Simple, Practical, and Inexpensive Diagnostics: They Tell Us More Than We May Think

As we navigate the rapid development and transformation of veterinary medicine, diagnostic tests are continuously emerging and evolving. Although these newer tests can be valuable and relevant resources, they often provide a narrow spectrum of information attached to a sizable price tag. As newer diagnostic tests become available, many older, practical, and affordable tests may be overlooked. Performing and interpreting these diagnostics are critical skills as these tests can provide comprehensive diagnostic, monitoring, and treatment information. This article provides cost (BOX 1), indications, methods, and interpretation for several key practical and cost-effective diagnostic tests.

Blood Smear

Blood smears are a fundamental part of a complete blood count (CBC). Although smears are inexpensive, training is required to prepare good quality slides, analyze them, and interpret the findings (BOX 2, FIGURE 1). A well-prepared blood smear remains paramount for:

• Verifying and providing accurate differential cell counts
• Assessing morphologic abnormalities (e.g., red blood cells [RBCs], leukocytes/white blood cells [WBCs])
• Identifying hemoparasites, neoplastic cells, and cellular inclusions

Cost: $

Materials needed: Microscope slides, Diff-Quik stain (Romanowsky stain variant), microscope with 4× to 100× objective lenses, immersion oil

Methods and interpretation: Evaluating blood smears requires a systematic approach. To assess the quality of the smear, the authors recommend initial evaluation under low magnification, followed by assessment of the density of RBCs and WBCs (10× to 20× objective). Slides are also assessed for rouleaux formation and/or agglutination. Under the same objective, the feathered edge is assessed for platelet clumps, microfilaria, or any large abnormal cells (e.g., blast cells, mast cells, macrophages).

To approximate hematocrit, monolayer examination of RBC apposition may be used. In anemic patients, RBCs are widely separated from one another; in nonanemic patients, RBCs are closely apposed (FIGURE 2). These estimates should be referenced against measured hematocrit or packed cell volume (PCV).

To approximate WBC counts under the 10× objective, count the number of WBCs in the monolayer (usually 18 to 50 cells). Each WBC corresponds to 330 cells/µL. For example, 13 WBCs/10× objective; 13 × 330 cells/µL = 4290 WBCs/µL. The number of cells per µL = (average no. cells/field) × (objective power).¹

A more definitive assessment of RBCs, WBCs, and platelets can be performed under higher power (100× objective). More specifically, a manual differential cell count can be performed to verify hematology analyzer results, concurrently assessing morphology, density, and presence of inclusion bodies or parasites (TABLE 1, FIGURES 3 AND 4). At 100× magnification, a platelet count can also be performed. Platelets over several fields (5 to 10/field) are counted and then averaged. The average platelet number is then multiplied by 15 000 to 20 000 to estimate platelets/µL (FIGURE 3).

Packed Cell Volume/Total Protein

This test is underutilized in veterinary medicine. When performed and interpreted correctly, the test provides valuable information for disease diagnoses and assists clinicians with medically guided treatment.

Cost: $

Materials needed: Determining PCV requires microhematocrit tubes, clay sealer, a microcentrifuge, a refractometer, and a PCV reading tool.

Methods and interpretation: PCV should always be interpreted in conjunction with total protein and assessed with patient history, clinical examination findings, and other diagnostic test results when available (TABLE 2). Reference ranges are 37% to 55% for adult dogs (most breeds) and 30% to 45% for cats. PCV is lower in young animals (4 weeks; 24% to 34%) than in adult animals and gradually increases with maturity.¹

Microhematocrit tubes also provide valuable information after blood is centrifuged and separated into layers.

1. Assess the plasma color, which is normally clear and colorless (TABLE 3).
2. Next, examine the buffy coat. The buffy coat consists of WBCs with a thin white layer of platelets on the top (FIGURE 5). If the bottom layer of the buffy coat is pink to red, it indicates presence of abnormal and immature RBCs. Buffy coat thickness is measured similarly to PCV; accurate measurements are difficult. The first 1% (0.01 L/L) is equivalent to 10 000 WBCs/µL (1 × 10⁹ WBCs/L), and each additional 1% represents 20 000 WBCs/µL (2 × 10⁹ WBCs/L).¹
   

   Figure 5. Blood-filled microhematocrit tubes after centrifugation. Note the red discoloration of plasma in the sample on the left, suggestive of free hemoglobin. The buffy coat (arrow) is easily visualized between the red blood cells and plasma.

3. Measure total proteins by using a refractometer. Reference total protein levels in dogs range from 5.9 to 7.8 and in cats from 5.9 to 7.5.²

Urinalysis

Complete urinalysis results play a vital role in evaluating urinary tract health and provide valuable information about the systemic wellness of patients. Urinalysis should be interpreted in conjunction with patient history and physical examination findings and is indicated for any patient having blood collected for a CBC and serum chemistry. Likewise, urine should be analyzed for patients that are systemically ill or have renal disease.

Cost: $

Materials needed: Refractometer, urine dipstick, centrifuge, pipette or syringe, conical or red top tube, microscope slides, microscope with 4× to 100× objective lenses, and immersion oil

Methods and interpretation: Urine can be collected by free catch, catheterization, or cystocentesis. Recording both the method and time of collection is recommended as these factors may affect results. Early morning urine samples are considered best for evaluating tubular function, whereas late morning to early evening samples are best for microbial culture and assessing cellular morphology.^3,4 To avoid temperature- and time-dependent effects on crystal formation, samples should be analyzed within 1 to 2 hours of collection³; if not analyzed during this window, samples should be refrigerated.

Assessment involves evaluation of physical characteristics, semiquantitative chemical analysis, and sediment.

Physical Characteristics

• Color: Normal urine color is light yellow to amber but may be influenced by diet, medications, and hydration status.⁵ Abnormal urine colors, when observed, should be investigated to determine the underlying cause. Pigmenturia may be differentiated from hematuria by centrifugation.
• Clarity: Urine clarity is influenced by particulate matter, which is subjectively graded from clear to flocculent. Turbid urine samples should undergo sediment evaluation to determine the cause.
• Specific gravity: Urine specific gravity (USG) measures the solute concentration of urine, providing an estimate of the ability of the renal tubules to concentrate or dilute the glomerular filtrate.^5,6 It should be interpreted in light of patient hydration status, electrolyte concentrations, blood urea nitrogen and creatinine concentrations, certain medications (e.g., diuretics, corticosteroids), and fluid therapy administration. USG obtained from a first morning urine sample can indicate optimum concentration ability but can vary up to 0.015 from day to day in healthy animals.⁷

Semiquantitative Chemical Analysis (pH, Blood, Glucose, Ketones, Bilirubin, and Protein)

Dry reagent strips are used in veterinary medicine to determine urine pH, protein, glucose, ketones, bilirubin/urobilinogen, and occult blood. Tests pads produce a colorimetric chemical reaction when interacting with specific substances in urine.⁸ Reagent strips are unreliable for measuring USG, WBCs, nitrite, and urobilinogen.⁹ Pigmented urine can interfere with test strip readings. Urine pH, a global estimate of acid–base status, may be affected by many renal and extrarenal factors (e.g., handling, diet, medications, bacterial infection, systemic illness).^5,10 Glucosuria, when detected, should be assessed concurrently with serum glucose.¹¹ Determining protein concentration by dipstick, which primarily detects albumin, is a good screening test for proteinuria. Results must be interpreted in light of USG, pH, and urine sediment examination.⁵ Negative reactions are usually reliable; samples with positive test reactions for patients with an inactive urine sediment should undergo quantitative confirmatory testing (i.e., urine protein:creatinine [UPC] ratio).

Sediment Evaluation (RBCs, WBCs, Organisms, Epithelial Cells, Crystals, and Casts)

Sediment provides definitive evidence of inflammation, infection, or hemorrhage within the urinary tract.⁵ Samples are centrifuged at low speed (1000 to 1500 rpm) for 5 minutes before most of the supernatant liquid is removed, leaving the sediment and a small volume (0.5 to 1 mL) of urine. The sample is then resuspended and 1 drop is placed on a microscope slide with a coverslip. Slides are analyzed at low power for crystals and casts and at high power for cells (RBCs, WBCs, transitional cells) and bacteria. Crystalluria is a common finding; certain crystals (e.g., amorphous, struvite) can be seen in healthy dogs.¹² More information on slide preparation and analysis is available in the Today’s Veterinary Practice May/June 2014 article, Urinalysis in Companion Animals, Part 2: Evaluation of Urine Chemistry and Sediment.¹³

Targeted Urine Testing

Targeted urine testing includes the urinary corticoid:creatinine ratio (UCCR) and UPC ratio. Testing is minimally invasive and can provide valuable information for screening and diagnosing disease. It may also be used for monitoring disease progression or treatment response. Proper patient selection helps ensure the most useful and cost-effective information. 

Cost: $$

Materials needed: Clean container for collecting urine

Urinary Corticoid:Creatinine Ratio

UCCR is a valuable screening test that has been used for decades for patients suspected of having hyperadrenocorticism.¹⁴ The test provides an indirect, integrated measure of circulating corticoid production over time by measuring renal excretion of cortisol (free and conjugated). The test therefore eliminates rapid fluctuations in plasma concentrations of cortisol, which can lead to a false-positive diagnosis.^15-17 This relatively inexpensive test is widely available to clinicians through commercial diagnostic laboratories.^18,19 The test is used to screen for hyperadrenocorticism in dogs but may also have diagnostic value as a screening test for hypoadrenocorticism (sensitivity 97.2% to 100%, specificity 93.6% to 97.3%).^14,16,18 It should not be used to monitor treatment of hyperadrenocorticism because it is not considered a reliable indicator of successful treatment.^20-22 The test is advantageous over other screening tests because clients can collect urine from clinically healthy animals in a stress-free home environment. As a screening test for hyperadrenocorticism, UCCR sensitivity is high (75% to 95%), but specificity is poor (reportedly as low as 20%).^17,18,23-29 The low specificity is attributed to increased corticoid release in stressed healthy dogs (e.g., during hospitalization) and dogs with nonadrenal-associated illness.^28,29 Therefore, UCCR can help rule out hyperadrenocorticism but should not be used to establish a diagnosis.

Urine Protein:Creatinine

UPC is another valuable urine test that measures the amount of protein loss through the kidneys. It can be valuable for diagnosing and staging disease as well as guiding clinical decisions and treatment response. UPC can also be a prognostic indicator; numerous studies have shown greater risk for death among patients with proteinuria.³⁰ This test is recommended for patients with documented proteinuria or suspected proteinuria based on an underlying disease process. The magnitude of UPC elevation can also help identify the location of protein loss (tubular versus glomerular disease).³⁰

Methods and interpretation: Because urine collected as a single sample, serial samples, or pooled samples yields similar results, single-sample collection may be preferred due to the ease of collection and analysis.³¹ This diagnostic test is available at reference laboratories and can also be performed in-clinic with some analyzers. UPC should always be performed in combination with urinalysis as abnormal urine sediment resulting from disease in the lower urinary tract can also increase the UPC (postrenal proteinuria).³⁰

Because UPC fluctuates widely day to day, for a single UPC measurement to be considered significant, it must vary substantially. For a safe assumption that proteinuria has increased, single samples must differ by up to 40% in dogs and 90% in cats.³⁰

A UPC of <0.2 is considered to be within normal range, with a UPC of 0.2 to 0.5 in dogs and 0.2 to 0.4 in cats considered to be borderline proteinuric. To investigate the underlying cause of proteinuria when the UPC is ≥2, additional diagnostics should be performed. UPC ratios of ≥0.5 (dogs) and ≥0.4 (cats) in stable patients should be monitored 2 to 3 times at 2- to 3-week intervals to determine if the proteinuria is progressing. For nonazotemic patients with a UPC ≥2, pathologic glomerular disease should be considered more likely. For patients with chronic kidney disease (i.e., azotemic patients) with UPCs ≥0.5 (dogs) and ≥0.4 (cats), further diagnostics should be performed to elucidate a cause for the proteinuria. When an underlying cause is identified, it should be treated.

Basal/Resting Cortisol

Basal/resting cortisol testing provides fast, simple, reliable, and cost-effective screening for animals that have clinical signs compatible with hypoadrenocorticism. Sensitivity is high (99.4% to 100%) when resting cortisol is <2 µg/dL (<55 nmol/L)^32-34; however, specificity is relatively low (63.3% to 78.2%), so it cannot be used as a confirmatory diagnostic test.^32,34

Cost: $$

Materials Needed: Serum separator tube

Interpretation: Basal/resting cortisol testing is better used as a test of exclusion for patients that have waxing and waning unexplained illness, have chronic gastrointestinal signs, or are highly suspected to have hypoadrenocorticism. That is, hypoadrenocorticism should be removed from the differential list for patients with resting cortisol levels of >2 µg/dL (>55 nmol/L). For patients with a resting cortisol of <2 µg/dL, an ACTH (adrenocorticotropic hormone) stimulation test should be performed to definitively diagnose hypoadrenocorticism.

Noninvasive Blood Pressure

Cost: $

Materials needed: Doppler unit and sphygmomanometer or oscillometric device, appropriately sized blood pressure cuff, ultrasonography gel (FIGURE 6).

Indications: Systemic arterial pressure is often referred to as the “fourth vital parameter” and provides valuable information about cardiovascular status for anesthetized patients, patients in emergent situations, and patients with associated diseases that pose a risk for the development of hypotension and hypertension. Arterial blood pressure plays a role not only in diagnosis but also in monitoring disease progression and guiding clinicians with therapeutic decision making.

Methods and interpretation: Indirect blood pressure measurement involves detecting arterial blood flow or vessel wall movement in a peripheral artery (palmar arterial arches of the forelimb or hindlimb or coccygeal artery of the tail). Oscillometric or Doppler sphygmomanometry techniques provide measurements of systolic arterial blood pressure.

Indirect blood pressure measurements can vary widely depending on the patient’s signalment and temperament, body condition, measurement technique, and operator experience. Several studies have identified a range of indirect systolic blood pressure measurements of 131 to 151 mm Hg for dogs and 115 to 162 mm Hg for cats (BOX 3).³⁵

Continuous Glucose Monitoring

Cost of sensor: $$$ (FreeStyle Libre; Abbott, freestylelibre.us)

Materials: Flash glucose monitoring sensor with corresponding applicator and reading device

Methods and interpretation: Historically, glycemic control in patients has been monitored via in-hospital and at-home blood glucose curves, spot blood glucose measurements, urine glucose measurements, and fructosamine measurement.³⁶ In recent years, flash glucose monitoring systems (FGMSs) have become more popular in veterinary medicine and provide an optimal, low-cost, low-stress way to monitor patients with diabetes. These factory-calibrated devices provide real-time estimates of blood glucose by constantly measuring the glucose concentration of the interstitial fluid through a disc-shaped sensor inserted into the subcutaneous space for up to 14 days. No blood sampling is required for patients with an FGMS; instead, the sensor has to be “flashed” with a portable reader or smartphone at least every 8 hours to retrieve, download, and store glucose measurements (FIGURE 7). The data can be uploaded to an online portal for remote interpretation by the clinician. Sensors are easily applied, are well tolerated, and have been validated in dogs and cats.^37-41

Several studies in dogs and cats have indicated good correlation between blood glucose measurements by FGMSs and by point-of-care glucometers and automated biochemistry analyzers.^38-44 Accuracy of FGMS measurements is lower for patients with diabetic ketoacidosis (DKA) and hypoglycemia (<70 mg/dL), most likely because of delayed equilibration of blood and interstitial glucose (i.e., rapid insulin-induced changes in blood glucose concentrations and dehydration) in DKA patients.⁴¹ Lag times between blood glucose and interstitial glucose have been reported as 5 to 10 minutes for dogs and 11.4 minutes for cats.^38,39

Ideally, sensors should be placed in areas that are difficult for the patient to reach and where skin thickness is greater than 5 mm and has limited subcutaneous movement. The dorsal neck and lateral chest wall are commonly used sites.^45,46 To lessen the chances of sensor displacement, the authors recommend placing 3 to 4 drops of cyanoacrylate tissue glue on the sensor surface adhesive after hair and skin site preparation but before sensor application. In cats, the authors prefer to place the sensor on the dorsal neck and to use Kitty Kollars (kittykollar.com) to further prevent the sensor from being dislodged.

After successful placement, the sensor is scanned to link the device to the reader; it takes 60 minutes before data can be collected from the sensor. Few complications are associated with FGMSs, the most common being premature detachment (up to 80%).^36,42 Superficial contact dermatitis has been reported and usually resolves within several days after sensor removal. Sensor malfunction has been reported, potentially caused by:

• Inadequate placement of the sensor
• Excessive movement of the sensor leading to damage of the sensor filament
• Local bleeding impairing the acuity of the system

FGMS sensor performance may also be affected by x-ray radiation and magnetic resonance imaging. While sensor accuracy following imaging has not been studied, the authors recommend placement of sensors after imaging has been completed. Clients should be counseled that although current new-generation sensors are designed for up to 14-day use, most devices provide data for only 7 days before sensor failure. It is also imperative that clients be instructed to not adjust insulin dosages based on their interpretation of the curve created by the device. When unlikely values are reported, it is recommended to spot check a blood glucose to ensure accuracy.

Cytopathology

In-house cytopathology is minimally invasive, rapid, and cost-effective. Its diagnostic value depends on the critical selection of appropriate lesions, good sampling technique, and quality sample handling.⁴⁷ Its usefulness is substantial as incorporation of ultrasonography into veterinary practices has made sampling of internal organs/tissue more accessible, and most patients do not require sedation or anesthesia.

Cytopathology is used to confirm initial clinical impressions, often providing a definitive diagnosis. It is also used to guide clinicians with regard to initial client communications about the need for additional diagnostic workup (e.g., staging) and testing (e.g., molecular diagnostics, special stains, culture for infectious agents, immunocytochemistry, histopathology).

Cost: $$$

Materials: Needles (21 to 27 gauge), syringe (6 mL), glass microscope slides, Romanowsky-type stain, microscope with 4× to 100× objective lenses, immersion oil. Needle size does not significantly affect cytologic adequacy; however, poorly exfoliative sites require larger gauge needles.

Methods and interpretation: After sample collection, slides are initially evaluated for sample adequacy. Initial evaluation is performed under low power. Samples are then evaluated for diagnosis. Slides are scanned at low power, which is transitioned to a higher power to identify pathology.

The authors recommend categorizing slides as normal, inflammatory, or neoplastic. Definitive diagnosis is possible, but samples may need to be sent to an external laboratory or may require digital image–based telemedicine for pathology review.

Agreement between cytology and histopathology depends on sample recovery, lesion visualization, tissue sampling location, and method of collection. Agreement reportedly ranges from 33.3% to 66.1% (TABLE 4).⁴⁸

Summary

Despite the ever-expanding assortment of diagnostic tests becoming available, do not overlook simple, practical, and affordable diagnostic tests. When performed correctly, these economical tests provide a wealth of information to facilitate diagnosis and treatment of patients.