Journal of the American Association for Laboratory Animal Science Vol 46, No 2 Copyright 2007 March 2007 by the American Association for Laboratory Animal Science Pages 59–64 Complete Blood Count, Clinical Chemistry, and Serology Profile by Using a Single Tube of Whole Blood from Mice Charles E Wiedmeyer,1,* Dawn Ruben,3 and Craig Franklin2 Clinical pathology is a valuable means for assessing specific organ pathology and a screening tool for general animal health. Routine clinical pathology evaluation in mice usually includes whole blood for a complete blood count (CBC) and a clinical biochemistry analysis. Acquisition and analysis of these samples can be problematic due to the small volumes of blood that can be obtained from a mouse. Typically, a complete blood count requires blood from a tube containing an anticoagulant, whereas a clinical biochemistry profile needs blood from a serum clot tube. Because of the small volume that can be obtained, splitting the blood from a single mouse into 2 different tubes may result in inadequate samples to perform the desired tests or introduce inaccuracies. We explored the feasibility of using a single lithium heparin tube for generation of a CBC, bio- chemistry profile, and serology profile. We also evaluated the consistency of CBC data, including the quality of a peripheral blood smear taken from a lithium heparin or EDTA tube after various storage times. We found that CBC, biochemistry, and serology profiles could be obtained more readily when blood samples were placed in a single lithium heparin tube than in 2 separate tubes. In addition, the quality of blood smears and CBC results from the lithium heparin tube were comparable (with few exceptions) to those from an EDTA tube after prolonged storage. Abbreviations: CBC, complete blood count; EDTA, ethylenediaminetetraacetic acid; HCT, hematocrit; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; RBC, red blood cells; WBC, white blood cells Clinical pathology evaluation of mice used for toxicity and Although clinical pathology testing may be very useful, the safety studies or biomedical research is relatively common and acquisition and analysis of samples from mice is problematic required by various regulatory agencies.2,4,5 Clinical pathol- due to the small sample volumes that can be obtained from a ogy data can identify disease in specific target organ systems, single animal.6,9,14,15 Pooling of samples is not recommended, provide a general health profile of the individual animal, and and interim (in life) testing is usually not possible for complete establish dose-response relationships.3 Guidelines for clinical clinical pathology evaluation of a single animal.2,15 Most clinical pathology testing on animals used for toxicity and safety studies pathology samples, especially whole blood and bone marrow have been established.4,15 The parameters recommended for a smears, are taken at termination. Typically, blood taken from core hematology evaluation include: a total leukocyte (WBC) the mouse at termination is allocated into 2 different tubes.6 One count, absolute differential leukocyte count, erythrocyte (RBC) tube, containing an anticoagulant such as ethylenediaminetet- count, hematocrit (HCT) or packed cell volume, hemoglobin raacetic acid (EDTA), is used for a complete blood count, and concentration, mean corpuscular volume (MCV), mean cor- the other, a serum clot tube, for serum chemistry analysis. For puscular hemoglobin (MCH), mean corpuscular hemoglobin coagulation studies, blood must be added to another specific concentration (MCHC), erythrocyte morphology, and platelet tube (that is, citrate).6,10 The use of 2 (or more) different blood count. A blood smear for a reticulocyte count and a bone marrow tubes may result in an inadequate amount of blood in each tube; smear for cytologic examination also are recommended.2,3,15 The an inadequate amount of blood in the tube may yield inaccurate recommended clinical chemistry profile includes: glucose, urea or unobtainable results.1,14 nitrogen, creatinine, total protein, albumin, calculated globulin, Moreover, mice requiring clinical pathology often are shipped calcium, sodium, potassium, cholesterol, and hepatocellular to diagnostic laboratories for necropsy and terminal blood and hepatobiliary tests.2,3,15 For hepatocellular evaluation, a collections. During shipping, mice typically are provided a minimum of 2 appropriate tests is recommended; these in- source of hydration other than water (that is, hydration gel). clude alanine aminotransferase, aspartate aminotransferase, This practice may lead to mild, subclinical dehydration that sorbitol dehydrogenase, glutamate dehydrogenase, and total further precludes obtaining sufficient blood volume for clinical bile acids.2,3,15 The hepatobiliary tests that may be evaluated pathology. Alternatively, rather than shipping mice for necropsy include alkaline phosphatase, gamma glutamyltransferease, 5' and blood removal, blood samples taken at the home facility nucleotidase, total bilirubin, and total bile acids.2,3,15 are shipped on ice to the laboratory. Therefore, transit time and temperature may alter the quality of clinical pathology results Received: 30 Jun 2006. Revision requested: 6 Sep 2006. Accepted: 19 Sep 2006. obtainable from these samples. 1Veterinary Medical Diagnostic Laboratory, College of Veterinary Medicine, and 2Research In this study we compared the use of a single heparinized Animal Diagnostic Laboratory, Department of Veterinary Pathobiology, University of tube for generation of an automated complete blood count, a Missouri-Columbia, Columbia, MO; 3Department of Molecular Comparative Pathobiol- ogy, Johns Hopkins School of Medicine, Baltimore, MD. 14-parameter chemistry profile, and a 19-parameter serology *Corresponding author. Email: [email protected] profile to using 2 separate tubes (that is, EDTA and serum clot Vol 46, No 2 Journal of the American Association for Laboratory Animal Science March 2007 Figure 1. Technique used for mouse cardiocentesis to obtain all blood samples. tubes). We also assessed whether the use of gel packs as a water source affects the performing of these tests. Finally, the qual- ity of CBC data, including that of smears, from blood placed in 2 different anticoagulants and stored for various lengths of Figure 2. Tubes used for this study. (A) Red-top serum tube. (B) EDTA tube. (C) Lithium heparin separator tube. time was examined. Our results indicate that providing mice with water rather than a gel pack for hydration and using a single tube for blood collection increase the amount of clinical water bottle, and 10 male and 10 female mice received a gel pack pathology data that can be obtained from a single mouse. CBC as a water source. For this study, mice were housed in same-sex data were comparable (with few exceptions) between blood groups of 5 under the same conditions as stated previously. stored in lithium heparin and EDTA at 4 pC after various inter- All mice were euthanized by CO2 overdose and weighed, and vals. However, the quality of blood smears deteriorated with whole blood samples taken via cardiocentesis with a 22-gauge prolonged storage; therefore smears should be created shortly needle immediately after euthanasia (Figure 1). Whole blood after blood collection. taken from 10 (5 male and 5 female) mice in each group was placed in 0.5-ml lithium heparinized gel separator tubes (Becton -ATERIALS¬AND¬-ETHODS Dickinson, Franklin Lakes, NJ; Figure 2) by removing the needle !NIMALS The population for the tube study comprised 20 from the syringe, placing the blood into the tube, and mixing male and 20 female DBA/2 mice (Mus musculus) from an in- thoroughly. The lithium heparin tubes used do not contain a house source. Mice were housed in rooms that were monitored visible amount of anticoagulant and are supplied with a gel by use of a quarterly sentinel mouse program. Sentinels were plug for facilitation of plasma separation from cells. Therefore, consistently free of mouse hepatitis virus, mouse minute virus, dilution of the blood sample with anticoagulant is negligible. mouse parvovirus, Sendai virus, Mycoplasma pulmonis, Theiler Whole blood from the other 10 mice in each group was treated in murine encephalomyelitis virus, epizootic diarrhea of infant the same manner but was used first to fill an EDTA tube (Becton mice, pneumonia virus of mice, reovirus type 3, lymphocytic Dickinson, Franklin Lakes, NJ) to the manufacturer-required choriomeningitis virus, ectromelia virus, and ecto- and endopar- amount (500 Nl),13 and the remaining amount of blood was asites. The study was conducted in accordance with guidelines placed in a red-top serum clot tube (Becton Dickinson; Figure set forth by the Guide for the Care and Use of Laboratory Animals8 2). Each sample was analyzed within 2 h of acquisition. and approved by the University of Missouri-Columbia Animal Shortly after the sample was taken, 1 femur from each ani- Care and Use Committee. Mice were housed in polycarbonate mal was removed and the marrow flushed from the bone by static microisolation cages (Thoren, Hazelton, PA) with auto- used of formalin-buffered saline and a syringe. The flushed claved pelleted bedding (Paperchip, Canbrands International, bone marrow was smeared on several glass slides by use of a Moncton, New Brunswick, Canada) and were allowed ad libi- squash technique. tum access to Irradiated Picolab 20, 5053 Rodent Chow (Purina Whole blood from the 18 mice for the blood-storage ex- Mills, Richland, IN) and autoclaved tap water. Environmental periment was collected in the same manner as previously conditions consisted of 10 to 15 air changes hourly, room tem- mentioned. The whole blood from 9 of the mice was pooled in perature of 21 q 2 pC, relative humidity of 30% to 70%, and a a 10.0-ml lithium heparinized tube, and blood from the other 9 12:12-h light:dark cycle. The mice were part of an unrelated mice was placed in a 10.0-ml EDTA tube. The 9 mice used for study and were slated for termination. The mice ranged in age each pooled sample were not discriminated by sex.