* Laboratory of Molecular Toxicology/National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina (RTP, NC); Laboratory of Experimental Pathology, National Institute of Environmental Health Sciences, RTP, NC;
Biostatistics Branch, National Institute for Occupational Safety and Health, Morgantown, West Virginia;
TNO Nutrition and Food Research, Zeist, The Netherlands; ¶ Laboratory of Computational Biology and Risk Assessment, National Institute of Environmental Health Sciences, RTP, NC; || Pathology and Physiology Research Branch, National Institute for Occupational Safety and Health, Morgantown, West Virginia; ||| Toxicology and Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan; and |||| Toxicology and Molecular Biology Branch, National Institute for Occupational Safety and Health, Morgantown, West Virginia
Received September 22, 2003; accepted November 28, 2003
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ABSTRACT |
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Key Words: immunology; pathology; spleen; thymus; lymph node; histopathology; immunopathology; risk assessment.
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INTRODUCTION |
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Other studies suggested that inclusion of a functional test, in addition to pathology endpoints, might be more sensitive at detecting potential immunotoxicants than was histopathology alone (reviewed in van Loveren et al., 1996). The use of enhanced/expanded histopathologic evaluation as a primary screen would be advantageous for two reasons. First, potential immunotoxicity could be assessed during routine toxicology studies, such as the 28-day rodent study, without the need for additional animals. Secondly, no specific expertise in immune-function testing or equipment would be required. For screening tests to be meaningful, however, it is important to identify both the limitations of the test, as well as the concordance (i.e., how accurately the test predicts the interest of concern). The latter usually involves more than a simple qualitative answer, and quantitative issues may need to be considered (i.e., sensitivity differences between the screening test and the interest of concern). In addition to sensitivity, potential inter- and intralaboratory variability needs to be considered.
The predictive value of various laboratory tests and their potential correlation with morphological change has been investigated for a number of other target organs/systems. The validation process for these studies has required the availability of relatively large databases of tested compounds, where the experimental procedures were uniformly applied. One example of such an effort is the comparison of serum enzyme levels that measure hepatic and renal function, microsomal enzymes, organ weight, and histopathological evaluation in the liver (Amacher et al., 1998; Travlos et al., 1996
). Waters et al. (2003)
recently described a large-scale initiative to investigate the correlation between altered gene expressions, as evaluated using microarray techniques, with specific parameters from standard toxicology studies including a thorough histopathological evaluation.
To help validate the enhanced/expanded histopathology, including grading of lymphoid organs in mice, a workgroup was formed consisting of four pathologists from academia, government, and industry. In the ICICIS study (1998), it was noted that training of the pathologists in the structured histopathology assessment scheme greatly added to the sensitivity of the histopathology. Thus, for all pathologists to have the same understanding of the evaluation criteria, the individual with the most expertise in immunopathology presented the details of standardization of grading of the tissues. Once the evaluation criteria were agreed upon, histopathology slides from 10 separate chemical studies were selected from those that had been conducted as part of the National Toxicology Program (NTP) testing efforts. We analyzed the histopathology results from these studies in order to evaluate the consistency among pathologists and the sensitivity of the individuals, and combined histological endpoints for hazard identification and dose-response assessment.
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MATERIALS AND METHODS |
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Within the NTP, preliminary dose-range studies are routinely conducted prior to immunotoxicity studies, and, for the reasons stated above, the highest dose is set slightly below the MTD and at doses where body weight changes would not be 10%. Histopathological examination and data analyses also included tissue from the positive controls, run as reference compounds for the immunotoxicity studies. Positive control chemicals, which included cyclophosphamide, methotrexate, and sodium arsenite, were examined at only one dose level and were performed in conjunction with 6 of the 10 chemicals in the data set; cyclophosphamide was used in four experiments. Detailed information on each of the chemicals and doses used in these studies can be found in Supplementary Appendix A, available online at the journal's Web site (www.toxsci.oupjournals.org). Additional details on the duration, route of exposure, and other study parameters can be found in the following references: Burns et al., 1994
; Cao et al., 1990
; Karrow et al., 2000a
,b
; NTP 1988a
,b
, 1989
; Phillips et al., 1997
; Sikorski et al., 1989
.
Tissue preparation and histological examination.
All tissues represented archived samples from previous NTP immunotoxicity studies using female B6C3F1 mice. Tissues were collected at the termination of each study under GLP guidelines, according to standard operating procedures developed under an NIEHS contract. All animals were weighed and then humanely euthanized, using carbon dioxide inhalation. Thymus, spleen, and the complete chain of the superior mesenteric lymph nodes were collected and fixed in 10% neutral-buffered formalin. The thymus and spleen were weighed prior to fixation. One middle cross-section from the spleen, both lobes of the thymus, and the mesenteric lymph nodes were embedded in paraffin, and 5-6 micron sections were prepared and stained with hematoxylin and eosin (H&E) for histopathological evaluation. For each pathologist, a standardized slide set was generated for each of the chemicals.
The pathologists did not know the identity of the test chemical at the time of evaluation. However, for each slide set, the pathologist received data identifying positive control, negative control, and chemical concentrations, as well as organ weights. As discussed above, a working group was convened for establishment and standardization of the endpoints to be evaluated. All of the participating individuals were toxicologic pathologists with extensive experience in laboratory animal studies. However, only one pathologist was considered to have specific expertise in immunopathology and this individual provided training in the evaluation of tissues for the study. Following subsequent microscopic evaluation of the lymphoid organs from treatment groups of two randomly selected compounds, telephone discussions among the pathologists ensured that every participant understood the usage of the criteria and provided an opportunity for necessary changes to be made.
A semiquantitative assessment was used to estimate the histopathological changes within different anatomical compartments of the lymphoid tissues. The diagnostic terms for identifying and evaluating the histopathologic changes were those recommended by Kuper et al. (2000; 2002
). The grading scheme consisted of ordinal categories ranging from "0" (no effect) to "4" (severe effect) and an indicator as to whether the effect was increased or decreased relative to normal tissue. Histopathological evaluations took into consideration changes in cell density or change in the anatomical compartment size. The pathologists were also instructed to add comments that were not quantifiable but considered important for proper histopathological assessment, such as "focal increased cellularity of outer thymic cortex" and "increased tingible body macrophages in the thymic cortex." Remarks were made on quality of the sections (i.e., plane of sectioning that influenced the size of lymphoid follicles, staining quality, thickness of section, and on quantity of tissue present in section), bleeding that may have influenced the morphology of the red pulp of the spleen or other compartments, and suggested usage of immunohistochemical staining for better characterization of the changes. Four compartments were evaluated in the lymph node: grade of cellularity in the follicles, paracortical areas, medullary cords, and sinuses. Five compartments were evaluated in the spleen: cellularity of periarteriolar lymphoid sheaths (PALS), lymphoid follicles, marginal zone, red pulp, and the total number of germinal centers in each section. Three compartments were evaluated in the thymus: cortex cellularity, medullary cellularity, and the cortico-medullary ratio. Following the microscopic examination, the coded data were transferred to an electronic format, and a formal quality control was conducted on the data entry of the entire set of findings.
Statistical Methodology: Analytic approach.
Although efforts were made that all pathologists would have the same level of understanding in the use of the expanded histopathologic nomenclature, complete agreement was not expected because of the subjective nature of the evaluation. This incomplete agreement, or variation among pathologists, can occur for several reasons. First, given the complex nature of pathological assessment, each pathologist is not going to weigh every facet of the tissue in exactly the same manner. The grading assigned to a given tissue section by a given pathologist will ultimately reflect that pathologists individual criteria, which are influenced by experience and biases. Furthermore, there may be slight variation between the actual set of tissue sections that was evaluated by each pathologist and a certain amount of random error, or unexplained variation, which will contribute to the incomplete agreement. Conceptually, agreement can be evaluated by examining the associations, biases, or tendencies to grade specimens systematically higher or lower among the pathologists. Given these multiple components of agreement, a single index of agreement cannot fully describe the data and statistical modeling has been advocated (Agresti, 1992; Uebersax, 1992
). Factor analysis methods (Dunn, 1989
) were used to estimate the associations and analysis-of-variance methods (Ubersax, 1992
) to evaluate biases. Factor analysis was used to estimate the association of each pathologist with a so-called latent factor. The latent factor can be thought of as representing the underlying trait that is being estimated, i.e., the pathological trait of a given tissue specimen, while each grade assignment can be viewed as an imperfect representation of the underlying pathology that is subject to the pathologist's biases and random error. A "common factor" analytical approach was used to assess the association among the pathologists with the underlying latent factor. This common factor is ultimately derived from the matrix of correlation coefficients among all of the pathologists. The factor loadings, or output, generated from the common factor approach, are essentially the correlation coefficients of each pathologist with the underlying common factor. Factor loadings that are positive and high (1 [one] is the maximum value) are indicative of high levels of agreement. The square of these factor loadings represents the proportion of variance in each pathologist's ratings that is accounted for by the underlying common factor. The higher these values, the more in agreement the pathologists are with the underlying common factor. These types of agreement statistics have been used successfully in other disciplines, most notably psychology and sociology, to predict changes in dependent variables using multiple explanatory variables (Hair et al., 1992
). Factor analysis has also been used to model relationships between immune-function endpoints and host resistance following exposure to the prototypical immunosuppressant, dexamethasone (Keil et al., 1999
, 2001
).
The agreement among pathologists on each tissue parameter was assessed by calculating the correlation coefficient between each pathologist and the common factor, as a function of the specific tissue parameter that was scored and as a function of the dose of the chemical that was applied. These analyses were all performed without regard to the specific test chemical. However, for some analyses, the positive controls were evaluated independently to assess agreement when effects would likely be the most severe.
To assess bias among the pathologists, we utilized an analysis-of-variance model that provided estimates of the mean grades of each tissue compartment as a function of the individual pathologist and the specific dose of each of the chemical compounds. These estimates allow assessment of particular biases, apparent by pathologist on a given tissue compartment, which would not be reflected in the above analysis examining associations. For example, the factor analytic approach would indicate very high agreement among pathologists, even if one pathologist consistently rated a given histological specimen one unit higher than another. Using an analysis-of-variance approach, this bias would be apparent in the plots of the mean grades. Given the large amount of data in this experiment and the high statistical power to detect small differences among pathologists and doses, a classic hypothesis-testing paradigm was not used; a more descriptive analysis of the results was applied. For these analyses, data from all test chemicals were analyzed together; however, each chemical was included in the model as a random effect to assess variability in scoring across different chemicals (Littell et al., 1996).
Data.
The pathologists' ratings were on a scale of 0 to 4, as an ordinal index of the severity of the lesion, with an additional categorical designation for either an increase or a decrease in grading. These data were converted for each compound to a scale in which those ratings with a decreased grade designation were assigned a negative value and those with an increased grade designation were assigned positive numbers. Thus, the data regarding pathological ratings are on a scale ranging from -4, to +4, with 0 indicating no effect. The high dose and the control groups were examined first for histopathological lesions. If there was no effect observed in any of the animals getting the high dose, a value of zero was assigned to animals in the low- and medium-dose groups. There were several instances of missing data where either the tissue section was unreadable or had been lost. Missing values are represented as blanks in the data set.
Computational methods.
The data were analyzed using SAS/STAT software, Version 8.2, of the SAS System for Windows (SAS Institute, Cary, NC). Factor analysis was performed utilizing Proc Factor, with the iterated principle factor method (prinit) and the number of factors set to 1. Correlations among the pathologists with the common factor scores for both tissue compartment and dose were calculated using Proc Corr. Mixed model analyses of variance were performed on each tissue parameter using Proc Mixed. Random effects in the mixed model included animal, since each animal was evaluated by each pathologist and for each test chemical. Nonparametric tests of each pathologist, for a trend to assess whether the individual tissue parameters would show dose-responsive effects of the compounds, and were performed using the Jonckheere-Terpstra option in Proc Freq.
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RESULTS |
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The analyses examining agreement as a function of chemical dose is shown in Figure 2. There is a general trend toward greater agreement for increasing doses of chemicals examined. Analogous to observations made when examining positive controls and test chemicals, this implies that the level of agreement increases with the severity of the lesion and that subtle changes are more difficult to detect. As demonstrated for the relative level of agreement between individual pathologists, when agreement was examined as a function of dose, pathologist #2 had the highest level of agreement with the common factor.
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DISCUSSION |
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Within the thymus, changes in cortical cellularity were readily detected and provided the highest degree of agreement. This would indicate that alterations in this endpoint are readily discernable or, as suggested by De Waal et al. (1997), that cells in the cortex are generally the most sensitive to injury. The thymus is a major generative organ of the immune system and orchestrates the development of the T-lymphocyte repertoire. The thymic lobules are divided into 2 zones: a peripheral, lymphocyte-rich cortex and a central, less densely populated medulla. Changes in thymus histopathology and architecture are considered to be of particular relevance for immunotoxicity screening (Schuurman et al., 1992
). After administration of immunosuppressive agents, depletion of lymphocytes, or reduction in cellularity can occur in a diffuse manner or be limited to either the cortex or medulla. Wachsmuth (1983)
has shown that the immunosuppressive effects of a number of different pharmaceutical agents are evident on histopathological examination of the thymus, and that histological findings correlate well with thymus weight and peripheral lymphocytic counts in both the rat and dog. Altered thymic cellularity may be reported for specific compartments, and together with remarks on the presence of cell necrosis or increased numbers of macrophages with tingible bodies, can provide some indication as to the mechanism of altered cellularity (Harleman, 2000
). Decreases in cellularity may be correlated with reductions in thymus weight, which has been shown to be a predictive indicator of immunotoxicity (Luster et al., 1992b
).
The spleen is generally composed of white and red pulp, and, while composed of discrete morphological structures, the analyses presented here suggest that group morphologic assessment is difficult. The white pulp is located around a central arteriole and comprises the periarteriolar lymphoid sheaths (T-cell area), adjacent follicles (B-cell area), and marginal zone. Nonimmune functions, such as extramedullary hematopoiesis in the splenic red pulp, may complicate the evaluation of this organ. Lymph nodes are organized structures, divided into capsule, cortex (B-cell zone, composed of follicles and germinal centers), paracortex (T-cell zone), and medullary sinuses and cords. Within the spleen, measures of follicle cellularity and germinal centers provided the best agreement. As observed in the spleen, the histopathological ratings for germinal center development had the strongest agreement for all parameters examined in lymph-node tissues. This is not surprising as germinal centers are distinct, highly active structures, formed as a direct result of immune activation and characterized by extensive lymphocyte proliferation and differentiation. An immunologically activated lymph node will show complex changes involving several of its anatomic subunits that may make it difficult to evaluate. The relatively poor agreement reached with the spleen and lymph-node assessment, compared with the thymus, may be related to the fact that these organs undergo subtle changes in the different zones (Greaves, 1990) and would suggest the need for a thorough analysis and awareness of the various functions and interactions of each zone.
From a histological standpoint, assessment of the mammalian immune system is neither routine nor simple. It is composed of multiple organs and tissues, some of which are also responsible for hematopoiesis (bone marrow and spleen), others for lymphocyte maturation (thymus), and others that generate responses to antigen (lymph nodes). In addition, there are specialized tissues located throughout the body that are responsible for responding to antigens or pathogens locally (e.g., skin- lung- and gut-associated lymphoid tissues). General and parameter-specific differences were observed and ranged from good agreement to poor agreement in detecting or not detecting pathological changes. Thus, ratings for the thymus cortical cellularity were highly consistent; pathology occurred with a significant number of the test chemicals and positive controls and occurred in a dose-dependent fashion. In contrast, significant pathological changes were seen in spleen red pulp but there was a lack of consistency in the ability of the pathologists to agree on the severity of the lesion or even the direction of the change. There was good agreement among the pathologists when examining spleen follicular cellularity for the positive-control data set, but not in the test-chemical data set, suggesting that subtle changes in this compartment may be difficult to read. There was also generally good agreement among the pathologists in the lymph node sinus, lymph node paracortical area, thymus cortico-medullary ratio, thymus medullary cellularity, spleen periarteriolar lymphoid sheaths, and spleen germinal centers. However, there was a lack of pathological change detected in both the experimental and positive-control groups for these endpoints, suggesting that the chemical agents did not target these tissue compartments, or that changes in these parameters are more difficult to discern. The spleen marginal zone and lymph node medullary-cord cellularity also appear to fall into this category, with only one individual reporting histological change in each parameter. The lymph node follicular germinal center-development measure appears not to be a very sensitive indicator, because it was not affected by any chemical exposure and showed poor agreement with the pathologists in the positive-control data set. While not assessed in the statistical evaluations conducted for this study, this type of semiquantitative examination of histological parameters should also consider a careful histological description of the types of lesions, including focal alterations, cell necrosis, granulomata, etc.
Summary
In summary, the ability to identify histopathological changes in lymphoid tissues was highly dependent on the severity of the specific lesion and the tissue compartment measured in these studies. Overall, histopathological changes were most frequently and most consistently reported in the thymus cortex and medulla, and in the spleen and lymph node follicles (cellularity and germinal center development). The ability to detect dose-response trends was not readily apparent in any tissue when all compartments in that tissue were considered. Similar to the ICICIS studies (ICICIS Group Investigators, 1998), specific training in expanded immunohistopathology was an important factor in the ability to detect subtle lesions in immune tissues. The correlation between expanded immunohistopathology and lymphoid organ weights and between traditional immune function tests and host resistance assays are addressed in an accompanying study (Germolec et al., in preparation).
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at the Laboratory of Molecular Toxicology, National Institute of Environmental Health Sciences, 111 Alexander Drive, P.O. Box 12233, Research Triangle Park, NC 27709. Fax: (919) 541-0870. E-mail: germolec{at}niehs.nih.gov
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