Appendix_1__Immunotoxicity_Study_Pathology_Specifications.pdf

NTP IMMUNOTOXICITY STUDY PATHOLOGY SPECIFICATIONS

Amended June 2011

I. Purpose and Background

1. The purpose of the NTP Immunotoxicity Contract is to 1) develop and validate methods to evaluate modulation and pathology of immune function 2) to evaluate the immunomodulatory potential of agents of concern using a tiered testing panel and 3) to use studies to define cellular and molecular events associated with modulation of immune function. The three areas of immune response that are of interest are 1) autoimmunity 2) allergy and 3) immunomodulation.

2. The purpose of the Pathology Specification document is to define the extent of pathology evaluation (necropsy and histopathology), pathology peer review, and archiving of pathology data for the NTP Immunotoxicity studies.

3. The tissues will be obtained from the NTP Immunotoxicology contract.

4. Estimated annual number and types of NTP immunotoxicity studies requiring pathology evaluation:

• Immunomodulation studies

i. 4 screening studies

a. necropsy and histopathology

ii. 2 definitive studies

a. necropsy and histopathology only if the dose is greater than the screening study or requested by NTP study scientist

iii. 4 developmental studies

a. necropsy and histopathology

• Autoimmunity studies

i. 1 compound

a. necropsy and histopathology not done unless requested by the NTP study scientist

5. Types of Studies

• Screening studies are designed to screen for immunomodulatory effects.

i. Standard design for a screening study

ii. One group of vehicle control animals

iii. Generally 5 treatment groups plus a vehicle control group

iv. One positive control group (cyclophosphamide)

v. One Anti Asialo GM1 group

vi. Each group generally consists of 8 animals

vii. All groups except the Anti Asialo GM1 group would be evaluated for pathology

NATIONAL TOXICOLOGY PROGRAM

U.S. Department of Health and Human Services

NATIONAL INSTITUTE OF

ENVIRONMENTAL HEALTH SCIENCES

111 T.W. ALEXANDER DRIVE

P.O. BOX 12233

RESEARCH TRIANGLE PARK, NC 27709

• Definitive (immunomodulation) studies are follow-up studies performed if positive results are detected in the screening studies.

i. Repeat of Humoral Mediated Immunity and Cell Quantification from screening studies plus additional tests to assess:

ii. Cell Mediated Immunity - MLR Response

iii. Non-Specific Immunity - Macrophage Function Assays

iv. Humoral Mediated Immunity - Antigen specific IgG

v. Host Resistance – Assay and endpoint dependent on the identified target in the screening studies

vi. Gross and histopathology at the request of the study scientist

vii. Standard design includes only 3 treatment groups with 8 animals per group (40 animals, 440 tissues)

viii. Pathology –only if the dose is different from the screening studies or if requested by NTP study scientist

• Developmental immunotoxicology studies are conducted to assess immunomodulation in animals exposed perinatally to compounds of interest.

i. Dosing begins on GD 6 and ends on PND 42

ii. Pathology (gross and histology)

iii. Clinical pathology – CBC

iv. Cell mediated immunity – CTL Assay

v. Humoral mediated immunity – AFC Assay

vi. Non-specific immunity – NK Cell Assay

vii. Cell quantification – B, T (total and subpopulations), NK, macrophages

viii. Standard design includes one group of vehicle control animals and

5 treatment groups

ix. One positive control group (cyclophosphamide) – add on adult animals of similar age, not from perinatal study

x. One Anti Asialo GM1 group - – add on adult animals of similar age, not from perinatal study

xi. Each group generally consists of 8 animals

xii. All groups except the Anti Asialo GM1 group would be evaluated for pathology

• Autoimmunity studies are compound-specific and are designed to assess whether compounds of interest induce or exacerbate disease in autoimmune prone rodent models. Study design and model selected vary with the compound and putative immune target. Gross and histopathology at the request of the study scientist.

6. Studies are grouped to obtain the maximum information from a given set of animals. All animals in a given study group are euthanized on the same day.

Measurement of body weights, organ weights, hematology, gross pathology and histopathology are conducted on a single set of animals.

II. Animals

1. Strains and source: The B6C3F1 (C57BL/6N X C3H/HeN MTV -) hybrid mouse and the Harlan Sprague Dawley rat will be the animals used in these studies, unless otherwise specified by NTP.

2. Specific mouse models (NZB mouse, NOD mouse, Brown Norway Rat) or strains (i.e. Balb/C) may be used occasionally at the request of the NTP.

III. Necropsy

1. Animal necropsies shall be performed according to the Mouse Necropsy

Procedure for NTP Immunotoxicity Studies (Appendix A).

2. List of tissues to be collected generally include: spleen, thymus, liver, lung, kidneys, adrenal glands, bone marrow (femur), GI tract (stomach to rectum) with Peyer’s patches, mesenteric lymph node chain, submandibular lymph nodes and popliteal lymph nodes.

IV. Collection of Blood for CBC

1. Refer to Blood Collection protocol (Appendix B).

V. Submission of Pathology Materials From Immunotoxicology Contact Lab to NTP and the NTP Pathology Support Contract Lab

1. All tissues will be collected and labeled at the immunotoxicology contract lab.

Wet tissues with animal identification (sex, strain, age) and study data [scientific protocol, study design, treatment group identification, list of tissues collected, gross abnormalities report (if any), Individual animal necropsy records (IANRs) body and organ weights, clinical pathology (CBC)] will be shipped to a contract lab designated by the Contracting Officer's Technical Representative (COTR) for NTP pathology support contracts and logged in and inventoried. Study data shall be uploaded to CEBS and the COTR for NTP pathology support contracts, NTP Archives, the NTP IT/Stat Support Contract User Support Group and the NTP Data Collection Unit (DCU) notified via email (Appendix C).

VI. Tissue Processing

1. Tissues will be trimmed and processed (trimming, embedding, sectioning and staining) according to the specifications in Appendix D.

2. Any gross lesions or other observations seen at trimming shall be recorded on the histology processing records.

3. The protocol for assigning histology numbers and labeling materials is specified in Appendix E.

VII. Histopathologic Evaluation

1. The initial histology evaluation shall be performed by a contract pathologist

(study pathologist) with the guidance of an NTP Pathologist. The COTR for the NTP Pathology Support Contract shall assign this study to a contract lab. The contract pathologist assigned to the study shall examine all submitted protocol-required tissues and gross lesions (Appendix A) microscopically for each animal (vehicle and cyclophosphamide controls and all doses) and render diagnoses, if present. All lesions shall be graded according to the criteria in Appendix F. The Anti Asialo GM1 group shall not be evaluated for pathology. When possible, it is preferable that the same pathologist conducts the histopathology on all phases of the same chemical being studied. Data shall be entered into current toxicology data management system such as TDMSE or Provantis), the NTP database and acquisition system.

2. Complete histology (Preparation of stained slides) and histopathologic evaluation shall be performed on early deaths or moribund sacrifices. Immunotoxicology contract lab personnel shall contact the NTP study scientist and COTR to determine when histopathology evaluation shall be done on early deaths.

Generally, histopathologic evaluation can be postponed to the end of the study, unless the NTP requests otherwise.

3. The following ancillary data shall be provided and considered to aid in histopathological evaluation and interpretation:

- Laboratory Protocol

- Body weight and organ weight

- Clinical Pathology (CBC)

- Gross abnormalities report (if any)

The following information or data may be available for some studies and, if so, shall be considered:

- Immunotoxicity report

- In-life data

- Individual animal necropsy records

- Toxicity assay results

4. For all lymphoid organs (thymus, spleen, lymph nodes, BALT, GALT, and bone marrow), the methods of enhanced histopathology as described in the STP position paper: Best Practice Guideline for the Routine Pathology Evaluation of the Immune System (reference 2) and recent publications (references 3-7) shall be employed as directed by NTP. Checklists to use for enhanced histopathology evaluation are provided in Appendix G. Non-lymphoid organs (liver, kidney, lung, adrenal gland, GI tract) shall have a standard histopathology review performed.

Evaluation of tissues with the NTP Pathologist shall be arranged.

5. A draft pathology report with checklists shall be prepared and submitted to the NTP Pathologist. After approval, the final report with checklists shall be verified to be accurate by quality control and shall then be submitted to DCU (electronically and hard copy).

6. Photodocumentation of representative lesions included in the report shall be acquired according to NTP guidelines (reference 1) and submitted to DCU on CD for storage in the NTP digital image database.

7. All pathology materials shall be sent to a pathology laboratory specified by the COTR for the NTP Pathology Support Contract where a pathology peer review shall be conducted.

VIII. Pathology Review Procedures

1. Overview: Pathology data and reports will be sent to a pathology contract laboratory for audit of pathology specimens (APS), pathology data review (PDR), Target Organ Letter (TOL), and a pathology quality assessment (PQA). These activities shall be performed under the direction of an NTP Pathologist.

2. For each study the following materials will be submitted as part of the pathology data review:

• Laboratory protocol

• Carcass (for studies commensing after Oct , 2008)

• Wet tissues, blocks, and H&E slides

• Histology processing records

• Decalcification records

• Gross abnormalities report (if any)

• TDMSE/Provantis summary tables

• Primary pathology lab's approved final report

• Body and organ weights

• CBC

3. The following may or may not be received:

• In-life data

• Individual animal necropsy record

• Toxicity assays results

4. Reviews:

• PDR/TOL: The NTP Pathologist and Study Scientist will review all the available immunotoxicology data and histopathology results. The tissues to be reviewed during the PQA will be determined at the PDR by the study scientist, NTP pathologist, and QA/PDR pathologist.

• APS: A modified APS of incidence data, slide-block match, adherence to NTP immunopathology guidelines, clinical pathology data and quality of slide preparation shall be done.

• PQA: If the functional assays are known to be negative and the histology was negative, then a “read down” PQA approach to histologic evaluation may be employed at the discretion of the study scientist and NTP pathologist. (i.e. start with the control and high dose groups and read until no effects seen). Otherwise, a routine PQA is performed according to the NTP Immunotoxicity Study Pathology Specifications.

• PPR/PWG: The findings from the PQA will determine if resolution of discrepancies requires a Pathology Peer Review (PPR) or convening of a Pathology Working Group (PWG). The PPR would be organized to include at least the PPR/PWG pathologist and NTP pathologist but may also include the Study Scientist (or representative), Study pathologist and QA pathologist, depending on study findings. If there are major discrepancies or no resolution at the PPR then a PWG of 7-10 pathologists will be convened. The PPR/PWG pathologist will work closely with the NTP pathologist regarding the issues presented to the PPR/PWG panel. After approval by the NTP pathologist, a written report of the findings will be verified by quality control and then submitted to NTP for redlining and finalizing the TDMSE/Provantis data for each study according to NTP guidelines. NTP will conduct an audit of final data.

IX. Data Entry

1. All diagnoses will be entered into TDMSE/Provantis by the Pathology Support

Contract Laboratory.

2. Peer reviewed diagnoses shall be verified by the NTP pathologist and NTP Data

Coordination Unit staff before being changed and/or made final. Redlining and final review and approval of the pathology data shall be performed by the NTP pathologist and DCU staff.

X. Submission of Pathology Materials to NTP Archives

1. Pathology materials [residual wet tissues, study inventory log (TDMSE report 15), blocks, H&E stained slides, study data, histology processing records, decalcification records, etc.] shall be submitted in accordance with the NTP specifications for submission to NTP Archives (Appendix C):

http://ntp.niehs.nih.gov/ntp/Test_Info/FinalNTP_ToxCarSpecsJan2011.pdf

References

1. Specifications for the conduct of studies to evaluate the toxic and carcinogenic potential of chemical, biological and physical agents in laboratory animals for the National Toxicology Program (NTP), available at http://ntp.niehs.nih.gov.

2. STP Position Paper: Best Practice Guideline for the Routine Pathology Evaluation of the Immune System (2005). P. Haley (chair), R. Perry (cochair), D. Ennulat, S. Frame, C.

Johnson, J-M Lapointe, A. Nyska, P. W. Snyder, D. Walker, and G. Walter. Toxicologic Pathology, 33, 404-407.

3. Elmore, S. A. (2006). Enhanced histopathology of mucosa-associated lymphoid tissue. Toxicol Pathol 34, 687-96.

4. Elmore, S. A. (2006). Enhanced histopathology of the bone marrow. Toxicol Pathol 34, 666-86.

5. Elmore, S. A. (2006). Enhanced histopathology of the thymus. Toxicol Pathol 34, 656-65.

6. Elmore, S. A. (2006). Enhanced histopathology of the spleen. Toxicol Pathol 34, 648-55.

7. Elmore, S. A. (2006). Enhanced histopathology of the lymph nodes. Toxicol Pathol 34, 634-47.

Appendix A

MOUSE NECROPSY PROCEDURE FOR NTP IMMUNOTOXICITY STUDIES

1.0 Principle: This procedure allows the evaluation of gross pathology and collection of selected tissues for histopathology for mice. The organ removal and evaluation covered in this appendix includes the spleen, thymus, liver, lung, kidneys, adrenal glands, bone marrow (femur), GI tract (stomach to rectum) with Peyer’s patches, mesenteric lymph node chain, submandibular lymph nodes and popliteal lymph nodes. Organs to be weighed may include liver, lung, thymus, spleen, adrenal glands.

2.0 Materials

• Necropsy table with ventilation system

• Examining boards and pins

• CO2 chamber

• Forceps

• Blunt and surgical scissors

• Single edge razor blades or scalpel

• Specimen containers

• Tissue Cassettes

• Balances and standard weights

• 4 x 4 gauze sponges

• 1 ml disposable syringes

• 23 and 25 gauge disposable needles

• 22-gauge gavage needle

• Suture material

• 10% neutral buffered formalin, pH 7.4

• Sheets of dental wax

• White index cards

• 100 mm x 15 mm plastic Petri dishes

• Absorbent pads

3.0 Procedure:

a. Prior to starting necropsy activate the fume recovery system.

b. Each necropsy station should be equipped with its own operating instruments as listed above.

c. All tissue storage containers and individual animal necropsy reports should be appropriately labeled in advance of the necropsy. All animals should be uniquely identified following AAALAC guidelines and these numbers recorded along with the protocol and study number. Both prosector and recorder together should confirm the animal number with the numbers on the storage containers and record sheets.

d. Accuracy of balances will be determined before weighing mice or organs by weighing at least three standard weights which covers the range of weights to be evaluated. Record the standard weights and the actual balance readings.

e. Examining boards and instruments must be cleaned with 3% hydrogen peroxide after each animal necropsy before the start of another animal necropsy.

f. Tissues will be placed in the appropriately labeled tissue containers containing approximately 200 mls of 10% neutral buffered formalin (pH 7.4). Lymph nodes and adrenal glands will be transferred to cassettes (without foam sponge).

g. Animals are sacrificed using a CO2 gas euthanasia chamber. There should be no other animals in the necropsy room other than the animal being euthanized. The animal should not be placed in the CO2 chamber until the necropsy team is ready to commence the necropsy upon the animal’s death. The order in which animals are necropsied shall be randomized.

Scheduled necropsies and necropsies of moribund sacrifice animals shall be initiated within five minutes after an animal is sacrificed in order to minimize post-mortem autolysis.

h. Once the animal is euthanized remove it from the CO2 chamber. Record body weight before the animal is given to the prosector. Examine the animal externally and record any gross abnormality on the record sheet.

Spray the animal with 70% ethanol, and wipe the fur with gauze to remove any excess. This will minimize fur contamination of surfaces.

i. Always use a sharp knife or razor blade in cutting tissue to avoid crushing artifact caused by scissors or dull knives. Handle all organs by edges, utilizing connective tissue whenever possible to minimize (eliminate if possible) forcep teeth marks on the organ. When an organ needs to be trimmed, divided or cut, place the organ on a dental wax sheet.

j. Remove a strip of skin from the symphysis pubis to the chin by using forceps and scissors. Use the animal’s weight to aid with this cut. Open the thoracic cavity to expose the heart. Exsanguinate the animal by use of a 23-gauge needle attached to a 1 ml syringe. Puncture the right ventricle and remove as much blood as possible using gentle syringe suction. One may need to rotate the needle if it seems that the bevel is against the heart wall.

k. After exsanguination remove both popliteal lymph nodes from each side of the animal and place into a tissue cassette (without foam sponge). Place the cassette into the tissue storage container. Then pin each animal by its extremities to the necropsy board using 23-guage needles. Further separate the skin from the musculature. Open the abdominal cavity.

l. Examine all organs in situ prior to removal. Record any abnormal findings.

If no gross abnormal findings are observed, the abbreviation “NF” (No Finding) is recorded in the comment section.

m. Obtain specimens of all grossly abnormal tissues in addition to those routinely taken.

n. When collecting Peyer’s patches and the mesenteric lymph node chain, remove the stomach, intestines and rectum as a unit. Gently separate the intestines to expose the mesenteric lymph node chain and its associated adipose tissue. Place the chain of nodes and surrounding tissue (flattened) onto an index card, trim away the intestine and place in the tissue storage container. For the Peyer’s patches, infuse the intestines from the duodenum to the rectum every 2cm with formalin using a 25-gauge needle and 1 ml syringe. Be careful not to over-inflate the intestines.

o. Remove the liver and take the transverse section of the left lateral lobe.

Take longitudinal sections of the right and left medial lobes, including the gall bladder.

p. Remove the spleen and pancreas as a unit. Separate the pancreas from the spleen. Weigh the spleen and place in the tissue storage container.

The pancreas is not retained.

q. Remove both adrenal glands and both kidneys. Make a single small longitudinal cut along the outer curvature in the left kidney with the razor blade or scalpel. A small crosswise cut is made in the right kidney. This serves to identify the organs. Place the kidneys in the tissue storage container and place the adrenal glands in a tissue cassette (without foam sponge).

r. Remove both lobes of the thymus (without mediastinal lymph nodes attached), weigh and place on an index card then place in the tissue storage container.

s. Remove the tongue, larynx, trachea, heart and lungs (pluck). Remove the heart from the pluck. Using a 22-gauge gavage needle, insert into tracheal opening and infuse the lungs with formalin. Be careful not to over-inflate. Tie with suture. Place the inflated lungs into the tissue storage container.

t. Remove the left femur. To remove the femur, a cut is made below the knee joint in the mid portion of the tibia. The knee and this section of the tibia are left attached to the femur. The femur is then separated from the hip joint. Using gauze, remove muscle and tissue from the femur and place in the tissue storage container.

u. Carcasses shall be saved once organs are collected.

References

1) Fehlert et al. Revised guides for organ sampling and trimming in rats and mice- Part 1.

2003. Exp Toxic Pathol 55: 91-106.

2) Kittel et al. Revised guides for organ sampling and trimming in rats and mice- Part 2.

2004. Exp Toxic Pathol 55: 413-431.

3) Morawietz et al. Revised guides for organ sampling and trimming in rats and mice- Part

3. 2004. Exp Toxic Pathol 55: 433-449.

APPENDIX B

PROCEDURE FOR BLOOD COLLECTION BY RETRO-ORBITAL BLEEDING FOR NTP

IMMUNOTOXICITY STUDIES

1.0 Principle: To collect blood samples from mice or rats for hematology.

2.0 Materials:

• BLUE tip (non-heparinized) Microhematocrit tubes (Fisher)

• Gauze

• 500 µl EDTA microtainer tubes (for HEMATOLOGY, Fisher)

• CO2 anesthesia chamber

- Procedure:

a. Remove a few BLUE tip microhematocrit tubes from the container and break each one into 3 or 4 small pieces. This will allow blood to flow quicker and decreases the potential to clot prior to reaching the tube.

b. Now place the tube in an easy-to-reach location.

c. Place your animal into the CO2 chamber and open the chamber valve for about 5 seconds to allow gas to enter.

d. After applying anesthesia, quickly remove your animal and insert the jagged edge of one of your small pieces of the hematocrit tubes into the inner canthus of either eye.

e. Upon inserting the microhematocrit tube, gently twist it about a half turn while at the same time pressing in a downward motion.

f. The moment the tube twists inward, a spontaneous surge of blood flows out. This is why you must have your blood collecting tube in an easy to reach position.

g. In most cases, the blood will at some point decrease in flow. This usually indicates that a clot has begun forming within the microhematocrit tube.

This is why it is important to have several pieces of microhematocrit tubes broken up prior to bleeding. In less than a few seconds, you can simply remove the clotted microhematocrit tube and quickly insert another. Only collect blood for a CBC analysis (EDTA tube).

APPENDIX C

SPECIFICATIONS FOR SUBMISSION OF HISTOPATHOLOGY MATERIALS (WET

TISSUES AND PATHOLOGY DATA) FROM THE IMMUNOTOXICOLOGY CONTRACT LAB

FOR NTP IMMUNOTOXICITY STUDIES

1. Histopathology materials shall be organized, packed, marked, and shipped prepaid to the contract lab as directed below.

a. Prior to shipping materials, the Inventory of Residual Material must be completed. The number and condition of wet tissues shall be shown on this form.

b. The scheme or SOP used to identify the animals in each study (including an appropriate figure or diagram) shall be submitted at the time that wet tissues are sent to the contract lab.

c. The study scientist shall provide the appropriate NTP Immunotoxicity experiment numbers that should be used as identifiers for the samples. Each study will have it’s own unique "I" number.

d. An email indicating intent to ship showing how many boxes of what kind of histopathology material(s) and on what date(s) shall be directed to the DCU with a copy to the COTR for NTP pathology support contracts, the NTP Archives, the NTP IT/Stat Support Contract User Support Group at least 7 calendar days in advance of shipment. This is required to aid in tracing lost or misdirected shipments, to allow time for the COTR to prepare a work assignment, and to notify the pathology support contract lab that the shipment is expected. DCU shall notify the pathology support contract lab designated to process tissues by the COTR for NTP Pathology Support Contracts.

e. An electronic copy of inventory and study details (scientific protocol, study design, treatment group identification, clinical observations, body and organ weights, list of tissues collected, clinical pathology, gross pathology, IANRs) shall be uploaded to CEBS, DCU notified, and then DCU shall send electronic copies to NTP Archives, the COTR for NTP Pathology Support Contracts, the NTP IT/Stat Support Contract User Support Group, and the Pathology Support Contract Lab designated by the COTR for NTP pathology support contracts (Appendix C). A hard copy should be submitted with the shipments.

f. For shipment, all wet tissues shall be placed in two separate heat sealed plastic bags (double bagged), each 4.5 mils thick, one inside the other so that there is no leakage, and organized by sex, species, strain, age, group, and animal number. Any tissue containing the unique animal identifier (tail tattoo, left ear, etc.) shall be included within the bag. A permanent ink label (not ballpoint pen) shall be placed between the two bags showing the study number, laboratory, group number, and animal number. A similar label shall be placed on the external surface of the outer bag. Once the bags are organized, they shall be packed in boxes. The boxes shall be marked on one end to show:

Name of contractor Contract Number Experiment number (I #) Animal group number(s) Histology numbers in that box

These tissues will then be shipped within 4 days following necropsy to a pathology support contract lab designated by the COTR for the NTP Pathology Support Contract and in compliance with all local, state and federal regulations.

g. Inventory and study details (treatment group identification, clinical observations, body and organ weights, clinical pathology, etc.) shall be shipped with the wet tissues.

2. Contact and Shipping Information

a. Charles River Laboratories –Pathology Associates

• Contact person Dr. Jim Morrison Senior Staff Pathologist Phone 919-206-7024 Fax 919-206-7001 James.Morrison@crl.com

• Shipping and mailing address

Charles River Laboratories – Pathology Associates Attention Histology 4025 Stirrup Creek Drive, Suite 150 Durham, NC 27703

b. Integrated Laboratory Systems (ILS)

• Contact person Theleria Hackett

Supervisor, Histology Laboratory Phone 919-281-1110 ext 730 Fax 919-281-1118 thackett@ils-inc.com

• Mailing address ILS, Inc

P.O. Box 13501 Research Triangle Park, NC 27709

• Shipping address ILS, Inc

Attention Histology (Theleria Hackett) 601 Keystone Park Dr., Suite 100, Bay #1 Durham, NC 27713

c. NTP Archives

• Contact person: Kylie Brockenfelt

Contractor: Experimental Pathology Laboratories, Inc.

phone 919.998.9009 ext. 646 fax 919.998.9008 brocken1@niehs.nih.gov

• Mailing address (NTP Program Archives) P.O.Box 13566 Research Triangle Park, NC 27709

• Shipping address (NTP Program Archives) 615 Davis Drive, Suite 400 Durham, NC 27713

d. NTP IT/Stat Support Contract

• Contact person: Mary Hicks NTP User Support Specialist NTP IT/Stat Support Contract

NIEHS

Contractor hicksm@niehs.nih.gov phone 919-313-7617

e. COTR for Path Support Contract

• Dr. Susan A. Elmore elmore@niehs.nih.gov phone 919-541-3474

• Alternate COTR Dr. Mark Cesta cesta@niehs.nih.gov phone 919-541-3229

f. Data Coordination Unit

• Annette Shambley shambley@niehs.nih.gov 919-541-1825

• Lorri Ezedin ezedin@niehs.nih.gov 919-541-1053

APPENDIX D

TISSUE TRIMMING PROCEDURE FOR NTP IMMUNOTOXICITY STUDIES

1. Tissue trimming shall be supervised by a Pathologist and/OR supervisor of the histology laboratory, although their continued presence is not required. A copy of the Individual Animal Necropsy Report (IANR) for each animal shall be available for the technician at the time of tissue trimming. Any additional gross observations identified during the trimming procedure shall be recorded.

2. All gross lesions, and all protocol required organs/tissues shall be trimmed, embedded, and H&E stained slides prepared. All early death animals shall be handled in the same manner at the time of trimming.

3. Fixation of tissues as soon as possible after death is preferred for optimal preservation. For screening studies, tissues shall be fixed in 10% NBF then shipped within 4 days to the assigned pathology support contract lab for immediate processing. The contract lab will be assigned by the COTR for the pathology support contracts.

Tissues are to be placed in blocks in a consistent manner so that the same tissues are in the same numbered blocks and placed in the same location within each block for all animals. A general blocking scheme and specific trimming instructions are presented below but may have to be modified for the larger rat tissues. Blocking scheme:

Block # Tissue 1 Lungs

Liver, left lobe 2 Liver, median lobe Kidneys 3 Adrenals

4 Thymus 5 Spleen 6 Mesenteric lymph node chain

Stomach Cecum Colon

Rectum Duodenum Jejunum

Ileum 9 Popliteal lymph nodes

10 Mandibular lymph nodes 11 Femur

4. Parenchymal organs, e.g., liver shall be free of adjacent tissues and trimmed to allow the largest cross-section surface area possible for microscopic examination. Hollow organs shall be trimmed and blocked to allow a cross-section slide from mucosa to serosa.

5. Tissues must be trimmed to a maximum thickness of 0.4 cm for processing. Small (less than 0.4 cm) endocrine organs and lymph nodes shall be submitted intact (whole).

6. Specific trimming instructions:

Spleen: A transverse section shall be taken at the largest extension of the organ and the cut surface placed down in the cassette. The remaining (longer) section shall be microtomed to the greatest width and the cut surface placed down in the cassette.

Large samples can be trimmed on one end to fit in the cassette and to allow placement on the slide.

Thymus: The whole thymus shall be placed in a cassette with the ventral aspect down to facilitate sectioning along the longitudinal axis of both lobes. This gives a standardized longitudinal section showing all anatomical structures of this organ.

Lymph Nodes:

The mandibular and popliteal lymph nodes and the entire mesenteric lymph node chain (Morawietz et al, 2004) shall be submitted whole (untrimmed) in separate labeled cassettes. Large samples can be trimmed on one end to fit in the cassette and to allow placement on the slide. It is important that a section is taken through the middle of the longitudinal axis of the lymph node at the area of greatest thickness in order to avoid a superficial cut and to be able to examine all major areas, such as cortex, paracortex and medulla.

Liver: A transverse section of the left lateral lobe and longitudinal sections of the left and right medial lobes, including the gall bladder (if present), shall be prepared (Ruehl- Fehlert et al, 2003).

Lung: The intact lung can be submitted in the cassette(s) oriented to obtain coronal (perpendicular to a sagittal plane and parallel to the long axis of the body) sections of both right and left lungs including main stem bronchi.

Kidney: The left kidney shall be sliced along the mid-longitudinal axis and the right kidney transversely through the entire cortex, pelvis and medulla and one half of each kidney shall be submitted.

Adrenal Glands: Both adrenal glands shall be embedded intact. Sections of adrenal gland shall include the cortex and medulla.

Bone Marrow (femur): After decalcification, the distal 1.5 cm (minimum length for rat) or 1 cm (minimum length for mouse) of the femur must be sectioned through the frontal plane to include the articular cartilage and articular surface, the femoral condyles with epiphyseal plate, and diaphysis with bone marrow. Sections of bone must include the joint surface and marrow cavity. A rapid decalcifier solution such as RDO Rapid Decalcifier (Apex Engineering Products Corp.) can be used for decalcification of the femur following suggested manufacturers recommendations for decalcification of the femurs of rats and mice.

Stomach: The stomach shall be opened along the greater curvature and the mucosa examined for gross lesions at trimming. The stomach must be cut first through the mid-sagittal plane thereby dividing it into two equal halves and both halves should be submitted for histology. Any gross lesions must also be embedded and sectioned.

Small and Large Intestines:

One transverse section from each part of the fixed unopened bowel shall be taken according to the localization guidelines provided below. Transverse sections that include all visible Peyer’s patches shall be taken. The remaining intestine shall be opened and examined; all observed gross lesions/abnormalities shall be sampled for histopathological examination.

NOTE - Peyer’s patches of the small intestine are mostly visible as slightly elevated lighter nodular areas in the wall of the intestine seen from the serosal surface and are prominent when activated.

Localizations:

Rat Duodenum:

• 1 cm distal to the pyloric sphincter in conjunction with a piece of adjacent pancreas.

Jejunum:

• central section containing Peyer’s patch.

Ileum:

• 1 cm proximal to cecum.

Cecum:

• central section (but due to larger diameter it is advisable to open section and then take a transverse section).

Colon:

• central section.

Rectum:

• 2 cm proximal to the anus; optional longitudinal vertical section to include the anus.

Mouse Duodenum:

• 0.5 cm distal to the pyloric sphincter in conjunction with a piece of adjacent pancreas.

Jejunum:

• central section containing Peyer’s patch.

Ileum:

• 0.5 cm proximal to cecum.

Cecum:

• central section (but due to larger diameter it is advisable to open section and then take a transverse section).

Colon:

• central section.

Rectum:

• 1 cm proximal to the anus; optional: longitudinal vertical section to include the anus.

7. All trimmed tissues shall be processed (dehydrated and infiltrated with paraffin) using appropriate chemicals, by an automatic tissue processor, sectioned and stained as indicated in the following sections.

8. After tissues are processed, they shall be embedded in paraffin blocks.

9. Tissues shall be sectioned in organ groups, in a random animal order, and by the same technician to prevent variation in tissue thickness. For example, all spleens shall be sectioned at one time, in random order, and by the same technician. Tissues shall be sectioned at 4-5 microns in thickness. Blocks shall be resealed using a warm spatula to melt the surface wax.

Blocks shall be clearly identified with the designated letter code, plus the laboratory's histology number and sub-numbered from 1 through n to indicate the block number for that animal.

10. Tissue slides shall be stained with hematoxylin and eosin, all slides stained on the same day to prevent staining variation, after which they shall be cover-slipped, with glass cover slips.

Each slide shall be permanently paper labeled, or labeled using an approved alternate method.

11. All slides, including stained and cover-slipped smears, when required, shall be subject to quality assessment before microscopic evaluation.

12. Slides shall be compared to the blocks (slide-block match-up) to ensure that all embedded tissues are represented on the slide and that the slide number matches the block number.

13. A Histology Processing Record shall be completed for each animal for which histology slides are prepared, and shall be submitted to the NTP with the Individual Animal Necropsy Record. The Histology Processing Record shall include, but is not limited to, the following information:

a. Header information to include test article, histology accession number, species and sex.

b. List of tissues trimmed, number of cassettes prepared and verification by trimming technician with initials and date.

c. List of tissues embedded, number of paraffin blocks prepared and verification by embedding technician with initials and date.

d. Number of blocks sectioned, number of slides prepared and verification by microtomy technician with initials and date.

e. Number of slides stained and cover-slipped with verification by technician with initials and date.

f. Number of slides checked out during quality control procedure and verification by technician with initials and date.

g. Number of block re-cuts and/or wet tissue re-cuts and verification by technician with initials and date.

h. Notes documenting deviations from protocol, missing tissues, missing gross lesions, problems, and/or comments.

i. Signature of histology laboratory supervisor indicating review and approval of Histology Processing Record.

References:

1. Ruehl-Fehlert et al. Revised guides for organ sampling and trimming in rats and mice- Part 1. 2003. Exp Toxic Pathol 55: 91-106.

2. Kittel et al. Revised guides for organ sampling and trimming in rats and mice- Part 2.

2004. Exp Toxic Pathol 55: 413-431.

3. Morawietz et al. Revised guides for organ sampling and trimming in rats and mice- Part 2004. Exp Toxic Pathol 55: 433-449.

APPENDIX E

ASSIGNING HISTOLOGY NUMBERS AND LABELING SLIDES FOR NTP

Each study will have a unique NTP I number. A unique histology number shall be assigned by the contract laboratory to each animal that is to receive histologic work-up. At the time of assignment, this number shall be entered in a permanent log and cross-referenced to the TDMSE/Provantis animal identification number.

• This histology number shall appear on the label placed on the tissue block, on the slide, and on the label between the two bags containing the wet tissues. This label shall also show the group and animal number.

• The format that shall be used for labeling slides is shown below. Each block and slide shall be sub-numbered from 1,2,3.....n to show which number that block/slide is for that animal.

• The label on the paraffin tissue block and the label between the tissue bags shall not have the contractor's name shown. Instead, the NTP designated letter code (acronym) for the laboratory shall precede the histology number.

FORMAT FOR SLIDE LABELS

Line l: Laboratory acronym/Pathology subcontractor acronym (if appropriate)/NTP [Acronyms shall be supplied by the COTR] Line 2: I number Line 3: 7 Digits of TDMSE/Provantis Number = Experiment (5) - Test (2) Line 4: Treatment/treatment group designation and individual animal number Line 5: Histology number - Slide number

Treatment/Treatment Group Designations The treatment/dose group designation shall consist of two letters. The first letter shall represent the treatment/dose group and the second letter shall be M for male or F for female. The letter designations for treatment/dose group are as follows:

28-day Screening Studies V= Vehicle Control X = Untreated, Chamber XP = Positive control A = Low Dose/Exposure Concentration B = Low medium Dose/Exposure Concentration C = Medium Dose/Exposure Concentration D = Medium High Dose/Exposure Concentration E = High Dose/Exposure Concentration

Definitive Studies V = Vehicle Control U = Untreated or Chamber Control XP = Positive control L = Low Dose/Exposure Concentration

M = Medium Dose/Exposure Concentration H = High Dose/Exposure Concentration

Developmental Immunotoxicology Studies V = Vehicle Control X = Untreated, Chamber XP = Positive control A = Low Dose/Exposure Concentration B = Low medium Dose/Exposure Concentration C = Medium Dose/Exposure Concentration D = Medium High Dose/Exposure Concentration E = High Dose/Exposure Concentration

Examples:

28-Day Screening Studies: CM035 (Medium dose male #035) Protocol Studies: HF389 (High dose female #389) Developmental Immunotoxicology Studies: EM400 (High dose male #400)

Sample Slide Label:

Immunotoxicology contract lab abbreviation/ILS/NTP or Immunotoxicology contract lab abbreviation/PAI/NTP I##### 05921-01

UF048

10-1

APPENDIX F

GRADING CRITERIA FOR ENHANCED HISTOPATHOLOGY EVALUATION OF NTP

In order to best assess and compare the immunomodulatory effects of the various chemicals being evaluated in the NTP immunotox studies, the following grading criteria shall be applied to each lymphoid tissue. These grades refer to relative increases or decreases in cell number, increases or decreases in lymphoid structures (i.e. germinal centers) and increases or decreases in the area of structures (i.e. PALS) per lymphoid organ. Since NTP is looking for subtle changes in lesions we have devised a modified grading scheme. There should be routine evaluation of non-lymphoid tissues.

For a given lymphoid tissue, this grading scheme would be applied after first evaluating all concurrent control slides of that tissue and determining the “range of normal”. So if a tissue from a treated animal has changes that are considered within normal limits, then it would receive a score of “0”. Approximate increases or decreases (%) in cell number, structure area or number would be graded as 1+ to 4+ according to the following criteria:

Within normal limits (0) 1-10% minimal (1+) 11-25% mild (2+) 26-50% moderate (3+) >50% marked (4+)

Examples of how to apply the grading criteria:

Spleen, periarteriolar lymphoid sheath

• Increased area, mild (2+) = The area of the PALS is increased 11-25% above the upper range of concurrent controls.

• Increased lymphocytes, minimal (1+) = The number of lymphocytes is increased 1-10% above the upper range of concurrent controls.

Spleen, marginal zone

• Decreased area, marked (4+) = The area of the marginal zone is decreased more than 50% below the lower range of the concurrent controls.

• Increased macrophages, moderate (3+) = The number of macrophages is increased 26- 50% above the upper range of concurrent controls.

• Lymphocytes- within normal limits (0) = Cannot be distinguished from concurrent controls

APPENDIX G

CHECKLISTS FOR ENHANCED HISTOPATHOLOGY EVALUATION OF NTP

Spleen: Enhanced Histopathology Checklist Chemical/Animal # Yes/⇑⇓/Severity grade No Periarteriolar lymphoid sheath

Increased/decreased area Increased/decreased lymphocytes

Marginal zone Increased/decreased area

Increased/decreased lymphocytes Increased/decreased macrophages

Follicles Increased/decreased numbers Increased/decreased lymphocytes

Increased/decreased germinal centers Increased apoptosis Increased Tingible body macrophages Red pulp

Increased/decreased area Increased/decreased lymphocytes Increased/decreased hematopoietic cells

Increased numbers Location Plasma cells

Apoptotic cells Tingible body macrophages Pigmented macrophages Dendritic cells Granulocytes/mast cells Granuloma/macrophage aggregates

Fibrosis Necrosis Other Comments

Thymus: Enhanced Histopathology Checklist Chemical/Animal # Yes/⇑⇓/Severity grade No Cortex

Increased/Decreased area Increased/Decreased number of lymphocytes

Increased numbers of apoptotic cells Increased numbers of tingible body macrophages

Necrosis Medulla

Increased/Decreased area

Increased numbers of Hassall’s corpuscles Necrosis Prominent epithelial cords and tubules

Cortex/medulla ratio Increased/Decreased

Epithelium free areas (EFAs): rats only Not evaluated Not present Increased/Decreased area necrosis Other (give location) Inflammation Cysts Pigment Extramedullary hematopoiesis (EMH)

Lymph Node: Enhanced Histopathology Checklist (Indicate lymph node location)

Yes/⇑⇓/Severity grade No Cortex Increased/Decreased area Number of follicles Increased/Decreased Germinal center development Increased/Decreased Increased/Decreased numbers Lymphocytes Increased numbers Apoptotic cells

Tingible body macrophages Plasma cells

Pigmented macrophages Granulocytes (indicate type)

Necrosis Granuloma/macrophage aggregates Interfollicular area (note changes) Paracortex Increased/Decreased area Prominent HEV Increased/Decreased numbers Lymphocytes Increased numbers Apoptotic cells

Tingible body macrophages Plasma cells

Pigmented macrophages Granulocytes (indicate type)

Necrosis Granuloma/macrophage aggregates Medullary cords Increased/Decreased area Increased/Decreased numbers Lymphocytes Macrophages Plasma cells Increased numbers Apoptotic cells

Tingible body macrophages Pigmented macrophages

Granulocytes (indicate type) Necrosis Granuloma/macrophage aggregates Subcapsular/transverse, medullary sinuses Increased numbers Lymphocytes Macrophages Plasma cells Pigmented macrophages

Granulocytes (indicate type) Other/Comments

MALT: Enhanced Histopathology Checklist (specify type: GALT, BALT)

Yes/⇑⇓/Severity grade No Follicles Increased/Decreased area or number Increased/Decreased numbers of

Increased/Decreased germinal centers Interfollicular area Increased/Decreased area

Increased/Decreased numbers of lymphocytes FAE ulceration Prominent HEV Increased numbers of Give location Apoptotic cells Tingible body macrophages Plasma cells Pigmented macrophages Granulocytes (type)

Granuloma/macrophage aggregates Necrosis

Bone Marrow: Enhanced Histopathology Checklist Chemical/Animal# Yes/⇑⇓/Severity Grade No Increased/Decreased number of cells

Myeloid Erythroid Myeloid/Erythroid ratio Ratio =

Megakaryocytes Adipocytes Reticular adventitial cells Macrophages Granulocytes (type) Maturation index abnormal?

Myeloid Erythroid Hemosiderin Increased Decreased Necrosis Hemorrhage Fibrosis Granuloma Neoplasia