Each veterinary diagnostic laboratory offers a unique set of diagnostic tests that is subject to frequent changes as better tests become available. The protocols for sample collection and submission are also subject to change. The practitioner and diagnostic laboratory staff must maintain good communication in order to complete their diagnostic efforts efficiently and provide optimal service to the animal owner. Practitioners must be specific and clear in their test requests. The laboratory staff can provide guidance when there are questions regarding sample collection and handling, as well as offering assistance in interpretation of test results. Most diagnostic laboratories publish user guidelines with preferred protocols for sample collection and submission, but the following broad recommendations are fairly standard.Regardless of the type of submission, a detailed case history should be included with the samples to assist laboratory personnel in determining a diagnosis. The information should include: owner, species, breed, sex, age, animal identification, clinical signs, gross appearance (including size and location) of the lesion(s), previous treatment (if any), time of recurrence from any previous treatment, and morbidity/mortality in the group. If a zoonotic disease is suspected, this should also be clearly indicated on the submission sheet to alert lab personnel. The submission form should be placed in a waterproof bag to protect it from any fluids that might be present in the packaged materials. Waterproof markers should be used when labeling specimen bags and containers.
Histology:
Microscopic examination of adequately prepared tissue sections is a valuable aid to diagnosis. Cellular changes in diseased tissues are often characteristic of a specific disease or group of diseases. Use of this relatively rapid and inexpensive diagnostic technique can often result in substantial savings in time, money, and animal life. The increasing number of immunohistochemical tests that can be applied to formalin-fixed tissue has further reinforced the utility of this diagnostic technique.Autolyzed tissues are generally useless for histopathologic examination; prompt necropsy examination and organ sampling are critical. Tissues collected for histologic examination should be representative of any lesions present and, if possible, should include some of the apparently normal surrounding tissue. Tissue should not be frozen before fixation. Samples of the various organs should be cut <1 cm thick (preferably 7 mm) and placed immediately into ≥10 times their volume of phosphate-buffered 10% formalin for fixation. Thin slices or cubes ensure adequate penetration of the fixative. Because the GI mucosa decomposes rapidly, short sections of gut must be opened lengthwise to allow adequate fixation. The tissues should remain in fixative for ≥24 hr; after this initial fixation, the samples may be placed in a smaller volume of fresh formalin for shipment. Samples should be shipped in unbreakable containers and packed in a manner that prevents spillage during shipment. Fixed tissues should be protected from freezing.If the animal exhibited CNS signs, the brain and portions of the spinal cord should be submitted. When the whole brain may be required, it should be placed in concentrated formalin (40% formaldehyde), to which water is added slowly and mixed until the brain sinks to just below the surface but not to the bottom. The brain should remain in this solution for ≥24 hr, after which it may be removed and placed in a solid container in 10% formalin and either mailed (suitably packed) or held until processing is desired. Often, the brain is halved longitudinally and one-half sent unfixed (fresh), properly refrigerated, for microbiologic tests.Biopsy samples should be fixed in the same manner as necropsy tissues. Small tumors (<1 cm) should be cut in half, and larger tumors into small pieces or several representative samples.
Microbiology:
Laboratory techniques and capabilities for microbiologic examination vary; available tests include bacteriologic culture, virus isolation, in-situ hybridization, PCR, fluorescent antibody tests, latex agglutination tests, and ELISA. Therefore, it is critical to obtain specific instructions from the diagnostic laboratory on sample collection and handling. Usually, unfixed specimens are submitted and should be collected aseptically, as soon as possible after death. If PCR testing is to be performed, it is particularly important to avoid cross-contamination between multiple animals in a submission; this applies to tissues, fluids, and even dissection instruments. Tissues for most microbiologic assays may be frozen before shipment, but freezing is undesirable if samples can be chilled and delivered directly to the laboratory in a short period. Adequate refrigerant should be provided so that samples will remain chilled until they reach the laboratory.
Toxicology:
If a known toxin is suspected, a specific analysis should always be requested—laboratories cannot just "check for poisoning." A complete description of clinical and epidemiologic findings may help differentiate poisoning from infectious diseases that can simulate poisoning. Appropriate samples for many of the more common toxicities are listed in Table:Guidelines for Submitting Samples for Toxicologic Examination.
Histology:
Microscopic examination of adequately prepared tissue sections is a valuable aid to diagnosis. Cellular changes in diseased tissues are often characteristic of a specific disease or group of diseases. Use of this relatively rapid and inexpensive diagnostic technique can often result in substantial savings in time, money, and animal life. The increasing number of immunohistochemical tests that can be applied to formalin-fixed tissue has further reinforced the utility of this diagnostic technique.Autolyzed tissues are generally useless for histopathologic examination; prompt necropsy examination and organ sampling are critical. Tissues collected for histologic examination should be representative of any lesions present and, if possible, should include some of the apparently normal surrounding tissue. Tissue should not be frozen before fixation. Samples of the various organs should be cut <1 cm thick (preferably 7 mm) and placed immediately into ≥10 times their volume of phosphate-buffered 10% formalin for fixation. Thin slices or cubes ensure adequate penetration of the fixative. Because the GI mucosa decomposes rapidly, short sections of gut must be opened lengthwise to allow adequate fixation. The tissues should remain in fixative for ≥24 hr; after this initial fixation, the samples may be placed in a smaller volume of fresh formalin for shipment. Samples should be shipped in unbreakable containers and packed in a manner that prevents spillage during shipment. Fixed tissues should be protected from freezing.If the animal exhibited CNS signs, the brain and portions of the spinal cord should be submitted. When the whole brain may be required, it should be placed in concentrated formalin (40% formaldehyde), to which water is added slowly and mixed until the brain sinks to just below the surface but not to the bottom. The brain should remain in this solution for ≥24 hr, after which it may be removed and placed in a solid container in 10% formalin and either mailed (suitably packed) or held until processing is desired. Often, the brain is halved longitudinally and one-half sent unfixed (fresh), properly refrigerated, for microbiologic tests.Biopsy samples should be fixed in the same manner as necropsy tissues. Small tumors (<1 cm) should be cut in half, and larger tumors into small pieces or several representative samples.
Microbiology:
Laboratory techniques and capabilities for microbiologic examination vary; available tests include bacteriologic culture, virus isolation, in-situ hybridization, PCR, fluorescent antibody tests, latex agglutination tests, and ELISA. Therefore, it is critical to obtain specific instructions from the diagnostic laboratory on sample collection and handling. Usually, unfixed specimens are submitted and should be collected aseptically, as soon as possible after death. If PCR testing is to be performed, it is particularly important to avoid cross-contamination between multiple animals in a submission; this applies to tissues, fluids, and even dissection instruments. Tissues for most microbiologic assays may be frozen before shipment, but freezing is undesirable if samples can be chilled and delivered directly to the laboratory in a short period. Adequate refrigerant should be provided so that samples will remain chilled until they reach the laboratory.
Toxicology:
If a known toxin is suspected, a specific analysis should always be requested—laboratories cannot just "check for poisoning." A complete description of clinical and epidemiologic findings may help differentiate poisoning from infectious diseases that can simulate poisoning. Appropriate samples for many of the more common toxicities are listed in Table:Guidelines for Submitting Samples for Toxicologic Examination.
If there is doubt about sample submission procedures, the laboratory should be contacted.Tissues or fluids for chemical analysis should be as fresh as possible and kept in a refrigerator or preserved chemically; packing with ice is preferred. A polystyrene refrigerator box, metal can, or stout cardboard box may be used for shipment. Packing must withstand breakage if the ice melts. Samples can be preserved for 72 hr if packed in a styrofoam box with dry ice. Packages containing dry ice must be so labeled on the outside and suitably vented to prevent pressure buildup. Adequate refrigeration is of special importance when submitting clean body fluids (such as those obtained from an eye) and material for nitrate or nitrite analysis; these salts are rapidly metabolized by microorganisms.The containers for packing and transporting specimens should be free of chemicals. Plastic containers, both bags and jars, are ideal; jars with metal screw caps should be avoided. Samples should be packed individually. Containers must be labeled with all information necessary to identify the sample and, if mailed, must conform to postal regulations.If legal action is a possibility, all containers for shipment should be either sealed so that tampering can be detected or hand-carried to the laboratory and a receipt obtained. The chain of custody must be accurately documented.
If feed or water is suspected as the source of poisoning, samples of these and any descriptive feed tag should accompany the tissue samples. If at all possible, a representative composite sample of the feed should be submitted from the suspect lot or shipment. In some instances, if an adequate amount of involved feed is available, some of it may be fed to experimental animals in an effort to reproduce the signs and lesions seen in the field cases.
Hematology:
Routine studies require anticoagulated whole blood and several blood smears. Blood smears should be prepared immediately after the sample has been collected to minimize cell deterioration. Anticoagulated blood should be kept refrigerated; blood smears should not. Ethylenediamine tetra-acetic acid (EDTA) is the anticoagulant of choice for a CBC because it best preserves the cellular components of the blood and prevents platelet aggregation. Blood for coagulation testing should be collected into a blue top tube, which contains sodium citrate. After mixing, the sample should be centrifuged for 5 min, and then plasma should be removed and transferred to a clean tube without anticoagulant. The plasma should be kept frozen until the time of analysis. Whole blood should not be frozen because this causes cell lysis and gross hemolysis, which interfere with testing.
Clinical Chemistry:
Most clinical chemistry tests require serum, but an occasional test may require plasma. Anticoagulants present in plasma may interfere with tests; therefore, serum should always be submitted unless plasma is specifically requested. Because lipemia can interfere with a number of chemistry tests, dogs and cats should be fasted for 12 hr before samples are collected. For serum samples, the blood should be drawn into a red top tube or a separator tube. The sample should be held at room temperature for 20-30 min to allow complete clot formation and retraction. Incomplete clot formation may cause the serum to gel due to latent fibrin formation. The clot should be separated from the glass by gently running an applicator stick around the tube walls ("rimming"). The sample should then be centrifuged at high speed (~1,000 g; 2,200 rpm) for 10 min. Rough handling of the sample or incomplete separation of erythrocytes from serum may promote hemolysis, which can interfere with certain tests. If the sample has been collected into a serum separator tube, centrifugation will cause a layer of silicon gel to lodge between the packed cells and the serum. The gel layer should be inspected to ensure the integrity of the barrier, and recentrifugation is recommended if there is a visible crack in this layer. If a red top tube has been used, the serum should be removed and transferred to a clean tube. Serum should be refrigerated or frozen until analyzed. Many commercial laboratories provide sample containers and mailers.
Serology:
Serology generally requires serum, but plasma is often satisfactory. Samples should be collected as described for clinical chemistry tests and should always be free of hemolysis. In some instances, paired samples may be required for an adequate diagnosis. The acute sample should be collected early in the course of the disease and frozen. The convalescent sample should be collected 10-14 days later, and both samples should be forwarded to the laboratory at the same time.
Cytology:
Air-dried smears are usually acceptable. Rapid air drying of smears minimizes cell distortion, thereby enhancing diagnostic quality. However, depending on the method of staining used, some laboratories prefer alcohol-fixed smears. Samples can be obtained by fine-needle aspiration or by scraping. Imprints (touch preparations) of external lesions can also be used, although these tend to have a greater degree of contamination. Aspirated material should always be smeared before air drying. Smears of fluid can be prepared using a traditional blood smearing technique. Highly cellular fluids may be smeared directly; fluids of low cellularity should be centrifuged to concentrate the cells. Thick material or viscous fluid is more readily smeared using a squash technique in which a second glass slide is placed over the aspirated material and then slid rapidly and smoothly down the length of the lower slide. Blood or cytologic smears should never be mailed to the laboratory in the same package with formalin-fixed tissues because formalin vapors will produce artifacts in the specimen.
Fluid Analysis:
Body cavity effusions should be analyzed. Fluid analysis usually includes determination of protein content and total cell count and cytologic examination. Other tests may be performed depending on the source (eg, synovial fluid) or appearance (eg, chylous fluid) of the effusion. A sample of effusion should be collected into an EDTA (purple top) tube for routine analysis. A second sample should be collected into a serum (red top) tube if any biochemical analyses (eg, triglyceride, cholesterol, lipase) are to be performed or if a bacterial culture is desired. Smears for cytologic examination should be prepared immediately after the sample has been collected to minimize cell deterioration and other in vitro artifacts.
If feed or water is suspected as the source of poisoning, samples of these and any descriptive feed tag should accompany the tissue samples. If at all possible, a representative composite sample of the feed should be submitted from the suspect lot or shipment. In some instances, if an adequate amount of involved feed is available, some of it may be fed to experimental animals in an effort to reproduce the signs and lesions seen in the field cases.
Hematology:
Routine studies require anticoagulated whole blood and several blood smears. Blood smears should be prepared immediately after the sample has been collected to minimize cell deterioration. Anticoagulated blood should be kept refrigerated; blood smears should not. Ethylenediamine tetra-acetic acid (EDTA) is the anticoagulant of choice for a CBC because it best preserves the cellular components of the blood and prevents platelet aggregation. Blood for coagulation testing should be collected into a blue top tube, which contains sodium citrate. After mixing, the sample should be centrifuged for 5 min, and then plasma should be removed and transferred to a clean tube without anticoagulant. The plasma should be kept frozen until the time of analysis. Whole blood should not be frozen because this causes cell lysis and gross hemolysis, which interfere with testing.
Clinical Chemistry:
Most clinical chemistry tests require serum, but an occasional test may require plasma. Anticoagulants present in plasma may interfere with tests; therefore, serum should always be submitted unless plasma is specifically requested. Because lipemia can interfere with a number of chemistry tests, dogs and cats should be fasted for 12 hr before samples are collected. For serum samples, the blood should be drawn into a red top tube or a separator tube. The sample should be held at room temperature for 20-30 min to allow complete clot formation and retraction. Incomplete clot formation may cause the serum to gel due to latent fibrin formation. The clot should be separated from the glass by gently running an applicator stick around the tube walls ("rimming"). The sample should then be centrifuged at high speed (~1,000 g; 2,200 rpm) for 10 min. Rough handling of the sample or incomplete separation of erythrocytes from serum may promote hemolysis, which can interfere with certain tests. If the sample has been collected into a serum separator tube, centrifugation will cause a layer of silicon gel to lodge between the packed cells and the serum. The gel layer should be inspected to ensure the integrity of the barrier, and recentrifugation is recommended if there is a visible crack in this layer. If a red top tube has been used, the serum should be removed and transferred to a clean tube. Serum should be refrigerated or frozen until analyzed. Many commercial laboratories provide sample containers and mailers.
Serology:
Serology generally requires serum, but plasma is often satisfactory. Samples should be collected as described for clinical chemistry tests and should always be free of hemolysis. In some instances, paired samples may be required for an adequate diagnosis. The acute sample should be collected early in the course of the disease and frozen. The convalescent sample should be collected 10-14 days later, and both samples should be forwarded to the laboratory at the same time.
Cytology:
Air-dried smears are usually acceptable. Rapid air drying of smears minimizes cell distortion, thereby enhancing diagnostic quality. However, depending on the method of staining used, some laboratories prefer alcohol-fixed smears. Samples can be obtained by fine-needle aspiration or by scraping. Imprints (touch preparations) of external lesions can also be used, although these tend to have a greater degree of contamination. Aspirated material should always be smeared before air drying. Smears of fluid can be prepared using a traditional blood smearing technique. Highly cellular fluids may be smeared directly; fluids of low cellularity should be centrifuged to concentrate the cells. Thick material or viscous fluid is more readily smeared using a squash technique in which a second glass slide is placed over the aspirated material and then slid rapidly and smoothly down the length of the lower slide. Blood or cytologic smears should never be mailed to the laboratory in the same package with formalin-fixed tissues because formalin vapors will produce artifacts in the specimen.
Fluid Analysis:
Body cavity effusions should be analyzed. Fluid analysis usually includes determination of protein content and total cell count and cytologic examination. Other tests may be performed depending on the source (eg, synovial fluid) or appearance (eg, chylous fluid) of the effusion. A sample of effusion should be collected into an EDTA (purple top) tube for routine analysis. A second sample should be collected into a serum (red top) tube if any biochemical analyses (eg, triglyceride, cholesterol, lipase) are to be performed or if a bacterial culture is desired. Smears for cytologic examination should be prepared immediately after the sample has been collected to minimize cell deterioration and other in vitro artifacts.
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