clinical techniques Victoria Hampshire, VMD, Column Editor
Optimizing research animal necropsy and histology practices Victoria Hampshire, VMD & Marian Rippy, MS, DVM, PhD, Dipl. ACVP
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© 2015 Nature America, Inc. All rights reserved.
Reduction in animal numbers is an important tenet of laboratory animal research. Research animals should undergo complete necropsy at the close of a study to yield valuable information about the effects and safety of experimental manipulations. Proper and thorough tissue collection, storage and assessment are paramount in attaining the most efficient use of animals in research. The US Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training1 are the building blocks of US policy on humane care of animals in research. Principle 2 states, “Procedures i nvolving animals should be designed and p erformed with due consideration of their relevance to human or animal health, the advancement of knowledge, or the good of society” 1. Sound necropsy and tissue s torage and assessment policies for research animals are needed to ensure that essential information is g athered so that study results can be c orrectly evaluated. In the same vein as establishing best practices for in-life assessment of research animals2, standard operating procedures (SOPs) for necropsy and pathology should be developed and implemented. Common standards for the collection and reporting of findings (or a lack thereof) in animal tissues have emerged in response to public outcries from the Belmont report that addressed human subjects protection and Good Laboratory Practice (GLP) guidance from the US Food and Drug Administration that established conditions for quality systems for reporting preclinical testing data3,4. Definitions Pathology tends to be used as a catch-all term for the collection and examination of tissues, but there are different types US Food and Drug Administration, Silver Spring, MD. Correspondence should be addressed to V.H. ([email protected]).
170 Volume 44, No. 5 | MAY 2015
of pathology experts with different levels of training. The American College of Veterinary Pathologists (http://www.acvp. org/) certifies competency in two fields: clinical and anatomic pathology. Clinical pathologists generally work in off-site laboratories from the animal facility and examine blood and other body fluids to diagnose disease. Anatomic pathologists specialize in dissection of whole animals, selection of organs or tissues for further analysis (e.g., histology or microscopy) and correlation of gross findings with histological or microscopic findings. Personnel Federal laws require that personnel engaged in laboratory studies be properly trained to carry out the assigned functions 1,4. The ideal SOP invokes the standard of care of the veterinary profession, which is that clinical veterinarians must determine, on the basis of the differential d iagnosis they assign to a particular condition2, which body fluids should be collected and subjected to clinical
or toxicological p athology examination. They or their designated technicians then collect the fluid or tissue of interest using proper techniques to yield optimal results. Board-certified veterinary pathologists or their designees should generally carry out gross dissection, document findings, procure representative specimens, and send specimens to a h istology laboratory for processing, embedding and sectioning specific to the organ, tissue or device of interest. Facilities and equipment GLP regulations require the use of SOPs for necropsy of animals, c ollection and i dentification of specimens, and histopathology. In particular, n ecropsy areas in laboratory animal facilities generally must include a vented stainless steel table with a surface grate to allow body fluids to flow to dedicated waste areas. Fumes should be vented downward and through the exhaust ventilation s ystem. Particular attention should be paid to design, construction and
TABLE 1 | Basic necropsy tools Small animals Small and large surgical scissors Small and large forceps Tissue trimming and grossing boards Scalpels (#11 and #10) Liston bone cutters Bone elevators Dental probes and picks Extra fine bone saw Surgical loupes (for mice) Personal protective equipment
Large animals Small and large surgical scissors Large forceps Pruning shears (ribs and sternum) Hatchet Small crosscut saw Boning knife Scalpels and straight razor blades Oscillating bone saw (for cranium) Hair clippers and blades Personal protective equipment www.labanimal.com
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© 2015 Nature America, Inc. All rights reserved.
clinical techniques
FIGURE 1 | Excerpt from a sample necropsy form showing dorsal and ventral views of a domestic pig with key wounds, lesions and sections noted.
aintenance of these areas. Good lighting m is necessary to thoroughly examine body cavities and their contents. Tables used for necropsy of rabbits and smaller rodents should be a ugmented with trays c ontaining standard necropsy tools including scissors and forceps (Table 1). Large animal dissection areas should have a selection of sharp knives, shears and electrical saws to ensure a clean atraumatic entry and r emoval of the cranium, sinuses or v ertebrae to e xamine the detailed s tructures of the head and spinal cord (Table 1). Personal protective
e quipment should be stocked and g owning and degowning protocols should be used as appropriate for the biosafety level of the facility. Appropriate receptacles for 10% neutralbuffered formalin and other special fixatives should be shelved above the vented areas, and plastic tissue specimen jars should be readily available and p roperly marked for receipt of identified tissues. Finally, the w ritten record should be enriched with labeled, high-quality digital images. A photography stand with good lighting will produce optimal images without glare and shadows.
FIGURE 2 | Excerpt from a sample necropsy form with an illustration of the liver for noting areas of interest. LAB ANIMAL
Methods The most important feature of a good necropsy is the use of a systematic p rocess. Before commencing a necropsy, the animal should be properly identified and the r elevant clinical history should be reviewed. The necropsy process may be slightly different depending on the goal: to diagnose the cause of an unscheduled death or euthanasia due to morbidity or to assess the effects of a particular e xperimental technique, drug, device or other product on target organs and associated systemic effects. A necropsy might be documented using a form containing ventral and d orsal views of the silhouette of the animal (Fig. 1) so that distinguishing marks and wounds can be drawn and described. Organ systems should be listed and systematically checked off as they are reviewed, and the areas from which sections were taken or where lesions were found should be i llustrated and istology referenced to sections taken for h (Fig. 2). There should be adequate room on the form for descriptive comments and measurements. Pathologists or their trained technical designees should have a working knowledge of the anatomy for each species they are examining, and general anatomy texts can be kept nearby as useful r eferences. A proper sequence of e xamination and sectioning of the thorax, abdomen and head is well described5–7. Areas s uspected to be infected should be identified and cultured using sterile t echnique and can also be marked on the necropsy form (Fig. 1). After the external carcass is thoroughly examined and any abnormalities documented, the animal should be placed on its back, its legs should be reflected l aterally and a midline incision should be made to allow examination of the entire t horacic and peritoneal cavities. Any abnormal findings should be described in situ before being removed i ndividually in a pre-specified order that should be driven by the particular goals of the n ecropsy. Digital photography of the explanted organs with lesions and organ weights might be useful additions to the necropsy record. During necropsies that are done to support development of ap harmaceutical agent, several organs that may be involved in drug m etabolism (e.g., liver, kidney) are c ommonly weighed to evaluate s ystemic effects of the test agent. In contrast, n ecropsies that s upport Volume 44, No. 5 | MAY 2015 171
evelopment of a device might not require d organ weights but might instead require a detailed d escription of the device, its l ocation and findings in s urrounding ecropsies are usually t issues. These n more targeted, but the e xamination of downstream organs might also be i mportant to evaluate the safety of the device. The pathologist should use a consistent method to describe normal and abnormal tissues and to indicate why any particular portions of an organ were selected for histologic analysis. In the investigation of devices, orientation and gross photography of tissue blocks are helpful references when examining microscopic slides, but these are not as useful in drug investigations because drug effects are usually systemic. Documentation A method for documenting and archiving necropsy findings is important to the p rocess of recording post-mortem f indings. Some software programs are now available for comprehensive n ecropsy, but most c ontract research facilities use a standardized form for d ocumentation. Forms might have
check boxes to d esignate a tissue or organ as normal or abnormal, but the pathologist should use declarative statements to describe the location, distribution, color, consistency, size, shape, smell and other characteristics of a particular tissue or organ and should also explain why a particular region of tissue was sampled. Photos of tissues containing lesions are helpful additions to the record. Conclusion A thorough necropsy done by a trained expert can be paramount to u nderstanding a disease etiology or systemic interactions. Failure to thoroughly document and c orrelate the gross, microscopic and ecropsy clinicopathological findings of a n can result in an i nability to attribute conditions to any particular cause. Thus, failure to carry out thorough and well documented necropsies involving proper procurement of s pecimens could result in additional expenses for repeat studies and costly delays in the development of potentially valuable therapies and devices. Periodic re-evaluation of necropsy SOPs and overall programmatic compliance
with this important last step of the research protocol contributes to an optimal animal reduction strategy.
1. Interagency Research Animal Committee. US Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training (US Department of Health and Human Services, Washington, DC, 1985). 2. Hampshire, V. A systematic process for physical examination in preclinical research. Lab Anim. (NY) 44,89–90 (2015). 3. The National Commission for the Protection of Human Subjects of Biomedical and Behavioral Research. Ethical Principles and Guidelines for the Protection of Human Subjects of Research (Department of Health and Human Services, Washington, DC, 18 April 1979). 4. Food and Drug Association. 21 CFR. Part 58. Good Laboratory Practice for Nonclinical Laboratory Studies. 5. Andrews, J.J., Van Alstine, W.G. & Schwartz, K.J. A basic approach to food animal necropsy. Vet. Clin. North Am. Food Anim. Pract. 2, 1–29 (1986). 6. Severidt, J.A., Madden, D.J., Mason, G., Garry, F. & Gould, D. Dairy Cattle Necropsy Manual (Colorado State University, Fort Collins, CO, 2001). 7. Purdue University Animal Disease Diagnostic Laboratory. Veterinary Necropsy Technique (Purdue University, West Lafayette, IN, 2014).
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© 2015 Nature America, Inc. All rights reserved.
clinical techniques
172 Volume 44, No. 5 | MAY 2015
www.labanimal.com
Optimizing research animal necropsy and histology practices Victoria Hampshire, VMD & Marian Rippy, MS, DVM, PhD, Dipl. ACVP
npg
© 2015 Nature America, Inc. All rights reserved.
Reduction in animal numbers is an important tenet of laboratory animal research. Research animals should undergo complete necropsy at the close of a study to yield valuable information about the effects and safety of experimental manipulations. Proper and thorough tissue collection, storage and assessment are paramount in attaining the most efficient use of animals in research. The US Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training1 are the building blocks of US policy on humane care of animals in research. Principle 2 states, “Procedures i nvolving animals should be designed and p erformed with due consideration of their relevance to human or animal health, the advancement of knowledge, or the good of society” 1. Sound necropsy and tissue s torage and assessment policies for research animals are needed to ensure that essential information is g athered so that study results can be c orrectly evaluated. In the same vein as establishing best practices for in-life assessment of research animals2, standard operating procedures (SOPs) for necropsy and pathology should be developed and implemented. Common standards for the collection and reporting of findings (or a lack thereof) in animal tissues have emerged in response to public outcries from the Belmont report that addressed human subjects protection and Good Laboratory Practice (GLP) guidance from the US Food and Drug Administration that established conditions for quality systems for reporting preclinical testing data3,4. Definitions Pathology tends to be used as a catch-all term for the collection and examination of tissues, but there are different types US Food and Drug Administration, Silver Spring, MD. Correspondence should be addressed to V.H. ([email protected]).
170 Volume 44, No. 5 | MAY 2015
of pathology experts with different levels of training. The American College of Veterinary Pathologists (http://www.acvp. org/) certifies competency in two fields: clinical and anatomic pathology. Clinical pathologists generally work in off-site laboratories from the animal facility and examine blood and other body fluids to diagnose disease. Anatomic pathologists specialize in dissection of whole animals, selection of organs or tissues for further analysis (e.g., histology or microscopy) and correlation of gross findings with histological or microscopic findings. Personnel Federal laws require that personnel engaged in laboratory studies be properly trained to carry out the assigned functions 1,4. The ideal SOP invokes the standard of care of the veterinary profession, which is that clinical veterinarians must determine, on the basis of the differential d iagnosis they assign to a particular condition2, which body fluids should be collected and subjected to clinical
or toxicological p athology examination. They or their designated technicians then collect the fluid or tissue of interest using proper techniques to yield optimal results. Board-certified veterinary pathologists or their designees should generally carry out gross dissection, document findings, procure representative specimens, and send specimens to a h istology laboratory for processing, embedding and sectioning specific to the organ, tissue or device of interest. Facilities and equipment GLP regulations require the use of SOPs for necropsy of animals, c ollection and i dentification of specimens, and histopathology. In particular, n ecropsy areas in laboratory animal facilities generally must include a vented stainless steel table with a surface grate to allow body fluids to flow to dedicated waste areas. Fumes should be vented downward and through the exhaust ventilation s ystem. Particular attention should be paid to design, construction and
TABLE 1 | Basic necropsy tools Small animals Small and large surgical scissors Small and large forceps Tissue trimming and grossing boards Scalpels (#11 and #10) Liston bone cutters Bone elevators Dental probes and picks Extra fine bone saw Surgical loupes (for mice) Personal protective equipment
Large animals Small and large surgical scissors Large forceps Pruning shears (ribs and sternum) Hatchet Small crosscut saw Boning knife Scalpels and straight razor blades Oscillating bone saw (for cranium) Hair clippers and blades Personal protective equipment www.labanimal.com
npg
© 2015 Nature America, Inc. All rights reserved.
clinical techniques
FIGURE 1 | Excerpt from a sample necropsy form showing dorsal and ventral views of a domestic pig with key wounds, lesions and sections noted.
aintenance of these areas. Good lighting m is necessary to thoroughly examine body cavities and their contents. Tables used for necropsy of rabbits and smaller rodents should be a ugmented with trays c ontaining standard necropsy tools including scissors and forceps (Table 1). Large animal dissection areas should have a selection of sharp knives, shears and electrical saws to ensure a clean atraumatic entry and r emoval of the cranium, sinuses or v ertebrae to e xamine the detailed s tructures of the head and spinal cord (Table 1). Personal protective
e quipment should be stocked and g owning and degowning protocols should be used as appropriate for the biosafety level of the facility. Appropriate receptacles for 10% neutralbuffered formalin and other special fixatives should be shelved above the vented areas, and plastic tissue specimen jars should be readily available and p roperly marked for receipt of identified tissues. Finally, the w ritten record should be enriched with labeled, high-quality digital images. A photography stand with good lighting will produce optimal images without glare and shadows.
FIGURE 2 | Excerpt from a sample necropsy form with an illustration of the liver for noting areas of interest. LAB ANIMAL
Methods The most important feature of a good necropsy is the use of a systematic p rocess. Before commencing a necropsy, the animal should be properly identified and the r elevant clinical history should be reviewed. The necropsy process may be slightly different depending on the goal: to diagnose the cause of an unscheduled death or euthanasia due to morbidity or to assess the effects of a particular e xperimental technique, drug, device or other product on target organs and associated systemic effects. A necropsy might be documented using a form containing ventral and d orsal views of the silhouette of the animal (Fig. 1) so that distinguishing marks and wounds can be drawn and described. Organ systems should be listed and systematically checked off as they are reviewed, and the areas from which sections were taken or where lesions were found should be i llustrated and istology referenced to sections taken for h (Fig. 2). There should be adequate room on the form for descriptive comments and measurements. Pathologists or their trained technical designees should have a working knowledge of the anatomy for each species they are examining, and general anatomy texts can be kept nearby as useful r eferences. A proper sequence of e xamination and sectioning of the thorax, abdomen and head is well described5–7. Areas s uspected to be infected should be identified and cultured using sterile t echnique and can also be marked on the necropsy form (Fig. 1). After the external carcass is thoroughly examined and any abnormalities documented, the animal should be placed on its back, its legs should be reflected l aterally and a midline incision should be made to allow examination of the entire t horacic and peritoneal cavities. Any abnormal findings should be described in situ before being removed i ndividually in a pre-specified order that should be driven by the particular goals of the n ecropsy. Digital photography of the explanted organs with lesions and organ weights might be useful additions to the necropsy record. During necropsies that are done to support development of ap harmaceutical agent, several organs that may be involved in drug m etabolism (e.g., liver, kidney) are c ommonly weighed to evaluate s ystemic effects of the test agent. In contrast, n ecropsies that s upport Volume 44, No. 5 | MAY 2015 171
evelopment of a device might not require d organ weights but might instead require a detailed d escription of the device, its l ocation and findings in s urrounding ecropsies are usually t issues. These n more targeted, but the e xamination of downstream organs might also be i mportant to evaluate the safety of the device. The pathologist should use a consistent method to describe normal and abnormal tissues and to indicate why any particular portions of an organ were selected for histologic analysis. In the investigation of devices, orientation and gross photography of tissue blocks are helpful references when examining microscopic slides, but these are not as useful in drug investigations because drug effects are usually systemic. Documentation A method for documenting and archiving necropsy findings is important to the p rocess of recording post-mortem f indings. Some software programs are now available for comprehensive n ecropsy, but most c ontract research facilities use a standardized form for d ocumentation. Forms might have
check boxes to d esignate a tissue or organ as normal or abnormal, but the pathologist should use declarative statements to describe the location, distribution, color, consistency, size, shape, smell and other characteristics of a particular tissue or organ and should also explain why a particular region of tissue was sampled. Photos of tissues containing lesions are helpful additions to the record. Conclusion A thorough necropsy done by a trained expert can be paramount to u nderstanding a disease etiology or systemic interactions. Failure to thoroughly document and c orrelate the gross, microscopic and ecropsy clinicopathological findings of a n can result in an i nability to attribute conditions to any particular cause. Thus, failure to carry out thorough and well documented necropsies involving proper procurement of s pecimens could result in additional expenses for repeat studies and costly delays in the development of potentially valuable therapies and devices. Periodic re-evaluation of necropsy SOPs and overall programmatic compliance
with this important last step of the research protocol contributes to an optimal animal reduction strategy.
1. Interagency Research Animal Committee. US Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training (US Department of Health and Human Services, Washington, DC, 1985). 2. Hampshire, V. A systematic process for physical examination in preclinical research. Lab Anim. (NY) 44,89–90 (2015). 3. The National Commission for the Protection of Human Subjects of Biomedical and Behavioral Research. Ethical Principles and Guidelines for the Protection of Human Subjects of Research (Department of Health and Human Services, Washington, DC, 18 April 1979). 4. Food and Drug Association. 21 CFR. Part 58. Good Laboratory Practice for Nonclinical Laboratory Studies. 5. Andrews, J.J., Van Alstine, W.G. & Schwartz, K.J. A basic approach to food animal necropsy. Vet. Clin. North Am. Food Anim. Pract. 2, 1–29 (1986). 6. Severidt, J.A., Madden, D.J., Mason, G., Garry, F. & Gould, D. Dairy Cattle Necropsy Manual (Colorado State University, Fort Collins, CO, 2001). 7. Purdue University Animal Disease Diagnostic Laboratory. Veterinary Necropsy Technique (Purdue University, West Lafayette, IN, 2014).
npg
© 2015 Nature America, Inc. All rights reserved.
clinical techniques
172 Volume 44, No. 5 | MAY 2015
www.labanimal.com
