Sources of Variation in Normal Values

Laboratory

One of the most commonly overlooked sources of variation in clinicopathologic data is the difference in results obtained by different laboratories in comparison with one another and especially to POCD.

Species

There is relatively modest variation among species for most clinicopathologic parameters. Notable exceptions are plasma

■ TABLE 22.1

Conversion of Conventional Units to International Units²

kPa, Kilopascal; Pθ₂, partial pressure of oxygen; PCO₂, partial pressure of carbon dioxide; TCO₂, total carbon dioxide.

electrolyte concentration; erythrocyte potassium concentration in some breeds of horse, pigs, llamas, cattle, and sheep; and serum bilirubin concentration, which is higher in horses than in other species.

Breed

Significant differences in hematologic parameters exist between hot- and cold-blooded horses. Hot-blooded horses include most of the athletic breeds of horses (Thoroughbred, Quarter Horse, Standardbred, and Arabian breeds). Cold-blooded horses include the pony and draft breeds. Cold-blooded horses have lower red blood cell (RBC) values both at rest and after exercise and maintain a slightly lower leukocyte count; they also have lower resting and fasting indirect bilirubin concentrations.

Age

Several important differences in hematologic and clinical chemistry findings exist between neonatal and adult animals within species. The effects of age have been studied most carefully in horses and cattle. In comparison to adult animals, suckling neonatal animals tend to have lower BUN, slightly lower total protein and globulin, moderately higher GGT and phosphate, and markedly greater alkaline phosphatase. Creatinine in foals can be a bit higher in well-muscled foals (up to 2.5 mg/dL; 221 μmol∕L) and occasionally very high (up to 8 mg/dL; 707 μmol∕L) shortly after birth and returning to normal values within 24 to 72 hours with maintenance fluid support, and it is thought to be due to defective placental transfer (spurious hypercreatinemia).¹⁰

Sex

With the obvious exception of sex hormone concentrations, there are few recognized differences in clinical chemistry values between sexes. In most domestic animals the intact male tends to have a slightly higher erythrocyte count, hemoglobin concentration, and PCV than the female or neutered male.¹¹ This sex-related difference has been demonstrated most clearly in the horse.

Factors Influencing Results or

Their Interpretation

Many factors influence the reliability and interpretation of results obtained by laboratory analysis. The main factors are quality of the sample (preanalytic phase), quality of the laboratory analysis (analytic phase), and quality of results dis­semination and record keeping (postanalytic phase).¹² Sample

■ TABLE 22.2

Clinical Chemistry: Normal Range for Large Animals

ALP, Alkaline phosphatase; AST, aspartate aminotransferase; BHB, β-hydroxybutyrate; BSP (t₁∕₂), bromsulphalein clearance halftime; GGT, γ-glutamyltransferase; CK, creatine kinase; LDH, lactate dehydrogenase; NA, not applicable; PCO2, partial pressure of carbon dioxide; SDH, sorbitol dehydrogenase; TCO₂, total carbon dioxide; TIBC, total iron-binding capacity.

Data from Kaneko JJ, Harvey JW, Bruss ML, editors.

■ TABLE 22.3

Serum Protein Electrophoresis: Normal Range for Large Animals

NA, Not applicable.

Data from Kaneko JJ, Harvey JW, Bruss ML, editors: Clinical biochemistry of domestic animals, ed 6, Burlington, MA, 1997, Academic Press.

■ TABLE 22.4

Recommended Anticoagulants for Hematologic or Clinical Chemistry Evaluation

PT, Prothrombin time; PTT, partial thromboplastin time; T₄, triiodothyronine; T₄, thyroxine.

Tube selection guide at: http://www.eclinpath.com/chemistry/sample-collection-chem/ and https://ahdc.vet.cornell.edu/docs/Blood_Tubes_and_Labeling _Guidelines.pdf.

Modified from Brobst DF, Parry BW. Normal clinical pathology data. In Robinson NE, editor: Current therapy in equine medicine, ed 2, Philadelphia, 1987, Saunders.

collection and handling are important. The sample collection site (e.g., jugular vein, mammary vein, tail vein, cephalic vein, lateral thoracic vein, ear vein or carotid artery, transverse fascial artery, metatarsal artery) can have an important effect on the results of tests such as blood gas evaluation, glucose, or ketones. The choice of anticoagulants depends on whether the samples are to be submitted for serum, plasma, or whole blood analyses. The specific sample requirements for the most commonly ordered clinical chemistry determinations are listed in Table 22.4. Serum is required for most chemistry analytes, and serum separator tubes work well in most settings. There have been some indications that results of some serum hormone assays may be influenced by collection of blood in serum separator tubes. Lithium heparin is the anticoagulant of choice for most chemical determinations requiring plasma. Historically fluoride-oxalate was the anticoagulant of choice for blood glucose determination because it did slow down glycolysis by the RBCs. However, fluoride may interfere with certain chemical procedures (specifically the glucose oxidase method for blood glucose determination) and should be used only for blood lactate determination or in selected circumstances in which glucose determinations are required and samples must be held for some period of time without refrigeration. Citrate is the required anticoagulant for clotting tests and blood typing. Ethylenediaminetetraacetic acid (EDTA) is the anticoagulant most often used for hematologic evaluation. Both citrate and EDTA are chelating agents, which will interfere with calcium determination and, in the case of EDTA, spuriously increase potassium.

Samples should be submitted as soon after collection as possible, but circumstances may require storage of some samples for 12 to 24 hours. For collection of serum, whole blood should be allowed to clot before refrigeration. Serum should be separated from the RBCs immediately after clot formation and then kept refrigerated. Serum samples should be stored in clean containers (e.g., plain tubes) free from exposure to sunlight, medications, or chemicals. If whole blood is left at room temperature for longer than 60 minutes, blood glucose will be falsely decreased as a result of RBC glycolysis. Storage of whole blood may result in in vitro hemolysis, with the potential for misleading increases in the serum or plasma enzymes AST and lactate dehydrogenase (LDH) and potassium and phosphorus concentrations as a result of hemolysis. In addition, failure to separate serum or plasma from the RBCs within an hour of collection may lead to leakage of erythrocyte potassium and a falsely elevated serum or heparinized-plasma potassium concentrations.

Stress, transportation, excitement, and handling produce physiologic responses in animals that affect a variety of hema­tologic and biochemical parameters. This is most evident in the horse, which shows marked increases in RBC mass and to a lesser extent plasma protein concentration in response to excitement, exercise, or catecholamine administration. The RBC count, hematocrit, and hemoglobin concentration can increase by as much as 50%, whereas plasma protein concentration may increase by 1 to 2 g/dL (10 to 20 g/L). Leukocytosis, mainly seen as neutrophilia, is induced as the marginating leukocyte pool is mobilized into general circulation. Prolonged stress results in the release of endogenous corticosteroids, which produce the typical “stress response” leukogram. A similar leukogram is found in racehorses some 4 to 6 hours after racing. The combination of catecholamine and glucocorticoid release associated with stress, transport, and excitement, as well as with many gastrointestinal catastrophes, may result in markedly elevated blood glucose concentrations (up to 400 mg/dL; 22 mmol/L). Modest elevations (twofold to fourfold increase) in muscle-derived enzymes occur in association with prolonged transport or endurance exercise.

Large losses or compartmentalization of sodium-containing fluid accompanies many systemic disorders, particularly digestive problems such as diarrhea, colic, displacement of viscera, excessive sweat losses, and some urinary tract diseases. These forms of dehydration lead to decreases in plasma volume, which are indicated by moderate-to-marked increases in the PCV and TP concentration. The concentration of other compounds dissolved in the plasma may also increase as a result of decreases in the plasma volume. The concentrations of compounds that are largely protein bound, such as calcium, are generally closely related to albumin concentration. Up to 50% of serum calcium is bound to proteins, predominantly to albumin. Increases or decreases in plasma protein concentration normally result in proportional changes in total serum calcium concentration, whereas the physiologically active ionized calcium may remain unchanged.

Diseases that cause a reduction in effective circulating fluid volume often also cause alterations in renal function. This so-called prerenal azotemia results in moderate to marked elevation in BUN and creatinine. Although this is generally considered primarily a prerenal azotemia, real pathologic changes in the kidneys often are associated with the systemic processes initiated by these disorders. Longitudinal reevaluation of renal function (urinalysis, serum BUN, and creatinine) in these patients during the course of disease is important because it assists with prognosis, assesses response to fluid administra­tion, and therefore the potential for nephrotoxicity and systemic toxicity of a variety of chemotherapeutic agents used in such patients.

Fasting laboratory data are important for evaluation of many disease conditions in human and small animal patients. Truly fasting conditions are rather difficult, given the large and complex gastrointestinal tract of most herbivores, and are thus seldom used. The feeding of animals in relation to sample collection, however, can have an impact on the data obtained. Hay feeding in horses is reported to affect sodium, potassium, and protein concentrations within the first few hours after feeding. Animals feeding on lush green pasture or large amounts of silage may have slightly different parameters from those fed high-concentrate rations. The anion-cation balance of the ration has an impact on relative serum electrolyte concentration, acid-base balance, and urine pH and urinary electrolyte excre­tion. Lactescent (cloudy from chylomicrons) plasma may be observed in samples from nursing foals or calves. The fluid intake of the normal nursing neonate may range from 100 mL/ kg/day to more than 250 mL/kg/day. This high fluid intake is reflected by a commensurately high output of urine with a low specific gravity and low osmolarity.

The administration of certain medications may have an impact on some laboratory parameters. Tranquilization may be necessary for restraint and safe sample collection. The practitioner should be aware that tranquilizers often decrease RBC mass and plasma protein concentration. This is particularly true of the phenothiazine-derivative tranquilizers when used in the horse. Xylazine administered to large animals produces a modest catecholamine release, which may be evidenced by the slight sweating response seen in many horses sedated with this drug. Glucose concentration will increase modestly in response to the xylazine-induced catecholamine release. Repeated intramuscular injections with certain antibiotics (especially erythromycin and tetracycline) or other preparations that are locally irritating may produce slight-to-moderate elevations in muscle-derived serum enzyme activities. Intra­venous administration of certain drugs and compounds such as dimethyl sulfoxide (DMSO) can produce intravascular hemolysis and hematuria. The amount of hemolysis in these circumstances is relatively small and of little consequence, except that it can cause confusion as to why hemoglobinuria occurred.