammatory response, with transcripts for multiple cytokines and acute phase proteins markedly induced 4 h after LPS challenge. Robust changes in the expression of transcripts involved with glucose homeostasis, biotransformation, and extracellular matrix remodeling were observed 24 h post-dose. In addition, the canine array identified several potential biomarkers of hepatic inflammation. Strong correlations were found between gene expression data and alterations in clinical chemistry parameters such as serum amyloid A (SAA), albumin, and alkaline phosphatase (ALP). In summary, this new genomic tool successfully detected basal canine gene expression and identified novel aspects of the acute phase response in dog that shed new light on mechanisms underlying inflammatory processes.

Alterations in hemograms and serum biochemical analytes of steers after prolonged consumption of endophyte-infected tall fescue.

The objective of the current study was to delineate changes that occur in serum analytes and blood cellular elements in cattle that graze endophyte-infested (Neotyphodium coenophialum) tall fescue. Tall fescue is grown on more than 35 million acres (14.2 million ha) of pasture in the United States, and three-fourths of the pastures are infected with the endophyte at a 60% or greater level. Tall fescue toxicosis caused by endophyte-produced ergot alkaloids continues to be the most important grass-related disease in the United States, in terms of economic loss to animal producers. However, the agronomic attributes of tall fescue make it an attractive forage species because of its ability to withstand cool temperatures, drought, poor soil conditions, and intensive defoliation from herbivore species, including insects. Tall fescue toxicosis is a complex disease and the need exists to understand the mechanisms of the toxic effects in order to institute effective, prophylactic control measures. Our group previously reported changes that occur in serum biochemical analytes of cattle that graze endophyte-infected tall fescue. An additional year's worth of data have been added, strengthening and corroborating these data. Consistent and significant changes associated with tall fescue toxicosis during the 3-yr study included decreased serum concentrations of cholesterol, globulin (increased albumin/globulin ratio), prolactin, total protein, and copper. The activity of alanine aminotransferase was decreased in serum, whereas an increase in serum concentrations of creatinine and total bilirubin occurred. The present report also documents comparative hemograms of cattle that grazed endophyte-infected or endophyte-free tall fescue over a prolonged period. The mean erythrocyte counts were increased in cattle that grazed endophyte-infected tall fescue, whereas mean corpuscular hemoglobin and mean corpuscular volume were decreased, as were mean eosinophil counts. Thus, repeatable changes have been identified that occur in serum biochemical and blood cellular values of cattle grazing endophyte-infected tall fescue that will aid in understanding the pathogenesis of the disease. In addition, these consistently altered parameters can be used to assess the effectiveness of potential prophylactic treatments.

Evaluation of plasma time-activity curves of technetium-99m-mebrofenin for measurement of hepatic function in dogs.

In this study, plasma time-activity curves of 99mTc-mebrofenin were used to quantify hepatic function in dogs before and after induction of hepatic damage using a hepatotoxic agent. Nine dogs were determined to be healthy on the basis of physical examination, laboratory data and hepatic imaging. Plasma samples were collected 1, 3, 5, 7, 9, 15, 20, 30, 40, 50, and 60 minutes following a peripheral venous injection of 111-222 MBq (3-6 mCi) of 99mTc-mebrofenin. The area under the plasma time-activity curve (AUC) was calculated using two different methods and compared to direct measurement of the hepatic extraction efficiency. First pass hepatic extraction efficiency of 99mTc-mebrofenin was calculated from differential equation analysis of a two-compartment model following mesenteric venous injection of the radiopharmaceutical. In 7 of the original 9 dogs and 2 additional healthy dogs, plasma clearance and hepatic extraction efficiency determination were repeated following induction of hepatic injury by thiacetarsamide (3 mg/kg IV twice daily for 1 day). In one additional dog, hepatic injury was induced using carbon tetrachloride (0.3 ml/kg IP). Plasma time-activity curves of 99mTc-mebrofenin had kinetics of a two compartment model. Area under the curve was highly correlated with hepatic extraction efficiency. The AUC integrated from 1-60 minutes (AUC60) had the best correlation with hepatic extraction efficiency (r2 = 0.978, p < 0.001). A formula for calculation of hepatic extraction efficiency was derived using linear regression analysis: hepatic extraction efficiency = 105.583 - 3.099 x 10(5) x AUC60. Plasma clearance of a peripheral venous injection of 99mTc-mebrofenin is a simple, non-invasive, convenient method to quantify hepatic function which can be performed without a gamma camera.

Radiographic, histologic, and cytologic lesions associated with mutations in the fitness1(4226SB) locus of mice.

BACKGROUND AND PURPOSE: Previous investigation of fitness1(4226SB) mice revealed growth retardation and microcytic, hypochromic anemia with functional iron deficiency. Serum biochemical analysis suggested protein-losing enteropathy and liver dysfunction. METHODS: Radiography was done to assess lumbar bone lesions in mice hemizygous for fitness1 (fit1) [c fit1(4226SB/Df(c Mod2 sh1)26DVT] and age-matched sibling controls [c(ch)+/c(ch)+] at 40 or 60 days of age. Macroscopic and microscopic lesions were evaluated at necropsy. Bone marrow was examined cytologically to evaluate hematopoietic lesions. RESULTS: Mice hemizygous for fit1 had radiographically evident lumbar vertebral abnormalities, including various degrees of vertebral body fusion, with loss of intervertebral disk spaces and mild, generalized osteopenia. All mutant mice had scoliosis. Several mutant mice had lordosis and/or kyphosis of variable severity and mild subluxation at the lumbosacral junction. Marked splenomegaly and mild cardiomegaly were evident, and bone marrow color ranged from normal to slightly pale. The spleen had marked extramedullary hematopoiesis; lumbar vertebrae contained microscopic lesions that corresponded to the radiographic lesions. Cytologic examination of bone marrow revealed normocellular to hypocellular status, with mild to moderate erythroid hypoplasia characterized by mild increase in the myeloid-to-erythroid cell ratio, decreased percentage of erythroid precursors, and slight increase in percentage of myeloid precursor cells. CONCLUSIONS: Mutations in fit1 directly or indirectly cause alteration(s) in blood, organs of hematopoiesis (bone marrow, spleen, and liver), heart, and vertebral column, and suggest that this mouse may be a good model for study of scoliosis and relationships between iron metabolism and bone growth.

Alterations in bovine serum biochemistry profiles associated with prolonged consumption of endophyte-infected tall fescue.

Tall fescue (Festuca arundinacea) is a forage grass that is widely used in pastures in the eastern US for cattle, sheep and horses. The endophytic fungus Neotyphodium coenophialum is endemic in tall fescue pastures in the US. The turfgrass industry intentionally infects fescue cultivars with strains of the fungus to impart desirable growth and disease tolerance qualities to the plants. In contrast, ergot and pyrrolizidine alkaloid toxins produced by fungus-infected plants have been incriminated causally in bovine tall fescue toxicosis, a poorly defined syndrome of morbidity that occurs in cattle that consume endophyte-infected tall fescue (E+TF). We compared the serum biochemistry profiles from cattle that grazed E+TF with those from control cattle that grazed on endophyte-free tall fescue (E-TF). Cattle were bled on 7 dates from April 1 to August 30, 1996 and on 5 dates from May 1 to July 30, 1997. Cattle that grazed E+TF retained rough winter haircoats and had lesser weight gains, typical of tall fescue toxicosis, compared to those grazing E-TF. They had decreased activities of alkaline phosphatase and alanine aminotransferase. Compared to controls, they had lower values for serum prolactin and globulin concentrations. The concentration of creatinine and the albumin/globulin ratio were increased in the cattle grazing E+TF. Isozyme determination of alkaline phosphatase indicated that the decrease in serum activity of cattle grazing E+TF was due to decreases in both intestinal and bone isozymes. Serum protein electrophoresis indicated that the decrease in serum globulin concentration was due to decreases in both alpha and gamma globulin fractions of this protein. The data collected in these experiments add to our understanding of the alterations that occur in the serum chemistry profiles when cattle consume E+TF for prolonged periods of time.

Lymphosarcoma with leukemic blood profile in a Savannah monitor lizard (Varanus exanthematicus).

A 6-mo-old male Savannah monitor lizard (Varanus exanthematicus) was presented for lethargy and anorexia of 7 days duration. Physical examination revealed a slightly raised subcutaneous mass (1 cm diameter) in the left scapular area. Fine-needle aspiration cytology of the mass revealed a population of immature, pleomorphic lymphoid cells consistent with lymphosarcoma. A hemogram indicated marked leukocytosis (465,000 cells/microl) characterized by extreme lymphocytosis and many circulating lymphoid blast cells. The lizard was euthanized at the owner's request. Necropsy revealed severe hepatomegaly and multiple raised, ulcerated mural masses in the gastrointestinal tract. There were many raised, poorly demarcated tan foci in all the parenchymal organs. Histopathologic examination confirmed infiltration of all parenchymal organs by neoplastic lymphoid cells. Transmission electron microscopic examination failed to identify viruses within the neoplastic cells. A literature review revealed few reports of squamate leukemia and lymphosarcoma and none in Savannah monitor lizards.

Chronic pulmonary hypertension--the monocrotaline model and involvement of the hemostatic system.

Monocrotaline (MCT) is a toxic pyrrolizidine alkaloid of plant origin. Administration of small doses of MCT or its active metabolite, monocrotaline pyrrole (MCTP), to rats causes delayed and progressive lung injury characterized by pulmonary vascular remodeling, pulmonary hypertension, and compensatory right heart hypertrophy. The lesions induced by MCT(P) administration in rats are similar to those observed in certain chronic pulmonary vascular diseases of people. This review begins with a synopsis of the hemostatic system, emphasizing the role of endothelium since endothelial cell dysfunction likely underlies the pathogenesis of MCT(P)-induced pneumotoxicity. MCT toxicology is discussed, focusing on morphologic, pulmonary mechanical, hemodynamic, and biochemical and molecular alterations that occur after toxicant exposure. Fibrin and platelet thrombosis of the pulmonary microvasculature occurs after administration of MCT(P) to rats, and several investigators have hypothesized that thrombi contribute to the lung injury and pulmonary hypertension. The evidence for involvement of the various components of the hemostatic system in MCT(P)-induced vascular injury and remodeling is reviewed. Current evidence is consistent with involvement of platelets and an altered fibrinolytic system, yet much remains to be learned about specific events and signals in the vascular pathogenesis.

Validation of deconvolutional analysis for the measurement of hepatic function in dogs with toxic-induced liver disease.

The extraction of the hepatobiliary radiopharmaceutical 99mTc-mebrofenin (Choletec) by the liver can be used to evaluate the severity of hepatocellular disease. The hepatic parenchymal cells extract mebrofenin from the blood by the same active transport mechanism as bilirubin. The ability of the liver to extract 99mTc-mebrofenin is a measure of hepatic parenchymal cell function. In this study, we induced hepatocellular disease by administration of a hepatotoxic drug and compared a direct method of determining the hepatic extraction of 99mTc-mebrofenin to hepatic extraction fraction derived from deconvolutional analysis. We also compared both methods of calculating the hepatic extraction of 99mTc-mebrofenin to liver histopathology. Hepatic extraction fraction derived from deconvolutional analysis correlated very well to the direct measurement technique (R=0.922, p < 0.001). Both methods of determining hepatic extraction correlated well to quantitative histopathology, having the same correlation coefficient and p values. (R=-0.833, p=0.003). As the hepatic extraction 99mTc-mebrofenin decreased, the severity of the histopathologic lesions of the liver increased in a linear fashion. There was a significant correlation of the hepatic excretion T1/2 to quantitative histopathology (R=0.949, p < 0.001). The hepatic excretion T1/2 increased as the severity of the histopathologic lesions of the liver increased. Hepatic extraction (HEF) and excretion of 99mTc-mebrofenin are good predictors of the severity of hepatocellular damage in toxic induced liver disease. This study helps validate the premise that HEF derived from deconvolutional analysis is a good predictor of the actual first pass hepatic extraction of 99mTc-mebrofenin.

Hepatic extraction efficiency of technetium-99m-mebrofenin in the dog with toxic-induced acute liver disease.

UNLABELLED: Extraction of the hepatobiliary radiopharmaceutical 99mTc-N-(3-bromo-2,4,6-trimethyacetanilide) iminodiacetic acid (mebrofenin; Choletec, Squibb Diagnostic, Princeton, NJ) by the liver may be used as an index of hepatocellular function. The hepatic parenchymal cells extract mebrofenin from the blood using the same active transport mechanism as bilirubin. METHODS: In this study, we induced hepatocellular disease by administering a hepatotoxic drug and compared the hepatic extraction efficiency (HEE), measured directly from an afferent injection of 99mTc-mebrofenin, to quantitative histopathology and to serum biochemistry analysis. RESULTS: The baseline HEE was 95.9% +/- 2.71% (mean +/- s.d.). Dogs that were affected by the hepatotoxic drug had reduced HEE. HEE correlated well to the severity of histologic lesions (r = -0.83, p = 0.003). HEE also correlated well to the increases in the activities of alanine aminotransferase (ALT; r = -0.85, p = 0.002) and aspartate aminotransferase (AST; r = -0.89, p = <0.001), the concentration of fasting bile acid (r = -0.97, p = <0.001), bilirubin (r = -0.92, p = <0.001) and, to a lesser degree, to the activities of alkaline phosphatase (Alk Phos; r = -0.73, p = 0.016). HEE had higher correlation coefficients to the serum biochemistry analysis than did the quantitative liver histopathology. CONCLUSION: Hepatic extraction of 99mTc-mebrofenin is a good predictor of the severity of hepatocellular damage in toxic-induced liver disease.

Essential thrombocythemia in a dog: case report and literature review.

A 10.5-year-old, castrated male shih tzu was presented for evaluation of weakness, pica, and pallor of the mucous membranes. A hemogram indicated an inflammatory leukogram and a regenerative anemia with spherocytosis and thrombocytosis. The dog responded well to conservative therapy for immune-mediated hemolytic anemia (IMHA). However, the thrombocytosis did not resolve. Serial hemograms were characterized by persistent thrombocytosis (platelet count, 577,000 to 1,200,000/microl) with abnormal platelet morphology. A systematic investigation ruled out causes of physiological and reactive thrombocytoses. A diagnosis of essential thrombocythemia was made by fulfilling the criteria of the Polycythemia Vera Study Group of the National Cancer Institute. The marked thrombocytosis was nonresponsive to hydroxyurea therapy. The dog remains healthy despite the marked increase in the number of circulating platelets. A review of causes of thrombocytoses in humans and animals is presented, and the criteria for diagnosis of essential thrombocythemia are examined.

Clinical, cytologic and histopathologic manifestations of protothecosis in two dogs.

Two cases of sudden onset of blindness associated with ocular protothecosis in dogs are reported. Both were adult, spayed female, mixed-breed dogs that lacked the usual clinical signs of systemic infection with Prototheca species. Physical abnormalities at the time of presentation were limited to the affected eyes which had serous discharge, hyperemic conjunctiva, and aqueous flare. The pupillary light reflexes were slow, and the menace reflexes were absent. Both dogs had glaucoma. Results of complete blood counts and serologic titres for antibodies to Blastomyces dermatitidis and Histoplasma capsulatum were within reference intervals. Protothecosis was diagnosed by cytologic analysis of vitreous humor and was confirmed at necropsy. These two cases were unusual because of their presenting signs and prolonged course of disease progression.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis of canine urinary proteins for the analysis and differentiation of tubular and glomerular diseases.

A preliminary investigation was performed to evaluate the use of a new, noninvasive technique for the localization of canine renal lesions by electrophoresis of urinary proteins. Urine specimens from six clinically healthy, nonproteinuric dogs and 12 dogs with persistent proteinuria were examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). Urine electrophoretic patterns of proteinuric dogs were classified as glomerular (n = 4), tubular (n = 2), or mixed (glomerular and tubular) (n = 6), based on the number and molecular weight of the silver-stained protein bands. Renal tissues from biopsies or necropsies were obtained from eight of the dogs with proteinuric disease. Interpretation of seven of eight electrophoretograms agreed with the histologic interpretation of renal lesions. We concluded SDS PAGE is a potentially valuable technique for detection and localization of renal lesions in dogs with proteinuric disease.

Repeated physical and cytologic characterizations of subcutaneous postvaccinal reactions in cats.

OBJECTIVES: To examine local reactions and short-term cytologic responses of cats to administration of rabies virus (RV); FeLV; and combined feline rhinotracheitis, calicivirus, and panleukopenia virus (FRCPV) vaccines. ANIMALS: 9 healthy 6- to 7-month-old specific-pathogen-free cats. PROCEDURE: One-milliliter doses of the aforementioned vaccines were administered SC (at different sites) to healthy, specific-pathogen-free cats. Each cat also received 1 ml of sterile saline solution SC as a control. Injection sites were visually examined and palpated daily for 4 weeks. Palpable lesions were measured by use of calipers. Temperature of the vaccination sites was measured weekly by use of a thermocouple. Aspirates were taken from vaccination sites weekly, and smears were submitted for cytologic analysis. RESULTS: There were no significant differences in lesion surface temperature among injection sites at any time. Injections of saline solution and FeLV vaccine resulted in no palpable lesions. The FRCPV vaccine elicited a minor reaction in 1 of the 9 cats. The RV vaccine caused palpable lesions in all cats. Smears of the aspirates from the sites of saline injection were poorly cellular. Cellularity of aspirates from the sites of FRCPV and FeLV vaccinations was moderate at week 1, and decreased with time. Inflammatory infiltrates were composed principally of lymphocytes, with fewer neutrophils and macrophages. In contrast, cellularity of aspirates from RV vaccination sites increased for 21 days and was characterized by increasing numbers of lymphocytes and macrophages. CONCLUSIONS: RV vaccine used in this study induced palpable lesions in many cats. In contrast, FRCPV and FeLV vaccines elicited less severe lesions. CLINICAL RELEVANCE: Subcutaneous administration of killed virus vaccines in cats may result in palpable lesions that are detected by clients or clinicians. Aspiration cytologic examination may reveal a different characteristic pattern of cells that is dependent on the individual vaccine and time elapsed from vaccination.

Bacterial endotoxin enhances the hepatotoxicity of allyl alcohol.

Lipopolysaccharide (LPS), or bacterial endotoxin, causes liver damage at relatively large doses in rats. Smaller doses, however, may influence the response to other hepatotoxicants. The purpose these studies was to examine the effect of exposure to relatively all doses of LPS on the hepatotoxic response to allyl alcohol, which causes periportal necrosis in laboratory rodents through an known mechanism. Rats were pretreated with LPS (100 micrograms/kg) 2 hr before treatment with a minimally toxic dose of allyl alcohol mg/kg), and liver toxicity was assessed 18 hr later from activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in plasma and from histologic changes in liver sections. Plasma ALT and AST activities were not elevated significantly in rats treated with vehicle, LPS, or allyl alcohol alone, but pronounced increases were observed in rats treated with LPS and allyl alcohol. Significant liver injury occurred as early as 2 hr after allyl alcohol treatment in LPS-pretreated rats and peaked at 6 hr. LPS treatment did not affect the activity of alcohol dehydrogenase and did not affect the rate of production of NADH in isolated livers perfused with allyl alcohol; thus, LPS does not appear to increase the metabolic bioactivation of allyl alcohol into acrolein. On the other hand, pretreatment with 4-methylpyrazole, an inhibitor of alcohol dehydrogenase, abolished the hepatotoxicity of allyl alcohol in LPS-treated rats, indicating that production of acrolein was needed for LPS enhancement of the toxicity of allyl alcohol. Pretreatment of rats with gadolinium chloride (10 mg/kg), a known inactivator of Kupffer cell phagocytic function, decreased LPS augmentation of the response to allyl alcohol. These data indicate that LPS markedly enhances the hepatotoxic response to allyl alcohol. Furthermore, the results suggest that the LPS-induced enhancement of allyl alcohol hepatotoxicity occurs through a Kupffer cell-dependent mechanism.

Gadolinium chloride pretreatment protects against hepatic injury but predisposes the lungs to alveolitis after lipopolysaccharide administration.

Exposure to lipopolysaccharide (LPS) can result in multi-organ failure and death. After an intravenous injection of LPS into rats, neutrophils (PMN) rapidly accumulate in the liver sinusoids and pulmonary vasculature, and PMN play a critical role in producing both hepatic and pulmonary injury. Kupffer cells (KC), the resident macrophages of the liver, phagocytose LPS and produce inflammatory mediators which may be chemotactic and stimulatory for PMN. The purpose of this study was to determine whether inhibition of KC function affects PMN accumulation and the development of parenchymal injury in the liver and lungs after systemic administration of LPS. Female, Sprague-Dawley rats (180-230 g) were pretreated with either gadolinium chloride-6H2O (GdCl3; 10 mg/kg, intravenously), to inactivate KC, or saline vehicle 24 h before receiving either LPS (4 mg/kg, intravenously) or saline vehicle. Rats were killed 1.5, 6, and 24 h after LPS administration. In a preliminary study, exposure to GdCl3 decreased uptake of carbon in the liver, indicating inhibition of phagocytosis by KC. Ninety minutes after administration of LPS, PMN accumulated in the livers of LPS-treated rats, and this effect was not altered by pretreatment with GdCl3. Similarly, exposure to LPS resulted in PMN accumulation in the pulmonary tissue, which was unaffected by GdCl3 pretreatment. Exposure to GdCl3 before LPS administration resulted in a significant increase in the number of PMN recovered by bronchoalveolar lavage at 24 h, indicating diffuse acute alveolitis. LPS-induced hepatic injury was prevented by pretreatment with GdCl3; however, the increased wet lung/body weight ratio observed after LPS administration was unaffected by GdCl3. These results confirm that inactivation of KC protects against hepatic injury and extend this finding by ruling out inhibition of hepatic PMN accumulation as a mechanism for this effect. The data also suggest that treatment with GdCl3 predisposes the lungs to alveolitis during systemic exposure to LPS.

Thrombin is a distal mediator of lipopolysaccharide-induced liver injury in the rat.

Previous results demonstrated that rats given Escherichia coli lipopolysaccharide (LPS; 4 mg/kg, i.v.) experience hepatocellular necrosis that begins within 4 hr and that prior treatment with anticoagulants (e.g., heparin) which target thrombin prevents the liver injury. In this study, hepatocellular injury, as marked by increased plasma alanine aminotransferase (ALT) activity and histologic changes, was prevented when heparin or hirudin was administered to rats shortly before the onset of injury. These results suggest that thrombin is a critical mediator that acts distally in the series of inflammatory events that culminates in hepatocellular damage. To explore further this hypothesis, livers isolated from rats 2 hr after LPS administration were perfused with various media. Perfusion of livers with medium comprising diluted blood from heparin-treated donors resulted in no release of ALT activity. By contrast, perfusion with similar medium anticoagulated with ancrod, which prevents clotting by depleting fibrinogen but does not inhibit thrombin, resulted in hepatocellular injury evidenced as a time-dependent appearance of ALT activity in the medium. Moreover, when livers from rats treated 2 hr previously with LPS were perfused with buffer to which thrombin had been added, injury resulted. No injury resulted when thrombin was omitted from the buffer or when livers from saline-treated rats were used. These results indicate that thrombin is a critical and distal mediator of LPS-induced liver damage and contributes to hepatocellular injury through a mechanism that is independent of clot formation. Furthermore, inflammatory events triggered by LPS exposure are a prerequisite for thrombin-induced injury.

The thrombin inhibitor, hirudin, attenuates lipopolysaccharide-induced liver injury in the rat.

The administration of gram-negative bacterial lipopolysaccharide (LPS) to rats results in hepatic parenchymal cell injury within 6 hr. The coagulation system is critical to the pathogenesis, but previously reported results suggested that its critical role is independent of insoluble clot formation and that thrombin may be a key mediator of liver injury. To test the hypothesis that thrombin is involved in LPS-induced liver injury, animals were treated with the selective thrombin inhibitor, hirudin. The hirudin treatment regimen effectively inhibited thrombin, as evidenced by prolonged activated partial thromboplastin time and by maintenance of plasma fibrinogen concentrations in LPS-treated rats. Treatment with hirudin prevented LPS-induced liver injury, assessed by plasma alanine aminotransferase activity and histological evidence of hepatocellular necrosis. Previous studies have shown that LPS exposure results in the accumulation of neutrophils and platelets within the liver and that both of these cell types are critical for the development of LPS-induced liver injury. Hirudin attenuated in part the decrease in blood platelet concentration that accompanied LPS administration, but did not alter hepatic platelet or neutrophil accumulation. These results support the hypothesis that thrombin is required for hepatic injury from LPS exposure, but that it does not act by promoting the accumulation of platelets or neutrophils within the liver.

Gadolinium chloride treatment attenuates hepatic platelet accumulation after lipopolysaccharide administration.

Intravenous administration of LPS to rats results in the accumulation of both neutrophils and platelets in the liver and the development of midzonal hepatocellular necrosis. The development of liver injury entails contributions from both cellular and soluble mediators, including neutrophils, platelets, Kupffer cells, tumor necrosis factor-alpha (TNF-alpha), and components of the coagulation system. Much remains unknown about the interactions among these mediators in the pathogenesis of liver injury in vivo. Accordingly, we conducted studies with gadolinium chloride (GdCl3), an agent that inhibits Kupffer cell phagocytosis, to evaluate the role of Kupffer cells in lipopolysaccharide (LPS)-mediated liver injury, elevation in plasma TNF-alpha activity, thrombocytopenia, hepatic platelet accumulation, and activation of the coagulation system. Female Sprague-Dawley rats were pretreated with GdCl3-6H2O (10 mg/kg, i.v.) or saline vehicle 24 h before the administration of LPS (4 mg/kg, i.v.) or saline vehicle. In a preliminary study, this GdCl3 treatment regimen decreased the clearance of colloidal carbon from blood, indicating inhibition of Kupffer cell phagocytosis. Pretreatment with GdCl3 attenuated LPS-induced liver injury, monitored as increased plasma alanine aminotransferase and isocitrate dehydrogenase activities and histologic analysis. Electron micrographs of livers from rats treated with LPS revealed platelets within the sinusoids as well as Kupffer cells with phagolysosomes containing material resembling platelets. Pretreatment with GdCl3 attenuated LPS-induced thrombocytopenia and hepatic platelet accumulation, as measured by radiolabeled platelets. Treatment with GdCl3 did not, however, alter the elevation in plasma TNF-alpha activity or the activation of the coagulation system, as evidenced by a decreased in plasma fibrinogen concentration. These results suggest that Kupffer cells contribute to LPS-induced hepatic platelet accumulation and raise the possibility that protection against LPS-induced hepatic injury by Kupffer cell inactivation may be due at least partly to decreased deposition of platelets within the liver.