Pathology Submissions
IHC Submissions
NecropsyMaximum number of animals (similar age and clinical problem) to be included in 1 accession:
Cattle: 1 adult or 2 calves
Cats: 1 adult or 3 kittens
Dogs: 1 adult or 3 puppies
Horses: 1 adult or 2 foals
Sheep: 1 adult or 3 lambs
Swine: 1 adult or 3 pigs
Abortion/Stillbirth: 3 fetuses w/placenta
Shipping containers for mail or courier services must be leak-proof and airtight-All species

-Carcasses must be held at refrigeration temperature until delivery to ADDL.

-DO NOT freeze unless interval between death and delivery > 3 days.

-Carcasses smaller than 150lbs can be delivered after hours to the walk-in refrigerator on the south dock (be sure to place red hold tag with owner's name, date and contact information on animal before placing in white cooler).

-After hours, if delivering an animal over 150lbs and will not fit in the white cooler please contact ADDL at 765-494-7440 to be put in contact with the pathologist on duty.

-Gross necropsy results will be reported in 1-5 working days; final reports are completed within 5-14 working days.

-ADDL does not euthanize companion animals, including horses.
Cosmetic necropsy1 animal-Prior arrangements with ADDL are required. However, cosmetic necropsy is discouraged because it prevents thorough postmortem examination and may hinder diagnosis.

-Animal must be in ADDL before 1 pm and will usually be returned to owner the same day. Additional charge applies.
Legal/Insurance Necropsy1 animalMust be designated as such prior to necropsy. Additional charge applies.
Field ConsultationCall to schedule. Field consultations are made only at the request of a veterinarian.
Practitioner-performed necropsy-Brain: whole or half

-Spinal cord: entire, with opened dura

-Eyes: whole

-Endocrine glands: small, whole; larger glands, bisected

-Heart, lung, liver, kidney, spleen: 5 mm thick slices (whole heart if cardiac defect is suspected)

-GI tract: 3-5 cm segments
10% formalin-Brain, heart, lung, liver, kidney and spleen plus any other tissues with gross lesions should be submitted from all necropsy examinations. Other tissues should be included according to history or clinical signs.

-Formalin-fixed specimens accompanied by fresh (unfixed) specimens for ancillary testing (microbiology, toxicology, virology etc) are processed as long-form accessions. Histologic findings are reported in 3-5 working days with final reports in 7-15 days.
Amputations-Small specimens, e.g. digits, may be submitted whole

-Larger specimens, e.g. limbs, should be refrigerated and delivered to ADDL within 24 hours
10% formalin -Refrigeration for large specimensBony specimens will require 1-10 day delay for decalcification. Additional charges apply.

-Limb amputations can be submitted as a "Biopsy Plus" for dissection and gross examination by a pathologist before histopathology. These will be charged as a necropsy.
HistopathologyVarious tissue specimensLeak-proof wide-mouth jar or Whirl Pak-Tissues should be fixed in 10% buffered formalin with 10:1 formalin to tissue ratio.

-Excess formalin can be decanted after 1-2 days fixation.
Endoscopic, pinch, core (Tru-cut), or punch biopsiesEntire biopsy specimen: if <3mm in any dimension, submit in histologic cassette to avoid specimen damage or loss10% formalinMultiple specimens should be submitted in individually labeled containers to indicate tissue site. Histologic findings from Neoplasm Submission Form accessions are reported in 1-3 working days. Histochemistry or immunohistochemistry requires another 1-5 working days.
Excisional biopsiesIf >2cm in diameter, slice accordingly to allow fixation10% formalinUse paint or suture tags to orient specimen or identify margins, if pertinent. Histologic findings from Neoplasm Submission Form accessions are reported in 1-3 working days.
Histopathology Submission Information
  • Please complete an appropriate Submission Form, available on the Forms page, and submit with sample.
  • Tissue samples should be no more than 0.5 cm thick and placed in a wide mouth jar containing 10% buffered formalin fixative.
  • For fixation, the formalin amount should be at least 10X the volume of the sample.
  • Alternatively, tissues may be fixed for 24-48 hours and then transferred to a Whirl Pak bag with enough formalin to prevent drying.
  • Tissues should arrive at ADDL already fixed in formalin.
  • If formalin is not used as the fixative, please indicate which fixative was used.
  • Before shipping, contact the postal service or courier service to be sure to comply with their regulations.
  • Histopathology mailers are available from ADDL. See the Supplies page.
Painting the Surgical Margins of a Tumor Biopsy
A common problem faced by pathologists is interpreting surgical margins of tumor biopsies. Although it is often easy to distinguish surgical margins from those produced during the trimming process in the histology laboratory, it is sometimes difficult and frustrating for both pathologists and clinicians. Diagnosticians know that the three most common questions posed by the clinician regarding a neoplastic process are "What kind of tumor is it?," "Is it benign or malignant," and "Are the margins free of neoplastic cells?"
In human medicine, surgical margins of a biopsy are commonly painted with a dye that adheres to tissue and is visible under the microscope. The procedure is simple and does not interfere with histologic evaluation. Its advantage is that it clearly distinguishes surgical margins from trimming margins. This is essential to assess complete/incomplete excision of a tumor.
Biopsy margin painting can be done on unfixed or fixed tissues; however, painting unfixed tissues is easier and preferred. There are several commercially available products for this purpose (call the ADDL for additional information, if needed). The use of different colors for different aspects of mass orientation is superior to using sutures of different colors. Painting biopsy margins is also inexpensive; a 20 ml bottle of dye will last several years.
Please check the Winter 2004 Newsletter for additional information.
How to Paint the Surgical Margins of a Tumor Biopsy
  1. Blot the biopsy margins of the mass.
  2. Select the dye color.
  3. With a wooden applicator stick or cotton swab, "paint" the biopsy margin.
Do not pour dye on the surface; apply as if painting.
  1. Let the dye dry for 5-10 minutes, and immerse the sample in regular fixative solution. Some of the dye will dissolve with the fixative; this will not affect the sample. If the sample is thicker than 4-6 mm, section it to improve penetration of fixative as you usually do.
Immunohistochemistry General Information
Immunohistochemistry (IHC) uses immunologic and histologic techniques to detect antigens in tissues. The antigen is recognized by a specific antibody that is added to the tissue section. The immunologic reaction is visualized under the microscope by adding an enzyme, a substrate to the enzyme, and a chromogen, producing a colored reaction. IHC is a very sensitive and specific technique. For diagnosticians, it is an important technique that allows us to colocalize antigens and lesions in the same tissue section.
Neoplastic and infectious diseases are the main focus of IHC in veterinary medicine. The ADDL IHC Service offers a variety of tests for both infectious and neoplastic diseases:
  • Diagnosis of neoplasia: Often, the tissue of origin of a tumor cannot be determined with routine histology. Using specific antibodies for different tissues or cells (e.g. cytokeratin for epithelium, vimentin for mesenchymal cells, lymphoid markers, etc), the origin of many tumors can be determined with IHC.
  • Diagnosis of micrometastases: Early metastasis can be difficult to detect using conventional histology. IHC highlights the presence of single or small groups of neoplastic cells in metastatic sites. Early detection of micrometastases increases the chances of survival with surgical removal of affected nodes or by modification of the treatment protocol.
  • Prognostic markers: Some proteins are expressed in neoplastic, but not in normal, mature cells (e.g., embryonal proteins), expressed in neoplastic cells in larger amounts than in normal cells (e.g. cycle-related proteins), or structurally modified in neoplastic cells (mutant p53 protein). These changes may have prognostic significance in specific tumor types. For instance, it has been reported that the immunohistochemical detection of c-Kit protein in mast cell tumors of dogs has prognostic significance. Some of these markers are being tested to determine their significance in veterinary cancers.
  • Diagnosis of infectious diseases: Detection of antigens of an infectious agent using IHC has etiologic significance. Antigen detection can be correlated with histopathologic changes and thus can confirm the significance of a particular microorganism detected by other methods.
How to Submit Samples for Immunohistochemical Testing
  • Please complete an appropriate Submission Form, available on the Forms page, and submit with sample.
  • We test samples that have been fixed in formalin, so you do not have to do anything special.
  • Submit the sample as you would for routine histopathology.
  • Do not hold fixed samples in your office longer than 2 days as prolonged fixation may destroy antigens. As soon as you place your sample in formalin, send it to the ADDL.
Infectious Diseases
Bovine respiratory syncytial virus
Bovine virus diarrhea
Coronavirus, bovine
Coronavirus, porcine
Coronavirus, feline
Cryptosporidium parvum
Distemper virus
Feline leukemia virus
Francisella tularensis
Herpesvirus 1, bovine
Herpesvirus 1, equine
Herpesvirus 1, feline
Influenza A
Mycobacterium bovis
Neospora caninum
Papilloma virus
Parvovirus, canine
Porcine circovirus 2
PRRS virus
Rotavirus A
Sarcocystis neurona
Toxoplasma gondii
West Nile Virus
Yersinia pestis
Tissue/Cell MarkerUse
Actin, muscleAll muscle
Actin, sarcomericStriated muscle
Actin, smooth muscleSmooth muscle
B-cell marker (BLA36)B-cell lymphoma, some histiocytic tumors
CD3T-cell lymphoma
CD10Renal tubular epithelium
CD11dSome histiocytic tumors
CD18Leukocytic tumors
CD20B-cell lymphoma
CD31Vascular tumors
CD45Leukocytic tumors
CD79aB-cell lymphoma
CD117 (c-Kit protein)Mast cell tumors
CalcitoninC-cell tumors of thyroid gland
CalretininRenal tubules, nerve tissue
E-CadherinLangerhan cells, epithelium, histiocytomas
Caspase-3Apoptotic cells
Chromogranins A+BGeneral neuroendocrine marker
COX-1Normal urothelium, endothelium
Cytokeratin 5Myoepithelium, basal cells
Cytokeratin 7Glandular epithelium
Cytokeratins (MNF and AE1/AE3)General epithelial marker
Cytokeratins high molecular weightSquamous epithelium, mesothelium, hepatocytes
DesminMuscle tumors
Estrogen receptorEstrogen receptor-positive tissues and tumors
Factor VIII-related antigenVascular tumors
Glial fibrillary acidic proteinNeural (glial) tumors
GlucagonPancreatic islet tumors
Glucose transporter (Glut-1)Peripheral nerves, kidney
Hepatocyte marker 1Hepatocytes and their tumors
Tissue/Cell MarkerUse
Immunoglobulin kappa chainsPlasmacytomas, immunocomplex disease
Immunoglobulin lambda chainsPlasmacytomas, immunocomplex disease
Inhibin-alphaSex-cord testicular and adrenal cortical tumors
InsulinInsulin-producing tumors
Ki-67Cell proliferation
Melan AMelanomas, steroid-producing tumors
MHC IIAntigen presenting cells, lymphocytes
Microphthalmia transcription factorMelanomas
MUM1Plasmacytomas, some B-cell tumors
MyoglobinStriated muscle
Myeloid-histiocytic markerMacrophages, myeloid cells
Natural killerPeripheral nerve cell tumors
Nerve growth factor receptorNeural tumors
NeurofilamentNeural tumors
Neuron specific enolaseGeneral neuroendocrine marker
p53p53 abnormal (mutated) detection only
p63Myoepithelium, basal cells
PGP 9.5General neuroendocrine marker
Progesterone receptorPositive tissues/tumors
S-100 proteinGeneral neural marker, neuroendocrine tumors
SomastotatinPancreatic islet cell tumors
SynaptophysinGeneral neuroendocrine marker
ThyroglobulinThyroglobulin-producing cells
Thyroid transcription factor 1Lung/thyroid tumors
TryptaseMast cell tumors
Uroplakin IIIUrothelium and its tumors
VimentinGeneral marker for mesenchymal tumors