TOPICAL REVIEW

Common Laboratory Artifacts Caused by Inappropriate Sample Collection and Transport: How to Get the Most out of a Sample
Shir Gilor, DVM, MS, Dipl. ACVP, and Chen Gilor, DVM, PhD, Dipl. ACVIM (SAIM)
Clinicians are frequently presented with laboratory test results that are not consistent with preconceived expectations for a given case. One important reason for such results is the occurrence of preanalytical errors. In this article preanalytical errors are discussed in 2 parts. The rst part covers the steps of sample collection, preparation, and transportation, in which preanalytical errors often occur. This part would be most useful if read in full before collecting a sample. The second part of this article includes a systematic review of preanalytical errors divided according to individual analytes or parameters or, when appropriate, groups of analytes and parameters that represent the same biological system. This part will hopefully serve the clinician as a quick and user friendly guide for identication of possible pitfalls when presented with unexpected laboratory test results for a given case. This article is limited to errors that can affect the complete blood count, chemistry, and coagulation panels. 2011 Elsevier Inc. All rights reserved. Keywords: CBC, biochemistry, coagulation, canine, feline

linicians are frequently presented with laboratory test results that are not consistent with preconceived expectations for a given case. Unexpected clinicopathological ndings might occur in a patient that lacks classical signs of a disease, with a disease that causes subtle or no clinical signs, and with interpretative mistakes such as disregarding normal physiological states (e.g., pregnancy anemia) or breed-associated abnormalities (e.g., microcytosis and hypekalemia in Akitas and thrombocytopenia in a Cavalier King Charles). Another important and often overlooked reason for unexpected results is preanalytical errors. Unfortunately, preanalytical errors are common and could affect the diagnostic quality of a sample, resulting in unreliable and inaccurate results. Clinical interpretation of such test results may be detrimental to the patient, cost-ineffective to the client, and frustrating to the practitioner. When preanalytical errors are suspected, a few simple corrective measures can be taken and repeat sampling might be helpful. Lastly, true laboratory errors (analyzer related, personnel related, etc.) should be considered. Therefore communication with the reference laboratory is important. If an in-house laboratory is used, assuring ongoing quality-control programs for the different analyzers is crucial to guarantee accurate, precise, and believable analysis results.
From IDEXX Laboratories LTD., Wetherby, United Kingdom. Address reprint requests to: Shir Gilor, DVM, MS, Dipl. ACVP, Clinical Pathologist, IDEXX Laboratories LTD., Grange House, Sandbeck Way, Wetherby, West Yorkshire LS22 7DN, United Kingdom. E-mail: ShirGilor@idexx.com. 2011 Elsevier Inc. All rights reserved. 1527-3369/06/0604-0171\.00/0 doi:10.1053/j.tcam.2011.02.003

Preanalytical errors can occur in any of the 4 major steps of sample collection and transport: 1) preparing the patient for sampling (fasting, effects of stress); 2) sampling; 3) collection tubes and preparation of the sample; and 4) transport to the referral laboratory. The rst part of this article presents common preanalytical errors in the 4 steps described above and discusses how to avoid them. The discussion will be limited to errors that can affect the complete blood count (CBC), chemistry, and coagulation panels. Additionally, although not considered preanalytical errors per se, the effects of commonly used drugs on the different analytes are briey mentioned when appropriate. This is important because patients might already be medicated when blood samples are collected. The second part of this article divides preanalytical errors according to individual analytes or parameters or, when appropriate, groups of analytes and parameters that represent the same biological system. This hopefully will serve the clinician as a quick and user friendly guide for identication of possible pitfalls when faced with test results that seem to be artifactual. Urinalysis, blood gas analysis, and other tests are beyond the scope of this article.

Preanalysis Step One: Patient Preparation for Sampling Fasting

Appropriate fasting of patients before sampling is often overlooked. Ingestion of food can have a considerable inuence on the composition of blood, plasma, and serum.1

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For standardization, reference intervals are typically constructed from test results of fasted animals. Similar conditions are necessary for a given patient to accurately use the reference interval for a given sample. For minimum database, including CBC and a biochemistry panel, a fasting period of at least 12 hours is recommended.1 Access to fresh drinking water should be provided during fasting. An important disadvantage of a nonfasting sample is the presence of lipemia. Lipemia is the presence of an excess amount of lipids in the blood. When these lipids include a large enough proportion of triglycerides (e.g., after ingestion of a fatty meal), the serum and plasma have a milky white appearance. Lipemia causes increased serum turbidity and may interfere with spectrophotometric-based tests (e.g., serum glucose, liver enzymes). The effect of lipemia on light transmission is unpredictable because the size and number of different lipoproteins may have opposite effects on light transmission. Lipemia can also falsely elevate plasma protein concentrations measured by refractometry.2 Lastly, it enhances hemolysis in vitro. Hemolysis might affect the CBC and biochemistry results and will be further discussed below. Different analytes are affected by different degrees of lipemia. The degree of lipemia is reported by many reference laboratories as lipemic index. Laboratories often omit the results of certain analytes when the lipemic index is suggestive of a degree of lipemia that interferes with the results. Note that values of the lipemic index might differ between laboratories. Lipemia can be partially cleared by ultracentrifugation techniques or by the addition of precipitating agents that can clear the sample. As a result, the laboratory might still be able to generate useful results from lipemic samples.3 The use of lipid-clearing products, however, might by itself induce artifacts if the analytes being measured bind to lipoproteins and are removed with them.4 A proper fasting before sample collection is necessary to avoid the analytical problems that are related to postprandial lipemia. It is also necessary for differentiating postprandial lipemia from fasting lipemia. Only the latter is clinically important and can occur in diseases that affect lipid metabolism (e.g., diabetes mellitus, pancreatitis, and hypothyroidism). Different types of meals may affect different analytes. A protein-rich meal may result in increased blood (serum) urea nitrogen, phosphorous, and ammonia concentrations. Because of a postprandial insulin surge, glucose concentrations might decrease. If large quantities of creatinine and noncreatinine chromogens found in meat are ingested, serum creatinine concentrations might increase.1 The effect of a carbohydrate-rich meal is not as profound as that of a protein-rich meal.5 Blood glucose and sodium concentrations might increase, whereas blood phosphorus and potassium concentrations might decrease (because of use by glycolytic enzymes and insulin-depended transportation, respectively). Prolonged fasting, due to food deprivation or, more commonly, anorexia, has profound effects on the body and results in signicant alteration to many analytes. Prolonged

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fasting, however, is not considered a preanalytical error and is therefore not discussed here. Oftentimes blood drawing in critically ill or acutely injured patients cannot be planned. This, however, is rarely the case in wellness examinations or in patients that are chronically ill. For the aforementioned reasons, patients should be fasted if blood drawing is planned.

Stress Reduction
Stress can have a signicant effect on CBC and glucose concentrations. In many patients (cats in particular) alleviation of stress, before and during sampling, is difcult. It is therefore important to recognize the potential effects of stress on laboratory test results. Increased blood cortisol concentrations lead to a stress leukogram. A stress leukogram is dened as one or more of the following: neutrophilia, monocytosis, lymphopenia, and eosinopenia. Lymphopenia is the most common change seen in dogs with a stress leukogram and is often the only change. An epinephrine response (also known as ight-or-ght response) is seen mainly in cats and typically includes neutrophilia and/or lymphocytosis.3 Hyperglycemia can be associated with either increased blood cortisol or epinephrine concentrations. Differentiation of stress hyperglycemia from persistent hyperglycemia (as in diabetes) can easily be done by measuring serum fructosamine concentrations. Stress responses can be quick and transient and not all animals are equally affected by equal conditions. It is not unusual to nd glucosuria without hyperglycemia in healthy cats that are stressed before arriving at the clinic but are more relaxed during the blood draw itself. In these cases, conrmation of stress-related glucosuria may be achieved by checking urine glucose in a free-catch urine sample that is collected by the owner at home.

Preanalysis Step Two: Sampling

Blood drawing techniques and inappropriate handling of the sample can have a major effect on blood test results. Drawing blood from small veins with small-gauge needles and multiple sticks may lead to excessive blood turbulence, hemolysis, and spurious activation of the coagulation system. Small (even microscopic) blood clots and platelet aggregations may lead to false results such as anemia, thrombocytopenia, and inaccurate coagulation test results. In cats, 21- or 20-gauge 1/2- to 1-inch needle with 5- to 10-mL syringe attached to it may be used. In dogs, 18- to 21-gauge 1-inch needle with 5- to 20-mL syringe attached to it is a good choice. A blood-drawing technique that is less than optimal can often result in hemolysis. Hemolysis can be visualized when hemoglobin concentrations exceed 20 mg/dL.6 Depending on severity, hemolysis may affect several analytes of the CBC, biochemistry, and coagulation tests (see discussion of individual analytes). Mechanisms include a direct inuence on spectrophotometric methods by affecting the absorbance reading, pH alteration of enzymatic reactions and, if severe, a

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slight dilution effect on those constituents present at a lower concentration in the erythrocytes than in plasma.3,6 The effect of different degrees of hemolysis on different analytes is to some extent laboratory dependent and is usually reported as hemolytic index. When collecting blood for coagulation tests, a clean venipuncture technique is crucial so that the sample is not contaminated with tissue factor from tissues surrounding the vein. Contamination of samples with tissue factor can initiate coagulation and shorten coagulation times. Atraumatic venipuncture that minimizes the activation of the clotting cascade is considered the gold standard for blood sample collection and results in the most accurate measurements.7-9 The use of intravenous (IV) catheters for collection of samples for coagulation analysis has been considered inappropriate. It was believed that the catheter can act as a foreign irritant to the blood vessel in which it is placed. Puncture of the vein and advancement of the catheter can cause the release of tissue factor followed by activation of the coagulating cascade in vitro and falsely shorter clotting times could be the result.7 Similar results may also be caused by the increased time needed to place an IV catheter compared with simple venipuncture. A longer period of venous congestion before sample acquisition while placing an IV catheter can activate the clotting cascade and cause a falsely shorter clotting time. Ideally, venous congestion should be limited before use of any method of sample collection to enable accurate prothrombin time (PT) and activated partial thromboplastin time (aPTT) measurements.7 On the other hand, collecting samples for coagulation panels from IV catheters might be an advantage in animals with clotting disorders if repeated sampling is necessary. A recent study reports a clinically acceptable agreement for measured PT, aPTT, and brinogen concentrations between a simple venipuncture and blood collected from a catheter.10 Time points evaluated in this study include 0 and 24 hours after catheter placement. The authors compared the collection of blood from 16-gauge IV catheters placed in a jugular vein or a lateral saphenous vein with a blood sample collected by direct venipuncture of the contralateral jugular or lateral saphenous vein or a cephalic or medial saphenous vein (20- or 22-gauge needle and 3-mL syringe). One shortcoming of this study was the limited time period examined. It is possible that a coagulation panel from a sample collected from a catheter that is placed in the vein for more than 24 hours would be in less agreement with a panel from a sample collected from a venipuncture. Another recent study looked at the diameter of the catheter and blood collection technique on platelet function and parameters reecting secondary hemostasis, physiologic anticoagulation, and brinolysis in dogs.11 Blood samples were collected with 20- and 18-gauge venous catheters immediately after catheters were inserted in a peripheral vein, through a 14-gauge central venous catheter that had been placed via the Seldinger technique (in which a trocar and insertion of a guide wire are used) in a jugular vein 30 minutes before sample collection, and through a 13-gauge central venous catheter placed via a catheter-through-the-needle technique 30 minutes before

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sample collection. The authors concluded that these sample collection techniques did not signicantly inuence any variable reecting primary (platelet function), secondary (PT, aPTT, brinogen concentrations, factor VIII activity), and tertiary (D-dimer concentrations) hemostasis or natural inhibitors of coagulation (activities of antithrombin, protein C, and protein S).

Preanalysis Step Three: Collection Tubes and Preparation of the Sample

Commonly used blood collection tubes are reviewed here. Most blood collection tubes available are vacuum tubes with a stopper. They either lack an anticoagulant or contain one to prevent the sample from clotting. The various anticoagulants dene the different tubes and dictate their use. The stopper color denotes the type of anticoagulant and/or other type of additive present and is fairly consistent from one manufacturer to another. Blood collected into a tube that contains one additive should never be transferred into another tube that contains a different additive because of potential interaction between the 2 additives and interference with their actions.

Ethylenediaminetetraacetic Acid Tubes (EDTA, Purple-top Tubes)

EDTA is a chelator of bivalent cations such as Ca2 and Mg2 . EDTA exerts its anticoagulant effect by binding calcium, making it unavailable for enzymatic reactions in the coagulation cascade. EDTA is the anticoagulant of choice for most routine hematology samples. It is also ideal for blood smear preparations because it minimizes cell clumping, helps to preserve cell morphology, and results in high-quality staining of blood smears. Plasma from EDTA samples can also be used for select serum biochemistry analytes (e.g., blood urea nitrogen, glucose, and total protein). This might be benecial when collection of large volumes of blood is difcult (e.g., from puppies or small cats) or when only minimum data are needed.

Clot Tubes/Serum Tubes (Red-top and Red/ Black Tubes)

There are 2 different serum collection tubes available: tubes containing a serum-separating material (SSTs, red/ black) and tubes without (red). SSTs are used to accelerate the coagulation process and reduce the time required to process the sample. They contain an inert gel material, silica, or glass particles that accelerate clotting. Centrifugation of the blood-lled tube displaces the gel, which settles like a disc to create a barrier between the cells and the supernatant. Release of intracellular components into the supernatant is prevented for several hours or, in some cases, for a few days.6 Whether SSTs or red tubes are used, centrifugation and collection of serum into another tube are recommended if transport to the reference laboratory is going to be prolonged (overnight or longer). This would minimize hemolysis and

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artifactual changes of certain analytes (see further discussion in the chemistry section). Serum is the standard sample submitted for chemistry proles, and it is also used for serologic titers, serum protein electrophoresis, and some endocrine tests. When using red tubes, serum should be harvested within 30 to 60 minutes of blood collection to allow for appropriate blood clotting and clot retraction. After harvesting, the serum should be transferred to another red-top tube if the sample has to be mailed to the laboratory. A proper clot retraction typically allows a sufcient amount of serum to be harvested. If serum is harvested in less than 30 minutes, a brin clot (which appears as a gel-like material) might be still present, making serum separation difcult and often resulting in an insufcient quantity for analysis. Poorly separated serum may be associated with several artifactual changes including hypoglycemia, hyperphosphatemia, and hemolysis. Because most assays are based on absorbance of light transmitted through the sample, hemolysis can markedly interfere with many tests, depending on the type of analyzer and methodology used. Hemolysis almost always occurs in unseparated samples over sufcient time but happens more quickly when the ambient temperatures are high; therefore samples mailed in the summer might be affected if not kept in an appropriate temperature.12

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Citrate Tubes (Blue-top Tubes)

These tubes contain sodium citrate solution at a concentration of either 0.109 mol/L (3.2%) or 0.129 mol/L (3.8%). They are widely used for routine coagulation tests because binding of citrate to Ca2 is easily reversed by the addition of Ca2 .17 Citrate tubes are used for some of the most common coagulation tests including PT, aPTT, thrombin time, and quantitative brinogen determination. Citrated plasma can also be used for measurement of D-dimers, specic coagulation factors (i.e., factor VIII activity), or Von Willebrand factor antigen concentrations. Appropriate lling of citrate tubes with 1 part citrate to 9 parts blood is extremely important. Underlling of these tubes will results in a relative excess of anticoagulant in the sample, which could prolong the PT and aPTT results. Overlling of the tube might result in decreased PT and aPTT. Blood collection tubes containing 3.2% sodium citrate, instead of 3.8% sodium citrate, became available recently in the United States. It has been shown that there is no clinically relevant difference between collection of blood into 3.2% or 3.8% sodium citrate for PT, aPTT, brinogen concentration, platelet count, or Von Willebrand factor antigen concentration.18

Heparin Tubes (Green-top Tubes)

Unlike other commonly used anticoagulants, heparin does not chelate calcium. Instead, heparin inhibits coagulation by acceleration of the action of antithrombin, which neutralizes thrombin (factor II) and several other enzymatic coagulation factors. Several heparin salts (e.g., sodium heparin, potassium heparin, lithium heparin) are used in commercially available blood tubes. Lithium heparin is the preferred type of heparin salt for most uses because it does not alter the concentration of routinely evaluated electrolytes. The main advantage of using heparinized plasma for biochemical analysis compared with serum is that it saves time. Blood collected into a red-top tube must sit for about 30 minutes to ensure proper clot formation and retraction. In contrast, heparinized blood can be immediately centrifuged, followed by plasma removal, allowing for more rapid processing. Major disadvantages are the higher cost, the temporary action, and the production of a blue background in blood lms that are stained with a Wrights stain. Heparinized plasma can be used to run a full chemistry prole, although the results of some analytes differ from the results generated from serum samples. Examples include higher albumin concentrations and lower potassium and ionized calcium concentrations in heparinized plasma compared with serum.13 Heparin has been advocated for use with blood samples collected from species with nucleated red blood cells (RBCs) based on the anecdotal observation of hemolysis in EDTA-preserved samples in some reptiles and birds.14,15 A recent study suggests, however, that EDTA is comparable with heparin when used for CBC and can be used in some birds and reptiles.16

Sodium Fluoride Tubes (Gray-top Tubes)

Sodium uoride (NaF) is considered a preservative for blood glucose but also acts as a weak anticoagulant. These tubes typically contain, in addition to NaF, potassium oxalate, a Ca2 chelator. Addition of potassium oxalate allows for a lower volume of NaF to be used. Fluoride is a potent inhibitor of enzymes involved in glycolysis and it stops metabolism of glucose by cells in the blood sample. NaF is poorly soluble; therefore blood must be well mixed before an effective antiglycolytic effect occurs. Blood samples collected in NaF tubes are used for the measurement of blood glucose without the need to separate serum or plasma from cells. NaF/potassium oxalate containing tubes can also affect other electrolytes concentrations. Lactate, when measured from samples collected in NaF tubes, is stable at room temperature in feline samples for at least 8 hours.19 It is stable for up to 4 hours in canine samples, whether they are stored at room temperature or refrigerated.20 Collection of blood in NaF tubes results in RBC shrinkage and lysis. The shift of intra-erythrocytic uid to plasma results in a decrease in hematocrit and subsequent dilution of some serum analytes, including glucose.19 There is little justication for the use of NaF tubes for clinical biochemistry tests except for lactate and glucose concentrations. This will be further discussed in the electrolyte section below. Inappropriate lling of a chosen tube or choosing the wrong tube would likely lead to poor sample quality (see discussion in appropriate sections). Inappropriate mixing of the tube and unsuitable centrifugation are additional problems that may be easily solved. EDTA and citrate tubes should be mixed gently immediately after blood drawing.

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Centrifugation protocols may vary between different centrifuges. The order in which tubes are lled is also important. It is recommended that citrate tubes used for coagulation tests are lled rst followed by EDTA. Lastly lled are serum or plain tubes used for biochemistry panels. This aims to decrease the chance of blood clot formation, which would affect CBC and coagulation tests but not serum-based tests. While allocating the sample between the tubes, small amounts of EDTA might be transferred to the serum tubes (i.e., EDTA contamination). This may affect several analytes concentration and will be discussed below for the specic analytes.

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tests may be stable for much longer periods.12 Studies on canine samples suggest that valid PT and PTT results can be obtained from citrated plasma (removed from RBCs) stored for up to 48 hours when refrigerated or even at room temperature.21,22 It is best to contact the reference laboratory for details regarding special sample storage during transporting.

Systematic Review of Preanalytical Error Divided by Tests Complete Blood Counts

Red Blood Cell Parameters Packed Cell Volume Inadequate mixing of the blood in the EDTA-containing tube before lling of the microhematocrit tube might lead to either increase or decrease in packed cell volume (PCV). Erythrocytes tend to settle if the anticoagulated blood is left standing in the tube rack, especially when pronounced rouleaux formation is present.23 Falsely decreased PCV would occur if the microhematocrit tube is lled with blood taken from the upper portion of the anticoagulated blood tube. Falsely increased PCV would occur if the microhematocrit tube is lled with the bottom portion.23 Improper centrifugation (lower centrifugation speed) could increase PCV because of poor packing of RBCs in the microhematocrit tube. Very small clots entering the microhematocrit tube may affect efcient erythrocytes packing, resulting in falsely increased PCV. Underlling of the EDTA tube results in excess of EDTA, especially when liquid EDTA tubes are used. Underlling of the liquid EDTA tube might lead to some degree of dilution artifact. In addition, EDTA is hypertonic and causes water to leave RBCs, resulting in shrinkage of the cells. The smaller erythrocytes occupy less volume and a signicant reduction of PCV is possible. Red Blood Cells, Mean Cell Volume, and Hematocrit Most automated analyzers calculate hematocrit by multiplication of RBCs and mean cell volume (MCV). Therefore, artifactual changes in RBC and/or MCV will affect hematocrit concentrations. Blood clots within the sample often lead to erroneously decreased RBC concentrations, resulting in decreased hematocrit. Blood clots might also cause an inappropriate sampling by the automatic analyzer or even clog the aspirating pipette of the analyzer. As mentioned above, underlling of an EDTA tube can lead to cell shrinkage. This will cause a false decrease in MCV and hematocrit concentrations, and a false increase in mean corpuscular hemoglobin concentration (MCHC). This is because MCHC is calculated by the formula: (hemoglobin 100)/hematocrit. It is important to note that these artifactual changes should be considered only when using instruments that determine hematocrit by blood electrical conductivity (e.g., Nova CCX). In most automatic analyzers, the effect of EDTA excess on cell volume is overcome by suspension of

Preanalytic Step Four: Sample Handling and Transportation to the Referral Laboratory
To avoid confusion and misreporting by the laboratory personnel, it is imperative to properly label the submitted tubes and clearly mark the requested tests. It is recommended to include the date of sample collection, especially for samples that are mailed. This would aid in minimizing misinterpretation of changes occurring during transportation. As a general rule, time elapsing between sample collection and sample processing by the reference laboratory should be minimized. During transportation, cells in the EDTA tube undergo apoptosis (programmed cell death) or lysis and may become pyknotic or fragmented. This would affect the quality of the blood smear reviewed by the clinical pathologist and might interfere with identication of cells by the automatic analyzers. Preparation and submission of fresh, air-dried, unstained blood smears are recommended. This is important for several reasons. First, cellular preservation in the submitted blood smear is often superior to that of the smears prepared in the reference laboratory from the submitted EDTA tube. Better cellular morphology in the submitted smears often enables better cytological interpretation. Second, blood smears are often used as a quality control measure for the results generated by the automatic hematology analyzer. Examples include conrmation of platelet counts when platelet aggregates are present, detection of left shift and/or toxicity signs in granulocytes and morphological evaluation of large abnormal (often neoplastic) cells. Minimizing some of the above processes is possible with adequate sample storage. EDTA tubes (but not the prepared blood smears) should be kept refrigerated but never frozen. Serum samples, as mentioned previously, should be centrifuge, with serum removed, and placed in a separate clean tube. Coagulation factors are degraded in vitro over time. It is recommended that citrated plasma used for coagulation testing be kept cool, separated from red blood cells, and assayed within 30 minutes to 4 hours of collection.12 If the sample is sent to a reference laboratory and the analysis is delayed in more than 4 hours, the separated plasma should be frozen and sent to the laboratory on a cold pack. Several studies, however, suggested that samples for PT and PTT

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erythrocytes in an isotonic diluent before analysis. This enables erythrocytes to regain their volume.23 Most laboratories require that EDTA tubes will be lled to at least 50% of the intended volume. Microcontainers that are designed to hold as little as 0.25 mL of whole blood are available. These are recommended if the sample size is limited to avoid the previously mentioned artifacts.12 Lysis of RBCs during blood collection or handling might result in decreased RBC and hematocrit concentrations and can also falsely increase MCHC. Mild hemolysis is unlikely to cause a clinically signicant change, but gross hemolysis would create a larger error. During transportation or in cases of late sample analysis (typically over 24 hours), sample aging can have a major effect on RBC parameters. The rate at which cells undergo degeneration can be quite variable and seems to depend on the type of cells present as well as ambient temperature.12 Erythrocytes may swell signicantly, leading to an increase in MCV and hematocrit concentrations and a decrease in MCHC.21,24 If anemia is present, a misdiagnosis of macrocytic hypochromic regenerative response is possible. Persistent and marked hypo-osmolality (secondary to hyponatremia) results in decreased intracellular erythrocyte osmolality. When these erythrocytes are placed in an isotonic solution before analysis, they lose water via osmosis and shrink. This results in decreased MCV and therefore decreased hematocrit. Opposite osmosis occurs when persistent hyperosmolality exists, resulting in falsely increased MCV and calculated hematocrit. White Blood Cell Parameters Changes in total leukocytes number and differential counts are more variable than changes affecting RBC parameters. Aging of the sample has an effect on leukocyte morphology. As previously mentioned, leukocytes often lose their morphological characteristics within time and can become apoptotic or karyorrhectic, resulting in a poor diagnostic quality of the blood smears. Storage temperature and time length are important factors participating in the aging process of the sample. Additionally, different methodologies used by different analyzers (or by the same analyzer) might be affected differently by these changes, yielding a different total leukocyte and differential count for the same sample. In one study using the Advia 120 analyzer, total white blood cell counts decreased during 48 hours of storage. A signicant decrease in lymphocyte count was also seen.21 These changes occurred in samples stored in 24C but not with samples that were stored at 4C. Additionally, granulocytes tended to be misclassied as mononuclear cells after storage of 48 hours in 4C, affecting the differential counts. This might be the result of decreased chromatin density in the deteriorating granulocytes.21 It is therefore recommended to store samples in 4C to improve the accuracy of CBC results. Samples containing blood clots containing samples may yield falsely decreased leukocyte counts, because leukocytes entrapped within the clots are not available for analysis by the automated analyzer.

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The effects of stress and ght-or-ight responses were discussed previously in the Preanalysis Step One section. Platelets One of the most important effects on automatic platelet counts and manual platelet number estimates is the presence of platelet aggregates/clumps. Platelet clumps often cause a false decrease in platelet numbers (pseudothrombocytopenia), which may have important consequences for patient care. Traumatic blood sampling is a common preanalytical cause for platelet activation and aggregation in many animal species. In cats, delayed analysis (e.g., mailing of samples), however, does not appear to be associated with lower automated platelet counts compared with counts obtained on the day of sampling.25 Feline samples are especially prone to platelet clumping, and clumps may occur in samples that are collected with minimal trauma. One study reported pseudothrombocytopenia due to platelet clumping to occur in as many as 71% of all feline samples, whereas only 3.1% of all samples were considered truly thrombocytopenic based on estimation of platelet counts from blood smears.25 Although this study used an impedance-based analysis, falsely decreased platelet counts also occur with laser-based cell counters because platelet clumps have a different light-scatter pattern than do individual platelets; therefore clumps are not counted as platelets.26 Given the high prevalence of pseudothrombocytopenia in automated platelet counts, careful examination of blood smears is required to identify the few cats with true thrombocytopenia. Storage time (i.e., aging of samples) and temperature are important factors participating in artifactual changes in platelet parameters. Platelet activation leads to their degranulation, resulting in reduced platelet density and decreased mean platelet component (MPC). In dogs, storage of 12 hours at room temperature caused a decrease in MPC. In addition, platelet counts were decreased and mean platelet volume was increased. This was probably due to platelet clumping and an enlargement of platelets in the stored samples.21 Because detection of activated platelets may have clinical applicability for the early detection of prothrombotic conditions,27 it is important to recognize the effect of sample aging, which may give the false impression of an early prothrombotic condition. Platelet parameters in samples stored at 4C are more stable than at 24C when measured up to 48 hours after collection.21 Platelet counts are consistently lower in citrate-anticoagulated blood compared with EDTA-anticoagulated blood in both dogs and cats, when laser-based analyzers are used.28,29 The lower platelet count in citrate-anticoagulated blood samples is attributed to platelet clumping, which occurs more frequently and to a greater extent in citrated samples. In contrast, higher counts (obtained by a hemocytometer and an impedance analyzer) and lower scores of clumping were seen in citrate compared with EDTAanticoagulated feline blood in another study, but only samples from 12 cats were evaluated.25 Although the difference in platelet counts with

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citrate-anticoagulated blood is signicant in dogs, it is unlikely to be clinically relevant. In blood smears made from citrated blood, platelets appear consistently larger and lighterstained compared with platelets in blood smears made from EDTA samples. Lastly, platelets in citrated samples have higher mean platelet volume and lower MPC compared with platelets analyzed from EDTA samples. Therefore, citrate is not recommended as an anticoagulant when accurate platelet counts are desired.28

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Potassium (K ) might be falsely elevated because of in vitro hemolysis or delayed separation (and leakage of K ) in Japanese breeds (e.g., Akita, Shiba) and their crosses. This is because in these dogs the concentrations of K are higher in erythrocytes than in the serum (because of the presence of Na/K pumps on erythrocyte membranes). In cats and in most breeds of dogs, the concentrations of K in erythrocytes and in the serum are equal and therefore hemolysis does not cause artifactual hyperkalemia in those animals.23 The supernatant does not have to be grossly hemolytic (red) for pseudohyperkalemia to occur. The use of plasma instead of serum for K measurement might cause an artifactual decrease in K concentrations. In this case, K concentrations would be expected to be lower in about 0.3 to 0.5 mmol/L compared with the serum sample because K is released from activated platelets during clotting.23 This, however, is unlikely to be of clinical signicance. The use of EDTA tubes for measurement of K would lead to pseudohyperkalemia (often 20 mEq/L). It does not require much potassium EDTA, however, to evoke spuriously abnormal results. Contamination of serum tubes with even small amounts of EDTA would lead to pseudohyperkalemia. This might occur when the EDTA tube is lled before the serum tube and the needle is not changed. Combination of hyperkalemia, hypocalcemia, and, when measured, hypomagnesemia, may raise a suspicion of sample contamination by EDTA. Angiotensin-converting enzyme inhibitors might also cause hyperkalemia because of the development of hypoaldosteronism. Only a mild hyperkalemia is expected if the kidneys are normally functioning.23 When indirect (but not direct) ion-selective electrodes or ame photometry methods are used to measure sodium (Na ) concentrations, lipemic or hyperproteinemic samples might yield falsely decreased Na concentrations (electrolyte exclusion effect). Another preanalytical cause for hyponatremia is the use of thiazide and furosemide diuretics or mannitol infusions. Plasma obtained from Na2 EDTA anticoagulant tubes should not be used because of pseudohypernatremia. Na heparin tubes do not contain a signicant amount of Na and may be used for Na measurement.23 When using the ion-selective electrodes method for measurement of chloride (Cl) concentrations, clinically signicant pseudohyperchloremia might be seen in animals treated with potassium bromide.31 Treatment with loop diuretics (e.g., furosemide) might lead to hypochloremia because they inhibit the luminal Na-K-2Cl symporter in the thick ascending limb of the loop of Henle. Depending on the assay used, pseudohypochloremia, similarly to Na , may be seen in lipemic samples. Glucose Glucose concentration decreases by approximately 10% per hour at room temperature if serum is left in contact with blood cells.12 This is because of consumption of glucose by erythrocytes. Other changes can also occur but are more variable.

Biochemistry Analytes
Urea A false increase in urea concentrations may be seen with hemolysis. This change is mild and often clinically insignicant (hemoglobin of 50 mg/dL will increase urea by 1 mg/dL).23 The use of NaF tubes for urea measurements by the enzymatic method is not recommended. This is because uoride inhibits urease activity used in the enzymatic assay, causing a false decrease in urea concentrations.23 Creatinine Plasma creatinine is increased for the rst few hours and remains increased for up to 12 hours after meals of raw or cooked meat. After ingestion of commercial food of undetermined creatine/creatinine content, increases, decreases, or no change in plasma creatinine have been reported.30 Electrolytes Calcium concentrations will falsely decrease when EDTA-, citrate-, and oxalate-containing tubes are used. As mentioned before, these anticoagulants function by calcium binding, making it unavailable for the coagulation cascade, resulting in inhibition of coagulation. Therefore, these tubes should not be used when calcium is assayed. Note that NaF tubes might also contain potassium oxalate and are not suitable for electrolyte measurements. If plasma is collected for calcium measurement, a heparin tube should be used. Although not considered a preanalytical error per se, certain drugs may affect blood calcium concentrations. Mechanisms include decreased urinary excretion of calcium (e.g., thiazide diuretics), excess urinary excretion of calcium (furosemide), and increased calcium binding leading to decreased gastrointestinal absorption (phosphate enemas).23 Serum inorganic phosphorous (Pi) concentrations might falsely increase because of delayed serum separation and/or hemolysis. In these cases, Pi is released over time from erythrocytes while cells metabolize ATP to ADP and Pi. Additionally, phosphate is released from phospholipids found in membranes and from the cytoplasm of erythrocytes.12,23 Iatrogenic causes that may increase serum Pi concentrations include phosphate enemas (increased gastrointestinal absorption) or oral phosphate urinary acidiers. Falsely decreased serum Pi concentrations may be seen in patients receiving glucose infusions and after insulin administration. This is because of a glucose-depended shift of Pi from the extracellular uid to the intracellular uid. Prolonged diuresis may also result in hypophosphatemia due to increased Pi urinary excretion.

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Blood collected in an NaF tube maintains a stable glucose concentration for at least 24 hours at room temperature and 48 hours when refrigerated.17 Glucose concentrations measured in NaF tubes are articially decreased compared with concentrations measured in serum in many species.17 In one study, glucose concentrations measured in NaF/Ox plasma were approximately 11% and 14% lower than glucose concentrations measured in serum samples from euglycemic and diabetic cats, respectively. The underestimated glucose concentration was attributed to RBC shrinkage and dilution of plasma by RBC cytosol, which contains a lower concentration of glucose. The authors recommended that glucose determination in cats would be performed on routinely processed serum, separated from the clot within 15 to 30 minutes or stored at 4C.19 Bilirubin Depending on the method, hemolysis can cause a false increase or a false decrease in bilirubin concentrations. Bilirubin is highly sensitive to ultraviolet light and when exposed to sunlight it undergoes quick degradation. Approximately 50% of bilirubin can undergo degradation within an hour of direct sunlight exposure. Bile Acids Hemolysis causes a false decrease in bile acid concentrations measured by spectrophotometric assays. Lipemia might also cause artifactual changes in bile acid concentrations (depending on the methodology used), and it is generally preferable to avoid it. In the bile acid stimulation test, however, fat is necessarily included in the meal so that gallbladder contraction is maximally stimulated and the test sensitivity is maximized. Because most commonly used bile acid assays are robust to the effect of lipemia, to avoid insufcient stimulation it is generally recommended that pets 5 kg of body weight eat at least 2 teaspoons of a commercial canned food, and those that weigh more eat at least 2 tablespoons of a commercial canned food.32 Bile acid concentrations should always be interpreted in light of the animals feeding state. Compared with the fasting state, a mild increase in bile acid concentrations is expected in healthy animals after a meal. One of the most common pitfalls in bile acid measurement is the indiscrimination between fasting and stimulated concentrations. Bile acid concentrations above a certain cutoff (typically 20 and 25 mmol/L in cats and dogs, respectively) are indicative of liver dysfunction, regardless of the feeding state. Concentrations lower than this cutoff should be interpreted in light of the animals fasting/feeding state. Maximal sensitivity of the bile acid test for detection of liver dysfunction is achieved when paired samples are obtained. The rst sample should be obtained after a 12-hour fast. The animal is then fed an amount of food that is sufcient to stimulate gallbladder contraction and a second sample is obtained 2 hours later. A small proportion of animals have higher fastingthan stimulated bile acid concentrations. Therefore, a single bile acid concentration below the diagnostic cutoff is insufcient to rule out liver dysfunction, whether it is mea-

Topics in Companion Animal Medicine

sured in a fasted sample, a stimulated sample, or a random (in relation to feeding/fasting) sample. Enzymes Most enzymes are stable for 24 hours in sera at 24C and 4C. The degree of deterioration that occurs after 24 hours varies among enzymes and in some cases among species.23 A poor serum separation technique would prolong contact with the clot, allow some enzymes (e.g., AST, LDH) to escape from erythrocytes, and might lead to their false increase. Unlike heparinized plasma, the use of plasma harvested from most anticoagulant tubes may lead to a false decrease in certain enzymes activities. This is because of chelation of divalent cations such as Ca2 , Mg2 , Cu2 , and Zn2 , which often serve as coenzymes and are vital for enzymatic activity. Examples include falsely decreased alkaline phosphatase, lipase, and creatine kinase.12 The effects on enzyme activity depend on the analyzer and the methodology used.13 Hemolysis may lead to a false increase in enzymatic activity because of the release of enzymes found within the cytoplasm of erythrocytes (AST and LDH and possibly ALT). Additionally, there are intracellular chemical constituents that may participate in the assays measuring enzymatic activity. Examples include glucose-6-phosphate, ATP, or adenylate cyclase for CK activity. Lastly, hemoglobin leakage from erythrocytes might interfere with light transmission and can cause false increases or decreases in spectrophotometric assays.23

Coagulation Panels
There are many preanalytical steps that, if not performed adequately, can artifactually change coagulation tests results. These include appropriate venipuncture technique, adequate lling of the tube, timely processing of the sample, and, if the sample analysis is delayed, adequate sample storage. A comprehensive discussion of these is presented in the rst section of this article.

Prolonged PT and aPTT

A common preanalytical cause of prolonged PT and aPTT is underlling of the citrate tube. Compared with other anticoagulant tubes, there is a relatively large volume of anticoagulant in the citrate tubes. It is critical that these tubes are lled to the proper level. There should be a 9:1 ratio of blood:citrate. If these tubes are underlled, there is a relative excess of anticoagulant in the sample, which could potentially prolong the PT and aPTT results. A similar phenomenon occurs in animals with polycythemia (erythrocytosis). If a patient has a markedly increased hematocrit, there is less plasma for a given volume of whole blood. Less plasma volume causes a relative excess of anticoagulant because of a relative excess of citrate. Another potential source for error occurs if blood is contaminated with a second anticoagulant. This might occur when blood is drawn from a heparin-containing catheter or

Volume 26, Number 2, May 2011

blood is placed in the wrong tube (e.g., EDTA or heparin) and then transferred to the citrate tube. In these cases, prolonged PT and aPTT are expected. Another mishandling of the sample is failure to promptly and gently mix the blood with the anticoagulant, resulting in prolongation of PT and aPTT. Examples include initial drawing of blood to a tube with no anticoagulant, failure to mix the blood with the anticoagulant and delayed transferring of the blood from the syringe to the anticoagulant-containing tubes.33

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ma-based Coagulation Assays; Approved Guideline, ed 5. Wayne, PA, Clinical and Laboratory Standards Institute, pp H21-A5, 2008 Maeckelbergh VA, Acierno MJ: Comparison of prothrombin time, activated partial thromboplastin time, and brinogen concentration in blood samples collected via an intravenous catheter versus direct venipuncture in dogs. Am J Vet Res 69: 868-873, 2008 Bauer NB, Er E, Moritz A: Inuence of blood collection technique on platelet function and coagulation variables in dogs. Am J Vet Res 72:64-72, 2011 Meinkoth JH, Allison RW: Sample collection and handling: getting accurate results. Vet Clin North Am Small Anim Pract 37:203-219, 2007 Ceron JJ, Martinez-Subiela S, Hennemann C, et al: The effects of different anticoagulants on routine canine plasma biochemistry. Vet J 167:294-301, 2004 Muro J, Cuenca R, Pastor J, et al: Effects of lithium heparin and tripotassium EDTA on hematologic values of Hermanns tortoises (Testudo hermanni). J Zoo Wildl Med: 29:40-44, 1998 Bouchelle HP, Raskin RE: The effects of tripotassium EDTA and lithium heparin on cell preservation and sample processing of loggerhead sea turtle blood [abstract]. Vet Clin Pathol 32: 1566, 2003 Harr KE, Raskin RE, Heard DJ: Temporal effects of 3 commonly used anticoagulants on hematologic and biochemical variables in blood samples from macaws and Burmese pythons. Vet Clin Pathol 34:383-388, 2005 Bermes EW: Young DS, Specimen collection and other preanalytical variables, in Burtis CA, Ashwood ER (eds): Tietz Fundamentals of Clinical Chemistry, ed 5. WB Saunders; Philadelphia, PA, 2001, pp 30-54 Morales F, Couto CG, Iazbik MC: Effects of 2 concentrations of sodium citrate on coagulation test results, von Willebrand factor concentration, and platelet function in dogs. J Vet Intern Med 21:472-475, 2007 Christopher M, ONeill S: Effect of specimen collection and storage on blood glucose and lactate concentrations in healthy, hyperthyroid and diabetic cats. Vet Clin Pathol 29:22-28, 2000 Ferasin L, Dodkin SJ, Amodio A, et al: Evaluation of a portable lactate analyzer (Lactate Scout) in dogs. Vet Clin Pathol 36:3639, 2007 Furtanello T, Tasca S, Caldin M, et al: Artifactual changes in canine blood following storage, detected using the ADVIA 120 hematology analyzer. Vet Clin Pathol 35:42-46, 2006 Smalko D, Johnstone IB, Crane S: Submitting canine blood for prothrombin time and partial thromboplastin time determinations. Can Vet J 26:135-137, 1985 Stockham SL, Scott MA: Fundamentals of Veterinary Clinical Pathology, ed 2. Wiley-Blackwell, 2008 Medailli C, Briend-Marchal A, Braun JP: Stability of selected hematology variables in canine blood kept at room temperature in EDTA for 24 and 48 hours. Vet Clin Pathol 35:18-23, 2006 Norman EJ, Barron RC, Nash AS, et al: Prevalence of low automated platelet counts in cats: comparison with prevalence of thrombocytopenia based on blood smear examination. Vet Clin Pathol 30:137-140, 2001 Zelmanovic D, Hetherington EJ: Automated analysis of feline platelets in whole blood, including platelet count, mean platelet volume, and activation state. Vet Clin Pathol 27:2-9, 1998 Moritz A, Walcheck BK, Weiss DJ: Evaluation of ow cytometric

10.

11.

Shortened PT and aPTT

Overlling of the tube might cause an in vitro hypercoagulable state (shortened PT and aPTT). Hemolysis or excessive agitation of blood with citrate will also result in articially shortened PT and aPTT. Traumatic venipuncture can cause contamination of samples with tissue factor, initiate coagulation, and shorten coagulation times.

12.

13.

14.

Other Coagulation Parameters

Thrombin time (used to assess brinogen concentrations), brinogen and plasminogen concentrations, and antithrombin activity increase, whereas Von Willebrands factor measurements (by Rocket immunoelectrophoresis) might be decreased in the presence of hemolysis.4

15.

16.

17.

References
1. Osborn CA: Bartges JW, Inuence of fasting and eating on laboratory values, in Bonagura JD (ed): Kirks Current Veterinary Therapy XII. W.B. Saunders Co., Philadelphia, PA, 1992, pp 20-23 2. Latimer KS, Mahaffey EA, Prasse KW: Duncan and Prasses Veterinary Laboratory Medicine: Clinical Pathology, ed 4. IA, Iowa State Press 2003, p 178 3. Meyer DJ: Burkhard MJ, Causes and effects of interference with clinical laboratory measurements and examinations, in Bonagura JD (ed): Kirks Current Veterinary Therapy XII. W.B. Saunders Co., Philadelphia, PA, 1992, pp 14-19 4. Meyer DJ, Harvey JW: Clinical chemistry, in Meyer DJ, Harvey JW (eds): Veterinary Laboratory Medicine: Interpretation and Diagnosis, ed 3. WB Saunders, Philadelphia, PA, 2004, pp 145155 5. Bermes EW: Young D, Specimen collection and processing: sources of biological variation, in Tietz MW (ed): Textbook of Clinical Chemistry. W.B. Saunders, Philadelphia, PA, 1986, pp 478-518 6. Burtis CA, Ashwood ER: Tietz Textbook of Clinical Chemistry, ed 3. Philadelphia, PA, Saunders, 1999 7. Mischke RN: Ingo JA, Hemostasis: introduction, overview, laboratory techniques, in Feldman BF, Zinkl JG, Jain NC (eds): Schalms Veterinary Hematology, ed 5. Lippincott Williams & Wilkins, Philadelphia, PA, 2000, pp 519-525 8. Papp AC, Hatzakis H, Bracey A, et al: ARIC hemostasis study development of a blood collection and processing system suitable for multicenter hemostatic studies. Thromb Haemost 61:15-19, 1989 9. Clinical and Laboratory Standards Institute (CLSI): Collection, Transport, and Processing of Blood Specimens for Testing Plas18.

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and automated methods for detection of activated platelets in dogs with inammatory disease. Am J Vet Res 66:325-329, 2005 28. Stokol T, Erb HNA: Comparison of platelet parameters in EDTA- and citrate-anticoagulated blood in dogs. Vet Clin Pathol 36:148-154, 2007 29. Omana MA, Silvestre-Ferreira AC, Torrent E, et al: Variation of platelet parameters obtained with the ADVIA 120 using three different anticoagulants [abstract]. Vet Clin Pathol 32:229, 2003 30. Braun JP, Lefebvre HP, Watson AD: Creatinine in the dog: a review. Vet Clin Pathol 32:162-179, 2003

Topics in Companion Animal Medicine

31. Rossmeisl JH, Zimmerman K, Inzana KD, et al: Assessment of the use of plasma and serum chloride concentrations as indirect predictors of serum bromide concentrations in dogs with idiopathic epilepsy. Vet Clin Pathol 35:426-433, 2006 32. Webster CR: Laboratory evaluation of hepatobiliary diseases, in Ettinger SJ, Feldman EC (eds): Textbook of Veterinary Internal Medicine, Diseases of the Dog and the Cat, ed 7. Elsevier, 2011 33. Marini JJ, Wheeler A: Clotting and bleeding disorders and anticoagulation therapy, in Critical Care Medicine: The Essentials, ed 4. Lippincott Williams & Wilkins, 2009, p. 542