Complete Blood Count and Clinical Biochemistry Analysis If a complete blood count (CBC) and clinical biochemistry panel

are desired from a single animal, it is recommended that the blood be placed in two tubes. First, for the CBC, whole blood should be placed in an appropriate tube that contains EDTA as an anticoagulant. A minimum of 50ul of whole blood is necessary for a CBC analysis. We recommend the microvet EDTA tubes from Sarstedt (www.sarstedt.com). Appropriately sized tubes with lithium heparin as an anticoagulant are acceptable for CBC analysis. An unstained, peripheral blood smear should also be submitted for each CBC sample because whole blood that is shipped may experience environmental or handling stresses that can negatively affect our ability to make a smear. Blood for clinical biochemistry analysis can be placed in an appropriately-sized tube without anticoagulant to allow clotting and collection of serum or can be placed in a tube containing lithium heparin and the plasma harvested following centrifugation. The minimum serum/plasma volume necessary for a full biochemistry profile (i.e. Glucose, Urea nitrogen (UN), Creatinine, Total Protein, Albumin, Globulins, Phosphorus, Sodium, Chloride, Potassium, Total CO2, Anion gap, Cholesterol, Triglycerides, Calcium, Total Bilirubin, ALP, ALT, GGT) is 200ul. Of course, the fewer number of analytes requested for analysis in the serum/plasma, the minimum volume of plasma required is lessened. For a renal (UN, Creatinine, Phosphorus, Sodium, Chloride, Potassium, Albumin, Calcium) or liver (Total protein, Albumin, ALP, GGT, ALT, Total Bilirubin, Cholesterol) profile, a minimum volume of 125ul of serum or plasma is required. For a muscle profile (AST, CK, ALT, LDH), a minimum of 100ul of serum or plasma is required. Therefore, for a CBC and full biochemistry profile, the minimum volume of whole blood needed is approximately 250ul separated into the tubes as mentioned above. As an alternative to splitting the blood into two tubes, the whole blood can be placed in a single lithium heparin tube and submitted for analysis. If you have questions as to the volume necessary for your desired analysis, which tubes are appropriate or procedures, please call or contact the RADIL customer service at (1-800-6690825 or radil@missouri.edu).

General guidelines for submission of whole blood for CBC analysis It is recommended that an unstained, peripheral blood smear be submitted for each CBC samples submitted. Occasionally whole blood that is shipped may experience environmental or handling stresses that can negatively affect the integrity of the blood. The submitted blood smear serves as a reference point in case the submitted blood is compromised. Also, if the blood samples submitted are inadequate for analysis, the pathologist may be able to offer interpretations of

pathologic hematologic processes from the submitted slide. In addition, the slide may be used to confirm automated CBC results. For a pictorial blood smear protocol, see RADIL STANDARD OPERATING PROCEDURE FOR MAKING A BLOOD SMEAR under Teaching Resources. Whole blood can be collected with EDTA or lithium heparin charged syringes (i.e. Saphenous bleeds or terminal cardiac sticks) and placed in the appropriate tubes. Once the blood is in the tube, roll the blood around in the tube to ensure contact with anticoagulant (prevents clotting which can alter CBC results). Samples can be shipped on ice but should not be placed in direct contact with the ice or ice pack, they just need to stay cool. Samples in direct contact might freeze which could alter the sample quality. Wrapping the blood tubes in a material to prevent direct contact with the ice or ice packs is highly recommended. If the blood cannot be shipped immediately after collection, storage for up to 24 hours at 4oC will most likely not alter the integrity of the sample. Samples for CBC should never be frozen or submitted in the same shipping container as formalin-fixed tissues. White blood cell counts, white blood cell differential counts, red blood cell parameters and platelet counts are measured on the Hemavet 950FS automated hematology analyzer. The submitted or prepared blood smear will be examined by a certified technician or pathologist to confirm automated results. All abnormal CBC findings are reviewed by a board-certified veterinary clinical pathologist. Consultation of results can be provided upon request. While there are "normal reference ranges" for mice and rat CBC values (we report those provided by Quesenberry, K.E. and J.W. Carpenter; Ferrets, Rabbits, and Rodents Clinical Medicine and Surgery; W.B. Saunders: Philadelphia, 2004), these ranges can vary with age, sex, strain, equipment and other factors. Therefore, these reference values should be interpreted as approximations. When using CBC values as data for research projects or genetically engineered rodent characterization (phenotyping), we recommend avoiding reference ranges and submitting samples from both experimental / mutant animals and appropriate control animals so that statistical comparisons can be made. Consultations of results can be provided upon request. General Guidelines for Serum/Plasma Biochemistry Analysis For clinical chemistry submissions, we recommend collection of blood in clot tubes (red tops) or lithium heparin tubes (green tops). Regardless of the collection tube used, be consistent; do not switch between tubes as analyte measurements may vary slightly between plasma (green tops) and serum (red tops). Once collected, the serum/plasma should be harvested by centrifugation (e.g. 5 minutes @ 15,000g) and submitted in a leak-proof container. As stated previously, the minimum volume of plasma/serum necessary for a full clinical biochemistry profile is 200ul. However, it is always wise to collect and submit as much sample as possible. This will prevent

occasional short sampling which can result in no tests and allows for follow-up testing (i.e. dilutions of samples with high value results) if needed. All clinical biochemistry parameters are measured on an Olympus AU680 analyzer. Normal range values from Quesenberry, K.E. and J.W. Carpenter; Ferrets, Rabbits, and Rodents Clinical Medicine and Surgery; W.B. Saunders: Philadelphia, 2004 are provided. Of course, the same reference range caveat described for hematology also applies for clinical chemistries. Aspirates, imprints, and smears All fluids for cytologic and fluid analysis should be submitted in an appropriately-sized tube containing EDTA. Generally direct or sedimentation smears should always accompany the fluid. All smears on slides for cytologic analysis should be submitted air-dried, unfixed, unstained and kept as dust-free as possible. Smears of bone marrow aspirates should be accompanied by current CBC results, or, preferably, by fresh smears and lithium heparin or EDTA-anticoagulated blood collected simultaneously with the bone marrow aspirate. Coagulation Profile Samples for a coagulation profile (i.e. prothrombin time (PT) and activated partial thromboplastin time (PTT)) must be collected in sodium citrate (3.2% or 3.8%) tubes in a one part citrate, nine part blood ratio. It is essential that that any tube used be filled appropriately. Once the tubes are filled, it is recommended that the samples be centrifuged and the plasma separated from the blood. This plasma can be frozen and shipped to the laboratory for analysis. Generally, whole blood shipped in sodium citrate tubes does not yield adequate results and best results are obtained from the above mentioned protocol. Small volume citrate tubes can be purchased from Sarstedt (www.sarstedt.com). Call 1-800-669-0825 or e-mail (radil@missouri.edu) the laboratory if you have questions about sample collection, processing, storage, or submission. See the Shipping Information web page for general shipping information.