Practitioners frequently submit tissue and fluid samples to pathologists—typically employed at academic institutions, state laboratories, or private diagnostic laboratories—to assist them in the diagnosis of infectious diseases, inflammatory or metabolic disorders, and neoplasia in veterinary patients. Pathologists typically provide reports that include a description and interpretation of their findings, the degree of confidence in the diagnosis, a list of differential diagnoses, and recommendations for additional tests or procedures. The common goal for the practitioner and pathologist is to arrive at a correct diagnosis or to help narrow the list of differential diagnoses.
Sample submission complications that can result in an unsatisfactory outcome for practitioners include delay in obtaining a diagnosis, receiving an incorrect diagnosis, or receiving an inconclusive report. Many of these complications can be prevented if samples are submitted correctly and the practitioner and pathologist communicate well.
In this article, we answer common questions associated with pathology sample submission for cytologic and histologic examination and address a few of the problems that arise in the collection, submission, and interpretation of samples. The goal is to improve communication between practitioners and pathologists at all diagnostic laboratories and improve outcomes for optimal patient care.
IS SUBMITTING A HISTORY THAT IMPORTANT?
Obtaining a thorough history is important in clinical medicine, and that applies to pathology as well. Unfortunately, it is relatively common for samples to be submitted with little, if any, patient history. Without a thorough history, practitioners may receive pathology reports that seem noncommittal or indicate uncertainty in the diagnosis. Pathologists may be reluctant to commit or express a high degree of confidence in a diagnosis if the cytologic or histologic findings are not straightforward and a thorough history is not provided. Treatment protocols and even the decision to euthanize a patient may be based on pathology reports. Thus, in the absence of key information about the case, pathologists may often provide a conservative interpretation.
At a minimum, patient signalment and any significant abnormalities identified from the results of physical examination, blood work, or diagnostic imaging should be provided. Furthermore, providing a working diagnosis or differential diagnoses list is often helpful to the pathologist in making an interpretation or ruling in or ruling out specific diseases. This information is crucial for successful communication between practitioners and pathologists.
SHOULD THE SOURCE FOR THE SAMPLE BE IDENTIFIED?
A misconception exists that pathologists can interpret sample findings without knowing the specific source (e.g. tissue, body cavity). Practitioners may assume that if a cellular sample is obtained or an adequate amount of tissue is submitted, the pathologist should be able to identify the tissue source and provide an accurate interpretation. While in some cases that assumption is correct, failure to indicate the sample source or providing incomplete information about the source may result in several problems.
First, the pathologist may be hesitant to interpret the sample findings without knowing the precise origin, which may lead to unnecessary delays while the laboratory or pathologist attempts to contact the practitioner to obtain this additional information.
Second, an incorrect or speculative diagnosis may result. For example, considerable overlap exists in the cytologic appearance of certain tumors that arise in various tissues and organs. Thus, the pathologist may be forced to speculate or simply provide a list of possibilities that may correlate with the microscopic findings.
A related problem occurs when samples are submitted with only a notation that a mass is present and without an appropriate description of the lesion. An adequate gross description of the lesion, such as a "rapidly growing, 3-cm firm mass in the skin over the right thigh attached to underlying tissue" instead of "skin mass," helps the pathologist accurately interpret cytologic and histologic findings.
Another important part of submission is to properly label all slides and containers with the patient name and sample source or tissue of origin. For
cytologic specimens, slides with a frosted edge are ideal, as this area can easily be labeled with a pencil. Labeling slides with a marker or attaching tape labels is discouraged. Tape labels may need to be removed for slide processing, and marker ink often dissolves during fixation and staining. Improperly labeled slides or containers can result in sample misidentification or unnecessary delays. All pertinent patient information and clinical and diagnostic findings should be included on the submission form.
Unfortunately, lawsuits in veterinary medicine occur, and a pathology report may play an integral role in the case management or outcome. If a sample was submitted improperly or requested information was not provided (history, sample source, or both), a potential liability exists. Proper sample submission is a simple task that increases the likelihood of receiving satisfactory results and protects the practitioner.
For example, a sample containing numerous small lymphocytes may be interpreted differently in the absence of an appropriate history and source. If the sample was obtained from a marginally increased popliteal lymph node, the findings might reflect a normal or slightly hyperplastic population. However, if the sample was obtained from a 6-cm lymph node, small cell lymphoma might be considered more likely. Another common example is a sample containing small numbers of spindle cells. If the sample was obtained from a small lesion of chronic duration, the cells might reflect traumatic injury with fibrosis or granulation tissue or both. If the sample was obtained from a baseball-sized mass, the pathologist is more likely to consider a mesenchymal tumor.
SHOULD I LOOK AT SLIDES BEFORE SUBMITTING?
Nondiagnostic cytology samples are commonly submitted for evaluation. This complication can delay patient treatment or prevent the practitioner from moving forward with additional diagnostics or procedures. Client frustration can occur over the cost of the test and time spent awaiting results, even if forewarned the procedure may be inconclusive.
When surgical biopsy samples are being submitted, practitioners are somewhat limited in assessing the sample quality. However, when samples are sent for cytologic evaluation, practitioners should stain at least one slide and examine it on low magnification (10X objective) to avoid submitting nondiagnostic samples.
Another potential problem that can result in a nondiagnostic sample is submission of slides that are understained or not properly fixed, which can make interpretation difficult or impossible. Slides that are not fixed adequately often cannot be restained and lack nuclear detail. For rapid Romanowsky-type stains (e.g. Diff-Quik—Dade Behring, Hema-Diff—Statlab), the recommended protocol for each product can be used as a general guideline. However, the time required for proper fixation and staining depends on the thickness of the preparation. In general, more time is required for highly cellular or proteinaceous specimens, such as liver aspirates or synovial fluid.
Well-stained nucleated cells should have dark-pink to purple nuclear chromatin, whereas nuclei from poorly stained cells may appear very pale blue or light pink. While understained slides can undergo additional staining at the laboratory, this may be of limited use for samples previously evaluated on oil magnification (100X objective). Immersion oil can interfere with additional staining, potentially rendering the sample nondiagnostic.
For specimens submitted for cytologic examination, submit multiple slides per site (three or four is generally sufficient), which may include stained and unstained specimens. Submitting only one or two slides, particularly if these have been previously stained, may limit cytologic evaluation depending on the sample quality.
IF THERE IS A DISCREPANCY OR PROBLEM, SHOULD I CONTACT THE PATHOLOGIST?
Contacting the pathologist is recommended if a practitioner has concerns about a pathology report (e.g. has questions about the diagnosis, prefers clarification of an interpretation, or needs help with narrowing the differential diagnoses) or the report correlates poorly with the clinical findings. However, if information such as the sample source, patient history, and pertinent clinical abnormalities was not included with the original submission form, be sure this information is readily available when the pathologist is contacted. Proper communication is essential to a positive outcome.
WHAT CAN I EXPECT WHEN I SPEAK WITH A PATHOLOGIST?
Practitioners may be apprehensive about contacting pathologists. One concern is that pathologists are too busy to discuss a particular case. However, inquiries from practitioners are relatively common, and pathologists typically correspond with practitioners in a timely manner. Another concern is that practitioners may think pathologists perceive questions as a challenge to their interpretation. Pathologists are generally receptive to discussing their interpretation, other possible differential diagnoses, and any additional test results that may strengthen the confidence in the diagnosis.
Conversely, pathologists may contact practitioners about a challenging case or if they identify a potential problem. Pathologists may also seek additional information to solidify a diagnosis or narrow their list of differential diagnoses.
Practitioners and pathologists benefit from a good working relationship established through proper communication and cooperation when challenging cases arise.
DO I NEED A SECOND OPINION?
Obtaining a second opinion is an option when a practitioner has concerns about a pathology report. However, before this option is elected, it is important to determine if the pathologist was provided an appropriate patient history, a sample source, and pertinent clinical findings. Contacting the pathologist before requesting a second opinion may resolve a case issue and alleviate concerns. Furthermore, the practitioner may find that the pathologist obtained a second opinion before completing the report, which is fairly common when dealing with challenging cases.
Another important factor to consider is why a second opinion is needed. If the interpretation does not correlate with the clinical findings, requesting a second opinion is reasonable. But when pathologists express concern that a sample is poorly representative of a lesion, obtaining a second opinion is unlikely to provide much additional information or result in a definitive diagnosis. For example, obtaining representative tissue samples can be particularly challenging with lesions involving bone or masses arising within the oral cavity (e.g. gingival or odontogenic tumors). It is not uncommon for core biopsies of bone masses to predominantly contain secondary reactive bone, with no clear evidence of the primary underlying lesion. With these types of cases, additional sample collection may be necessary to obtain a diagnosis.
COMMENTS AND RECOMMENDATIONS
Pathology reports often include a section containing comments about the case. Depending on the sample and information provided by the practitioner, the comments may be case-specific or relatively generic. In the absence of key information, when the sample quality is not ideal, or when faced with complicated cases, the pathologist may be hesitant to provide a definitive diagnosis or express a high degree of confidence in his or her interpretation. In these cases, it is common for the practitioner to receive a list of differential diagnoses and recommendations for other tests or procedures (e.g. histologic examination, special stains, serology, culture, molecular assays, or collection of additional tissue samples).
AVOID THESE COMMON PROBLEMS
Below are several tips that may be useful in preventing common sample submission and interpretation problems.
Problem: Poor tissue exfoliation or low cellularity cytology samples
Avoid using small syringes (1 ml or 3 ml) and select a 20- to 25-ga needle. A 22-ga needle with a 5-ml or 6-ml syringe seems to work well in most situations.
Certain lesions may exfoliate poorly regardless of the technique used (e.g. mesenchymal neoplasms). Aspirates from lipomas are often of low cellularity when lipid material is lost from the slide during routine processing.
Aspiration of fluctuant areas of a mass can also be problematic, as many different lesions can be cystic. Cells from the cyst wall may not exfoliate in significant numbers into the fluid, and evaluation of fluid alone is unlikely to be diagnostic. A fluctuant or cystic area could also be due to tissue necrosis or secondary inflammation that might not be representative of the lesion. Consequently, aspiration or biopsy of the lesion wall (more solid area) is recommended.
Problem: Blood contamination of cytology samples
Some practitioners prefer to collect cytology samples by using an aspiration technique (e.g. 22-ga needle attached to a 5-ml syringe). For this method, the needle should be inserted into the mass or tissue and the plunger rapidly withdrawn once to develop negative pressure. Excessive withdrawal of the plunger will not result in a better sample but will increase the likelihood of blood contamination and cell rupture. This negative pressure is maintained while the needle is partially withdrawn and carefully redirected into several areas for aspiration. Redirection should be done carefully to avoid excessive tissue trauma and bleeding. The negative pressure is then gently released before fully removing the needle, to avoid loss of cells into the syringe. After removal, the needle is detached from the syringe, and air is drawn into the barrel. The needle is then reattached to the syringe, and the sample is expelled onto the slide. The sample is then prepared for staining using techniques discussed in the next section.
A sewing machine, or woodpecker, technique, which uses only the needle hub without syringe aspiration, is an excellent alternative to the aspiration technique described above. The needle is placed into the lesion followed by a controlled sewing machine motion for cell collection. The needle may be partially or fully withdrawn and redirected to sample additional areas of the lesion. The needle is then attached to an air-filled syringe, and the sample is expelled onto a slide for further preparation.
Keep in mind that certain lesions such as hemangiomas, hemangiosarcomas, thyroid tumors, and mast cell tumors are frequently associated with marked blood contamination because of the intrinsic vascular nature of the lesion.
Problem: Poor cell preservation of cytology samples
Several excellent veterinary textbooks address collection and preparation of cytologic specimens in detail (see the suggested reading list below). Additionally, taking the following steps will increase the likelihood of obtaining a high-quality specimen by maintaining cell preservation.
1. Avoid using small-gauge needles (< 25 ga), as cells may be destroyed during aspiration or when they are expelled from the needle onto the slide. (Note: Some pathologists discourage use of 25-ga needles, whereas others think these are acceptable for obtaining diagnostic samples).
Slides can also be prepared for staining by using the same technique used for blood smears. The blood smear technique is often useful for cystic or fluctuant lesions or body cavity effusions.
Yet another option for slide preparation is the starfish technique. This technique is performed by carefully dragging a needle through the sample to create multiple thin strips of material radiating away from the center. The starfish technique can be particularly useful if cell rupture is a recurring problem when using the squash prep technique.
If impression smears are being made (e.g. from a surgical biopsy), it is often useful to lightly blot the tissue sample with gauze or absorbent paper to remove excess blood. The surface of interest is then lightly touched to the slide several times, creating a row of imprints. Excessive force or smearing of the tissue should be avoided to minimize cell rupture.
3. Never allow specimens collected for cytologic evaluation to come into contact with formalin or formalin fumes. Such contact also results in severe artifact and often renders the sample nondiagnostic (Figure 3). This is a potential complication when unstained slides and biopsy samples are shipped in the same package.
Problem: Inconclusive histologic findings or unsatisfactory results
Collect as large a sample as possible (within reason) to increase the likelihood of obtaining representative tissue sections. Collect multiple samples if necessary, particularly if there appear to be large areas of hemorrhage or necrosis.
It is important that a sufficient amount of formalin is used for the size of the sample and container. At a minimum, a ratio of 10:1 of 10% neutral-buffered formalin-to-tissue volume should be used, though some pathologists recommend a 20:1 ratio. The rate of formalin penetration and fixation reduces dramatically with increasing thickness of tissue, and unfixed tissue will undergo autolysis, which will impair or prevent diagnostic interpretation. So practitioners need to be aware of the thickness guidelines and adhere to them whenever possible. This holds particularly true for vascular, often congested tissues such as the spleen, liver, and kidney.
Sample sizes 0.5 to 1 cm thick are ideal for optimum fixation and should be provided for representative lesions in which complete excision is not achieved or is impractical. For larger submitted specimens, incision into the tissue may be necessary to allow the formalin to completely penetrate the tissue (e.g. splenectomy with a mass lesion). For very small specimens, submission of the sample in a tissue cassette is recommended, with sponges used for specimens smaller than cassette grate size.
For excisional mass lesions when border evaluation is necessary (e.g. mast cell tumors, soft tissue sarcomas), the specimen should be submitted in full. Designate borders appropriately (e.g. ink, sutures), and provide an explanation to orient the technician or pathologist for tissue sectioning. For example, write, "Two sutures are placed at the cranial border, and one suture is placed at the caudal border." Aside from ink labeling or placing suture for orientation, the borders of the specimen should not be dissected or incised.
For endoscopic biopsies, samples should be numerous (eight to 10 per location) to offer the best opportunity for evaluation of full-thickness mucosa and submucosa. Samples of only superficial mucosa are less diagnostic.
Use care when collecting and handling tissues to avoid crush artifact. This complication is common with skin biopsies. The interface of lesions and normal tissue is important for the pathologist in general, but particularly with skin samples. Such interfaces should always be included with skin biopsies. If skin lesions are large or multifocal, multiple biopsies are recommended to help ensure that the samples are representative. Avoid sampling of only ulcerated areas, as an intact epidermis is often critical to a diagnosis. Traumatic removal of surface crusts should be avoided; any crusted debris removed should be included in the submission.
The tissues should be submitted in jars or containers labeled with patient name and tissue source. When specimens from multiple sites are collected, each container should be labeled individually with patient name and source. Tissues to be submitted for histologic examination should never be frozen, unless specifically requested by the laboratory or pathologist for special testing. As always, provide patient history and information or results pertinent to the case on the submission form.
Proper sample submission and better communication between practitioners and pathologists will increase the likelihood of a positive outcome. This will enhance patient care, improve client satisfaction, and reduce the chance of complications.
Seth Chapman, DVM, MS, DACVP (clinical pathology)
300 E. Wilson Bridge Road
Worthington, OH 43085
Jason Roberts, DVM
West Tennessee Animal Diseases Diagnostics Laboratory
The University of Tennessee
Martin, TN 38238
1. Campbell TW, Ellis CK. Avian and exotic animal hematology and cytology. 3rd ed. Ames, Iowa: Blackwell, 2007.
2. Cowell RL, Tyler RD, Meinkoth JH, et al. Diagnostic cytology and hematology of the dog and cat. 3rd ed. St. Louis, Mo: Mosby, 2008.
3. Cowell RL, Tyler RD. Diagnostic cytology and hematology of the horse. 2nd ed. St. Louis, Mo: Mosby, 2002.
4. Raskin RE, Meyer DJ. Canine and feline cytology: A color atlas and interpretation guide. 2nd ed. St. Louis, Mo: Saunders, 2010.
The authors would like to thank the following individuals for their insight and contributions:
MedVet Medical and Cancer Center for Pets, Worthington, Ohio: Lisa Fulton, DVM, DACVIM (oncology), and Eric R. Schertel, DVM, PhD, DACVS
Charles River Laboratories, Department of Pathology, Reno, Nevada: David V. Calise, DVM, MS, DACVP (anatomic pathology), and Angela Wilcox, BVSc, MS, DACVP (clinical pathology)
Texas A&M College of Veterinary Medicine, Department of Pathobiology, College Station, Texas: Mark C. Johnson, DVM, DACVP (clinical pathology)
IDEXX Laboratories, Inc.: Stephanie Corn, DVM, DACVP (clinical pathology), Worthington, Ohio; Dean Cornwell, DVM, PhD, MT, Dallas, Texas; and Rick L. Cowell, DVM, MS, MRCVS, DACVP (clinical pathology), Stillwater, Okla.