Catherine J. Benson, Jed A. Overmann *, Consulting Editors Proper test choice and sample acquisition, preparation, and submission of specimens to a reference laboratory are crucial to making diagnostic decisions to aid in patient care and treatment. Numerous preanalytical factors such as incorrect labeling of samples, presence of interfering substances, use of incorrect collection tubes for a certain test, sampling the wrong tissue for cytologic evaluation, and samples with low cellularity can all impact data results and interpretation. Hematologic and cytologic sample quality can also be affected by the ambient storage temperature and the time interval between collection and evaluation. Care should be taken to eliminate or minimize sources of preanalytical errors to ensure high-quality laboratory results. Hematologic data and cytologic results should always be interpreted in light of the clinical history, physical examination findings, and other diagnostic testing such as imaging or histopathology results. If there are questions regarding sample collection, submission, or handling, contact the reference laboratory. Venipuncture site should be based on where it is safe to access a vein that will not collapse when blood is withdrawn.1 In horses and goats, the jugular vein is most commonly used for blood collection. Alternative but often less recommended sites for venipuncture in horses resulting from safety concerns include the facial sinus, cephalic vein dorsal to carpus, and the saphenous veins. In cattle, the jugular and coccygeal (tail) vein can be accessed. Less often, the mammary vein is used due to the risk of personal injury and of hematoma formation at the blood draw site. Depending on the venipuncture site, species, and size of the animal, an 18- to 22-gauge needle and syringe or a double-ended needle and sheath or Vacutainer system (Becton Dickinson and Co., Franklin Lakes, NJ) with collection tube can be used to collect blood after the site of venipuncture is cleaned with an absorbent and 70% isopropyl alcohol and the vein is digitally occluded. After venipuncture, digital pressure should be applied over the site where the needle is removed to prevent hematoma formation.2 Good technique is important because traumatic venipuncture can result in platelet activation and clumping of platelets. This can falsely decrease the measured platelet count generated by hematology analyzers. Samples for complete blood cell counts (CBCs) should be submitted in ethylenediaminetetraacetic acid (EDTA) tubes, filled to the proper amount indicated on the container, and inverted several times to ensure proper mixing of the blood with the anticoagulant. Too little blood may result in a falsely decreased pack cell volume (PCV) caused by shrinkage of red blood cells (RBCs). When collecting for both a CBC and serum biochemical profile, blood should first be put in an EDTA tube and then in a serum tube that does not contain anticoagulant. Care must be taken to avoid contaminating the blood remaining in the syringe with retrograde flow of EDTA blood, since this can alter calcium and potassium values on the serum biochemistry.1 Samples for coagulation testing (prothrombin time and activated partial thromboplastin time) should be placed in a sodium citrate tube at a proportion of 1 : 9 with blood. Concurrently sending blood from a clinically healthy animal can serve as a control to help assess artifacts if there is a delay in sample transit. Samples for plasma fibrinogen concentration can be submitted either as whole blood in EDTA tubes or in sodium citrate tubes.3 Consultation with the reference laboratory is advised because the technique used by a specific laboratory will dictate how the sample should be submitted. Blood smears for microscopic evaluation should be made immediately to minimize artifacts from deterioration of cells. Blood smears should also routinely be made for manual differential counting and to evaluate for hemoparasites and atypical cells that would otherwise not be detected. Hematologic results are affected by the ambient storage temperature and the time interval between collection and evaluation. If samples cannot be run immediately on a hematology analyzer, they should be refrigerated. In cattle and goats, if blood samples are handled appropriately, hemoglobin concentration, RBC count, and total white blood cell (WBC) count are stable for the first 24 hours after collection. PCV is stable for up to 12 hours with mild increases at 24 hours.4 Blood should not be frozen because this will rupture the cells.1 Shipping and packaging of samples for hematologic and cytologic evaluation should follow guidelines from the reference laboratory. Proper labeling of slides will help ensure accurate record keeping and help minimize error. Slides that have one edge that is frosted should be labeled with a pencil with the patient identification and sample site before the sample is put on the slides. This is preferred to “permanent” ink marking pens because the preparation stains and fixatives used in the staining process will often wash off the ink.5 When submitting samples to a diagnostic laboratory, it is important to fill out a complete history including the patient signalment, tissue sampled, location, duration of the mass or illness, a description of the mass or lesion (color, three-dimensional size for masses, shape, texture, consistency, etc.), other important clinical findings (bloodwork, surgical, imaging results), and differential diagnoses. Minimizing the amount of blood present on slides is also important, especially in cases in which inflammation is suspected. In these cases, cytologic findings may need to be interpreted in conjunction with CBC data to aid in determining if inflammation is present or if leukocytes are blood associated. Samples can be obtained by fine needle aspirate, impression smears, scrapings, and swabs of lesions. Depending on the sample, blood smear, squash, or roll preparations can be made to spread cells on the slide. Once air-dried samples are made, they can be stained with Romanowsky type stains (Wright-Giemsa, Diff-Quik, etc.). Although stained slides may be submitted, it is also important to include some unstained slides when submitting samples to a diagnostic laboratory. Keep unstained cytology samples away from formalin when histopathology samples are also being prepared and submitted because the formalin fumes can render cytology samples nondiagnostic.5
Collection and Submission of Samples for Hematologic and Cytologic Evaluation
Venipuncture Site and Technique
Handling and Transportation of Samples
General Principles for Submitting Cytology Samples
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Collection and Submission of Samples for Hematologic and Cytologic Evaluation
Chapter 23
