Toxicologic pathology

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Toxicologic pathology

• Standardization

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Importance of standardization

• Organs defined in various guidelines
• BUT guidelines usually do not mention which part
  of an organ should be examined histopathologically
  
• Probability of detecting lesions related to the
  amount of the tissue examined
• For larger organs (like lung or liver) it is
  necessary to define the number of sections and
  the specific lobe / area sectioned
• Cutting direction (longitudinal or transverse),
  is in particular of importance for hollow organs
  (like the urinary bladder, uterus) in order to
  provide comparable areas of tissue for examination

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Confusion in terminology example rodent liver
tumors

• Nonmalignant masses that arise from proliferating
  initiated hepatocytes have been variously called
• neoplastic nodules,
• benign hepatoma,
• hepatoma,
• hepatocellular adenoma
• nodular hyperplasia

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STP and RITA

• Initiatives were started in the late 80s in the
  United States by the STP (Society of Toxicologic
  Pathologists)
• and in Europe by the RITA data base group
  (Registry of Industrial Toxicology Animal-data)

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Harmonization of nomenclature of proliferative
lesions in the rat

• After finalization of the WHO/IARC publication
  "International Classification of Rodent Tumours"
  and the STP publication "SSNDC Guides for
  Toxicologic Pathology" the Rat Nomenclature
  Reconciliation Subcommittee was formed in 1998
  together with the U.S. STP and other
  international societies of toxicologic pathology
  in order to eliminate differences in both systems
  and to establish a common nomenclature. For more
  details, please click here.

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RITA and NACAD

• RITA Registry of Industrial Toxicology
  Animal-data.
• Abbott GmbH Co KG, Ludwigshafen, Germany
• ALTANA Pharma AG, Hamburg, Germany
• AstraZeneca, Södertälje, Sweden and Macclesfield,
  England
• Aventis Pharma Deutschland GmbH, Hattersheim,
  Germany
• BASF AG, Ludwigshafen, Germany
• Bayer HealthCare AG, Wuppertal, Germany
• Boehringer Ingelheim Pharma KG, Biberach,
  Germany
• Fraunhofer Institute of Toxicology and
  Experimental Medicine, Hannover, Germany
• Hoffman-LaRoche AG, Basel, Switzerland
• Merck KGaA, Darmstadt, Germany
• Novartis Pharma AG, Basel, Switzerland
• Pfizer, Amboise, France
• Pharmacia, Nerviano, Italy
• Syngenta CTL, Macclesfield, England

NACAD North American Control Animal Database.
3M Corporate Toxicology, St. Paul, MN,
USA Adolor Corporation, Malvern, PA, USA Bayer
CropScience, Stillwell, KS, USA Pfizer, Inc.,
Groton, CT, USA Pfizer, Inc., Ann Arbor, MI,
USA Pharmacia, Inc., Kalamazoo, MI, USA R.W.
Johnson Pharmaceutical Research Institute, Spring
House, PA, USA Schering-Plough Research
Institute, Lafayette, NJ, USA
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Neoplastic lesions (tumors) - Rat

• International Classification of Rodent Tumours,
  Part I The Rat
• The diagnostic criteria of tumors and
  pre-neoplastic lesions in all organ systems are
  presented in a series of 10 fascicles in a
  compact and easy-to-use format together with
  numerous images which show the typical appearance
  of a lesion. The classification contains besides
  the light-microscopic features criteria for the
  differentiation of hyperplasias, benign and
  malignant tumors. The diagnostic criteria have
  been reviewed prior to publication by
  internationally recognized experts in the area of
  toxicological pathology and/or veterinary
  pathology.The series has been published by
  WHO/IARC (IARC Scientific Publications, No. 122).
  (1992-97)

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Neoplastic lesions (tumors) - Mouse

• The corresponding publication on lesions in the
  mouse has been prepared in a joint initiative
  between the RITA group and members of many
  societies of toxicologic pathology (like ESTP
  (GTP), STP, BSTP, JSTP). Springer Press has
  published the comprehensive results under the
  title "International Classification of Rodent
  Tumors, The Mouse" in April 2001.

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Control animal data (carcinogenicity)

• NACAD North American Control Animal Database 1994
• RITA Registry of Industrial Toxicology
  Animal-data 1995
• both use the same data base structure and the
  data is stored on the same Fraunhofer Institute
  for Toxicology and Experimental Medicine (ITEM)
  data base server in Hannover, Germany

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RENI (Registry Nomenclature Information)
                                                                          
Optimum localization for tissue
preparation Sample size Direction of sectioning
Number of sections to be prepared.
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Sample size

• Definition the size (area) of an organ or part
  of an organ which is sampled in a cassette for
  processing.
• determined by the size of an organ
• for optimal fixation, sample thickness should not
  exceed 3-5 mm
• the examined area should be as large as possible
  and should contain the relevant anatomical
  structures
• the tissue can be adapted to the size of
  cassettes by trimming the margins off.

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Plane of section

• transverse in a 90 angle to the long axis of an
  organ or part of an organ
• longitudinal vertical in the direction of the
  long axis of the body, an organ or part of an
  organ in the dorsoventral axis
• longitudinal horizontal in the direction of the
  long axis of the body, an organ or part of an
  organ, perpendicular to the dorsoventral axis
         

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Example of trimming guidance

• KIDNEY, RENAL PELVIS and URETER
• SpeciesRats and Mice
• Organs Kidney Renal pelvis UreterLocalizationsK
  idney both in the median, through the tip of
  papilla and renal pelvis.Ureter transverse
  section midway between kidneys and
  bladder.Optional adjacent to the renal pelvis
  (not shown in the image).Number of sections2 (1
  per side)DirectionKidney one side
  longitudinal, other side transverseUreter
  longitudinal adjacent to kidney or transverse
  with adipose tissueRemarksKidney Capsule
  should not be removed.Fixation can be improved
  by an incision at necropsy.

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• Number of sections
• Usually one per specimenif gt1 take same amount
  of sections in all animals/groups to obtain
  comparable results. Staining Standard HE
  stain
• Other histological stains and immunohistochemistr
  y can be applied as a routine or on a case by
  case basis in addition to the HE stained
  sections.

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Fixation

• Tissues must be promptly and appropriately fixed
  by immersion.
• Adequate fixation time is necessary before tissue
  processing commences
• In literature a volume ratio of tissue to
  fixative of 120 is often mentioned.
• Less fixative may be sufficient, especially if a
  shaking device is used for freshly fixed tissues
  and/or fixative is replaced once.

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Literature
Rat
Mouse MARONPOT RR, BOORMAN GA, GAUL BW (eds)
Pathology of the mouse. Reference and atlas.
Cache River Press, Vienna, 1999 HASCHEK et al.
(2002).

• HEBEL R, STROMBERG MW Anatomy and embryology of
  the laboratory rat. BioMed, Wörthsee,
  1986.-detailed anatomical description of the
  organ systems
• BOORMAN GA, EUSTIS SL, ELWELL MR, et al. (eds)
  Pathology of the Fischer rat. Reference and
  atlas. Academic Press, San Diego, New York,
  London. - Embryology, anatomy, histology and
  pathology of the Fischer rat, for some organs
  also with trimming proposals
• KRINKE GJ (ed) The laboratory rat. Academic
  Press, San Diego San Francisco New York, 2000.

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Lesions

• - Undisputable lesions
• Increase or decrease in severity or incidence of
  background lesions/noise
• Background noise can only be determined when all
  observations have been made

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Severity grading

• application of numerical severity scores of
  specific lesions
• semiquantitative - relies on estimates of
  severity rather than actual measurements.
• primarily determined by the extent and magnitude
  of lesions
• No standardized guidelines for grading
  nonneoplastic lesions.

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• the severity grade scheme used has a great
  bearing on the NOAEL
• Usually 4-5 severity gradesminimal
  mild-moderateor grade 1,2,3
• Automated or semiautomated image analysis and
  manual stereology unbiased, consistent (still
  limited usefulness)

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Some commonly used severity grading schemes

• 0 Not present
• 1 Minimal (lt 1)
• 2 Slight (125)
• 3 Moderate (2650)
• 4 Moderately Severe/high (5175)
• 5 Severe/high (76100)

A B Grade 1 Minimal (lt10) (0-25) Grade 2
Mild (10-39)(26-50) Grade 3 Moderate (40-79)
(51-75) Grade 4 Marked (80-100)(76-100)
Grade 1 Minimal Grade 2 Slight (same
as mild) Grade 3 Moderate Grade 4 Marked
(same as severe) Grade 5 Massive (same as very
severe)
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Other technical procedures

• Instillation of fixative
• Decalcification
• Type of fixative used for particular organs
• influence the probability of detecting lesions
  in the final histological slide
• A thorough understanding of the anatomic
  features (sub-sites) is important to ensure an
  adequate histologic evaluation of all potential
  target sites in a given organ.

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Standardization of organ sampling and trimming

• Ruehl-Fehlert C, Kittel B, Morawietz G, Deslex P,
  Keenan C, Mahrt CR, Nolte T, Robinson M, Stuart
  BP, Deschl U (2003) Revised guides for organ
  sampling and trimming in rats and mice Part 1.
  A joint publication of the RITA and NACAD groups.
  Exp Toxic Pathol 55 91106
• Kittel B, Ruehl-Fehlert C, Morawietz G, Klapwijk
  J, Elwell MR, Lenz B, O'Sullivan MG, Roth DR,
  Wadsworth PF (2004) Revised guides for organ
  sampling and trimming in rats and mice Part 2.
  A joint publication of the RITA and NACAD groups.
  Exp Toxic Pathol 55 413431
• Morawietz G, Ruehl-Fehlert C, Kittel B, Bube A,
  Keane K, Halm S, Heuser A, Hellmann J (2004)
  Revised guides for organ sampling and trimming in
  rats and mice Part 3. A joint publication of
  the RITA and NACAD groups. Exp Toxic Pathol 55
  433449

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http//www.item.fraunhofer.de/reni/trimming/index
.php