Laboratory Testing

Basic laboratory work – complete blood count (CBC) and biochemical panel – in combination with a urinalysis is often performed during the work‐up of a respiratory patient and can help support the presence of an underlying respiratory tract disease. With local infectious or inflammatory disease processes such as rhinitis and tracheobronchitis, hematologic changes are typically absent. In contrast, in animals with nasal neoplasia and those with parenchymal diseases such as bacterial or aspiration pneumonia or pyothorax, a neutrophilic leukocytosis is often found, and a left shift on the leukogram supports an infectious bacterial process. Peripheral neutrophilia or eosinophilia is reported in eosinophilic pneumonia/bronchopneumopathy and in feline bronchial disease. Fungal pneumonia is anticipated to result in neutrophilia and monocytosis, reflecting the chronic nature of the disease. It is unusual for chronic hypoxemia to result in polycythemia (hematocrit >65%), with one study reporting no correlation between hypoxemia and any red blood cell parameters (Holopainen et al. 2022). Polycythemia should be considered more suggestive of a right to left cardiac shunt than with a respiratory cause of hypoxemia.

Biochemical abnormalities in respiratory diseases are usually non‐specific. Hyperglobulinemia can result from chronic antigenic stimulation in cases of feline bronchial disease, fungal pneumonia, chronic foreign body or aspiration pneumonia, or bronchiectasis, and concurrent mild hypoalbuminemia is occasionally present as a negative acute phase response.

Molecular diagnostics are increasingly used to document the presence of nucleic acids from an infectious organism, such as feline herpesvirus‐1, Bordetella, or Mycoplasma, in either upper or lower respiratory tract disease; however, there are important limitations to the interpretation of these results (see the sections on specific diseases). It is critical to understand that a positive molecular assay indicates only the presence of nucleic acids of the organism and does not confirm that the organism is responsible for the clinical disease identified. While culture techniques can be similarly misleading because many organisms are part of the normal flora of the respiratory tract, positive culture results reflect the presence of a multiplying, viable organism within the sample. Culture also allows assessment of susceptibility to antimicrobials.

Various studies have investigated the utility of biomarkers for differentiating cardiac from respiratory disease in animals presenting for cough or respiratory difficulty. The most common biomarker evaluated is plasma N‐terminal pro‐brain natriuretic peptide (NT‐BNP), which is produced in response to ventricular strain or stretch. A commercially available enzyme‐linked immunosorbent assay (ELISA) test is available for dogs and cats that provides a quantitative measure of NT‐BNP. This biomarker is reliably elevated in dogs with congestive heart failure in comparison to dogs with respiratory disease; however, there is overlap between groups and in some studies, the non‐cardiac causes of respiratory distress are poorly defined. It is unclear whether NT‐BNP is of added benefit when compared to combined assessment of history, physical examination, thoracic radiographs, and cardiac ultrasound in dogs. Also, the utility of measurement has not been assessed in dogs that have concurent cardiac and respiratory disease.

A point‐of‐care test is available for assessing feline NT‐BNP, and this can be applied to serum or pleural effusion fluid. Positive BNP tests in combination with appropriate cardiac and lung ultrasound examination could reliably establish congestive heart failure as the cause of respiratory distress, although the plasma BNP test was elevated in over 25% of cats with respiratory distress that was not due to cardiac disease (Ward et al. 2018). Renal disease can also increase BNP levels, therefore caution is warranted in relying on BNP alone for a diagnosis.

Testing for Hemorrhage

Animals presented for epistaxis, hemoptysis, and hemothorax require additional considerations when completing a diagnostic work‐up because of the concern for worsening the animal’s clinical presentation with invasive procedures in the presence of a bleeding disorder. Therefore, prior to performing diagnostic testing for local causes of bleeding, systemic causes of bleeding should be ruled out. Disorders of primary hemostasis (deficits in platelet number or function and vasculitis), secondary coagulopathic disorders (defects in clotting factors), and hypertension should all be considered. Epistaxis is most commonly seen with defects of primary hemostasis, and especially in animals with severe thrombocytopenia, although occasionally it will be seen in animals with hypertension. Vasculitic disorders or hyperviscosity can also result in nasal bleeding or in pulmonary hemorrhage, as is believed to occur in leptospirosis and leishmaniasis. Disorders of secondary hemostasis, and rodenticide intoxications in particular, lead to respiratory distress in association with parenchymal bleeding and can also cause mild to moderate pleural effusion.

A CBC will provide accurate assessment of platelet numbers, although determination of platelet function requires additional tests. A von Willebrand factor antigen assay is commercially available, but more specific tests of platelet function are typically only available at academic or research institutions. However, a buccal mucosal bleeding time (BMBT) can be performed in hospital practices to estimate in vivo platelet and vascular function. This test requires a compliant dog or a heavily sedated cat, because of the need for gentle restraint and for working in the region of the mouth.

To perform a BMBT, the animal is restrained in lateral recumbency and the lip is gently restrained upward with a strip of gauze to expose the buccal mucosa. Multiple squares of paper towel or filter paper should be available to gently blot the region below the incision into the mucosa. A spring‐loaded device containing a retractable blade (Surgicutt®, Accriva Diagnostics or JorVet®, Jorgensen Laboratories, Loveland, CO, USA) is used to make a standardized incision on the mucosa opposite the premolars. Blood can be blotted from below the incision line, but the clot should not be disturbed in order to obtain an accurate bleeding time. In normal cats, a clot will develop in 1–2 minutes and in dogs, a clot will be observed in 2–4 minutes.

For animals with hemoptysis or hemothorax, a disorder of secondary hemostasis should be investigated by performing a coagulation panel (Figure 2.1). The one‐stage prothrombin time (OSPT) provides an assessment of the extrinsic coagulation pathway and vitamin K‐dependent factors (II, VII, IX, X), while the activated partial thromboplastin time (APTT) evaluates the intrinsic and common pathway. In an emergency room, the activated clotting time (ACT) is often used to assess the intrinsic and common pathway (Table 2.1).

Additional tests of coagulation include D‐dimer and thromboelastography. D‐dimer measures the breakdown product of cross‐linked fibrin and is a reliable indicator that clotting and fibrinolysis have occurred. While this is a highly useful test in assessing the likelihood of pulmonary embolism in human patients, the test is commonly elevated in dogs with a variety of disease processes. Thromboelastography evaluates the kinetics of clot formation and breakdown, and thus can identify both hyper‐ and hypocoagulable states that could require intervention (Kol and Borjesson 2010). This test can be used to assess the need for anti‐thrombotics and for monitoring use of anticoagulants (see Chapter 8).

Testing for Allergy

An allergic etiology is often presumed when airway eosinophilia is documented, such as with eosinophilic airway disease/asthma in cats and eosinophilic lung disease in dogs, or with nasal signs in dogs or cats because of the tendency to correlate animal diseases with human diseases. Owners can ascribe seasonality to clinical signs based on their own diseases and fail to take into account the role that irritants in the environment might play. For example, animals that develop clinical signs as the weather warms might be responding to dusts or molds dispersed by air conditioning units rather than to environmental allergens.

Much remains unknown about the immune systems of veterinary species, and wide exposure to endo‐ and ecto‐parasitism that can cause eosinophilia hampers investigations into the role of potential hypersensitivity in naturally occurring respiratory diseases. Options for testing include total serum IgE, allergen specific IgE levels, and intradermal testing (IDT), but few tests have been fully validated in cats and dogs. Serologic tests can provide conflicting results depending on the methodology used, and IDT is notoriously difficult to interpret in cats. The effect of age on the immune response also has to be taken into consideration with well‐designed clinical investigations. Thus, the role of allergy in naturally occurring respiratory diseases remains controversial and routine allergy testing is not currently recommended for respiratory diseases.

Pulse Oximetry

Pulse oximetry provides an estimate of hemoglobin saturation with oxygen and is inexpensive, non‐invasive, and easy to perform. However, one problem with the technique is that it has low sensitivity and specificity for identifying normal versus abnormal arterial oxygenation in awake patients (Farrell et al. 2019). Methodology relies on detection of differential light transmission through oxygenated and deoxygenated hemoglobin, which have different absorption spectra, as blood passes through the circulatory system. Therefore, measurement is impacted by the pigment of overlying tissue and potentially by the amount of light in the area where the test is being performed. The sensor subtracts the signal between pulses from the height of the pulse wave to determine oxygenation of inflowing blood only. Because of this feature, pulse oximetry can provide a falsely low measurement in a hypotensive patient with weak pulses or in an animal with anemia. Patient movement can hamper detection of the impulse. Finally, this technique cannot differentiate between methemoglobin and oxyhemoglobin and will be inaccurate in any animal with a dyshemoglobinemia.

A pulse oximeter reading below 95% correlates with a partial pressure of oxygen (P_aO₂) of less than 80 mmHg (Figure 2.2). When such a reading is obtained, an arterial blood gas analysis should be performed, if available, to confirm the presence and degree of hypoxemia. Sites that can be used to obtain a pulse oximetry measurement include the lip, tongue, between the toes, on the ear pinna, on the vulva or penis, and sometimes on the flank fold. The probe can be applied to various sites several times to obtain a signal, and detection of a strong pulse rate suggests that the reading is likely accurate. It is important to remember that the pulse oximeter measures only oxygenation, not ventilation, which is assessed by measuring P_aCO₂. A venous blood gas analysis can be used as an approximation of this parameter.

Because of limitations in technology and various patient factors, direct measurement of P_aO₂ indicates that hypoxemia is absent in up to 7% of dogs with abnormal pulse oximeter readings and can be present in a similar proportion of dogs with pulse oximetry readings above 95% (Farrell et al. 2019). Therefore, an arterial blood gas would be advised whenever possible to ensure an accurate assessment of the patient, although in a practice setting this is rarely possible.

Despite its limitations, pulse oximetry can be useful prior to anesthetizing the patient for a respiratory procedure, as it provides a baseline for comparison during recovery. Pulse oximetry could also be considered somewhat helpful in determining response to therapy in hypoxemic patients, because improvements in oxygenation occur prior to radiographic changes. However, because of the sigmoidal relationship between hemoglobin saturation and arterial oxygen, oximetry remains a crude estimate of lung function. Also, this curve is shifted to the right (reducing the affinity of hemoglobin for oxygen and enhancing tissue delivery) by conditions associated with exercise and disease, such as increased temperature, decreased blood pH, increased P_aCO₂, and increased 2,3‐diphosphoglycerate (DPG) produced in red blood cells during glycolysis. Thus, changes in disease status can hamper the interpretation of results.

Other clinical features can impact interpretation of the oxyhemoglobin saturation curve. Stored red blood cells have lower concentrations of 2,3‐DPG, and transfusion with this product increases the affinity of hemoglobin for oxygen and reduces oxygen delivery to the tissues. It appears that this is primarily a concern during the first 4–12 hours after transfusion, until 2,3‐DPG concentrations rebound. Finally, hypophosphatemia also decreases 2,3‐DPG which would impact interpretation of pulse oximetry readings.

Arterial Blood Gas Analysis

Arterial samples are obtained by direct puncture of the dorsal metatarsal artery in dogs and the femoral artery in cats and small dogs, using a small gauge (25 gauge) heparinized syringe. In anesthetized animals, the lingual artery can yield a sufficient sample for analysis. It is easiest to use a self‐filling, pre‐heparinized syringe to obtain an arterial blood gas. The artery is palpated and stabilized with two fingers of one hand while the syringe is firmly placed through the wall of the artery. Typically, the syringe will fill quite rapidly when the artery is entered (versus the vein), although low pulse pressures in sick animals can make this difficult to interpret. When there is uncertainty about whether or not an arterial sample has been obtained, a venous sample can be submitted for comparison. Approximately 0.5 ml blood is needed for analysis and the sample must be tightly stoppered to prevent entrance of air into the sample and stored on ice until evaluated. After withdrawal of the needle from the artery, firm pressure is applied to the vessel for 3–5 minutes to prevent hemorrhage. An arterial blood gas analysis measures P_aO₂, pH, total carbon dioxide (CO₂), and hemoglobin saturation with oxygen, and allows calculation of bicarbonate, base excess and deficit, and oxygen content (Table 2.2). A P_aO₂ < 80 mmHg indicates hypoxemia and a P_aCO₂ > 45 mmHg is consistent with hypoventilation.

Alveolar–Arterial Oxygen Gradient and PF Ratio

The alveolar–arterial (A–a) oxygen gradient calculates the difference between the calculated alveolar oxygen level expected for the animal given its ventilatory status and the measured arterial oxygen level. Thus, the A–a gradient corrects for the level of ventilation performed by the animal and allows comparison of blood gas data throughout the course of disease that is not impacted by the effect of an increase or a decrease in P_aCO₂ on P_aO₂. The A–a oxygen gradient is calculated as:

where F_iO₂ is the fraction of inspired oxygen (0.21 on room air), PB is the barometric pressure (in mmHg), PH₂O is the water vapor pressure (47 mmHg at 37°C), and R is the respiratory quotient (ratio of CO₂ production to O₂ consumption, usually assigned a value between 0.8 and 1.0). P_aO₂ and P_aCO₂ are obtained from blood gas analysis. The normal value for the A–a oxygen gradient is <15.

The PaO₂/FiO₂ ratio (PF or oxygenation ratio) provides a measure of the ability of the lung to oxygenate as the fraction of inspired oxygen changes from room air to 100% oxygen. This is calculated by dividing arterial oxygen by FiO₂ (ranging from 0.21 to 1.0). Normal animals have a PF ratio of ~500 at sea level. Values between 300 and 500 indicate mild impairment of oxygenation, while values <200 indicate serious decrements in oxygenation. This ratio can be followed during treatment of a hypoxemic patient to determine therapeutic response, although the FiO is typically approximated by a ventilator setting rather than being measured directly. A PF ratio <200–300 is one of the criteria for a diagnosis of acute respiratory distress syndrome.

Radiography

Radiography is often the key to creating an appropriate list of differential diagnoses for the respiratory case and for determining the type of sampling method that is most likely to achieve a final diagnosis, such as endoscopy, fine‐needle aspiration (FNA), or an airway wash (Table 2.4). It will also help determine the need for advanced imaging, including fluoroscopy, ultrasound, nuclear scintigraphy, or computed tomography (CT). The widespread use of digital radiography has enhanced the evaluation of pulmonary patterns. Diagnostic imaging features for each disease are presented in the relevant sections.

Orthogonal views are always recommended for evaluation of thoracic contents, and assessment of the thorax is improved by obtaining both left and right lateral views, as well as a dorsoventral or ventrodorsal image. Lateral images provide an optimized view of the lung closest to the radiographic unit, therefore a left lateral projection would be more likely to identify infiltrates in the right middle lung lobe in a patient with aspiration pneumonia. A right lateral projection might be preferred to investigate airway collapse at the left cranial lobar bronchus (cranial and caudal segments). The dorsoventral view provides better imaging of the cardiac silhouette and pulmonary vessels, although the ventrodorsal view allows better detection of small volumes of pleural effusion, as well as infiltrates in the ventral or lateral portions of the lung. Importantly, attempts should be made to confirm the presence of pulmonary nodules on both a lateral and an orthogonal view.

In some patients, cervical radiographs can provide valuable information on the extrathoracic respiratory tract. Loss of the nasal air column from the nasal cavity into the nasopharynx, elongation or thickening of the soft palate, the suggestion of laryngeal edema or mass, air in the laryngeal ventricles, or caudal retraction of the larynx are features to investigate. These could reflect the presence of an upper airway obstructive lesion that might contribute to nasal discharge through nasopharyngeal regurgitation, disordered breathing associated with obstruction, or a lower respiratory tract process caused by mishandling of food and aspiration injury. Obtaining a lateral cervical view in addition to both lateral thoracic images can also enhance detection of tracheal collapse as well as intrathoracic airway or bronchial collapse.

Ultrasound

Ultrasound of the larynx can be used to evaluate patients for laryngeal paralysis or mass lesions, and cervical tracheal collapse can also be identified with ultrasound, although these studies can be technically challenging because soft tissues are adjacent to air‐filled structures, which results in marked attenuation of the ultrasound beam. However, valuable information can be gained by an experienced ultrasonographer that is then confirmed by endoscopy.

Thoracic ultrasound is highly useful in detecting pleural fluid and evaluating parenchymal mass lesions, and has also proven effective in identifying pulmonary infiltrates, particularly in the emergency situation. Fluid or thickening in the alveolar–interstitial space results in the artifact of comet tails, referred to as B‐lines. With alveolar–interstitial syndrome, the difference in echogenicity of fluid filled alveoli and surrounding gas generates hyperechoic wedges that extend to the pleura and move with respirations. In dogs and cats with pulmonary edema due to congestive heart failure, there is an increase in the number of detectable B‐lines visible in multiple lung fields (Lisciandro et al. 2017; Ward et al. 2017), while the absence of B lines can essentially rule out congestive heart failure. Because this technique relies on attenuation of the ultrasound beam by thickening of alveolar‐interstitial junctions, other diseases that result in this process and extend to the lung periphery could lead to the generation of B‐lines on ultrasound. Therefore, the use of lung ultrasound in large numbers of patients with respiratory conditions such as interstitial lung disease, pneumonia, and non‐cardiogenic pulmonary edema requires additional study. Nonetheless, it appears that ultrasound of the lung could be used to evaluate patients for fluid overload or congestive heart failure and to assess response to therapy. This technique would likely be less stressful to the patient than recheck radiographs and would result in less radiation exposure for the health‐care team.

Cardiac ultrasound can also be useful in providing preliminary assessment of cardiac structure and function in the emergency setting. Pericardial effusion is readily apparent as hypoechoic fluid constrained by a hyperechoic rim of pericardium that surrounds contracting myocardium. Decreased cardiac wall motion can be documented in dogs with dilated cardiomyopathy, although it is more challenging to make a diagnosis of hypertrophic cardiomyopathy in the cat. In dogs, the left atrial to aortic (LA‐Ao) ratio can be assessed through a right parasternal short axis view to determine the likelihood of congestive heart failure. A LA‐Ao ratio that exceeds 1.7 in this short axis view should be considered supportive of congestive heart failure in an animal with acute respiratory distress and pulmonary infiltrates. The LA‐Ao ratio can be used similarly in cats.

Fluoroscopy

Airway fluoroscopy uses air as the contrast medium to evaluate dynamic changes in the luminal diameter of the airways during normal breathing and during cough. Fluoroscopy is typically performed in lateral recumbency. It most readily recognizes cervical tracheal collapse because of the large airway diameter but can also provide information on intrathoracic airway and lobar bronchial collapse. A fluoroscopic study performed in right lateral recumbency will highlight the left lung lobes, while a study in left lateral recumbency will examine the right lobar bronchi. Right lateral recumbency appears to be used most commonly in the clinical situation. Dorsoventral positioning would likely be required to identify collapse of the accessory lobar bronchus. Fluoroscopy has poorer resolution than radiographs and the lower image quality can hamper interpretation of collapsing airways and pulmonary infiltrates. Extrathoracic airways will collapse on inspiration while intrathoracic collapse on expiration or cough.

Given the intricate relationship between neural pathways of the respiratory and digestive tracts as well as their close anatomic association, videofluoroscopic swallowing studies have a role in investigating the underlying etiology of some respiratory diseases as well as assessing the contribution of reflux to existing disease processes. Such specialized studies suggested occult aspiration injury as the sole cause of cough in some dogs and identified swallowing disorders contributing to cough in up to 75% of dogs with airway disease that lacked gastrointestinal signs (Grobman et al. 2019; Howard et al. 2023). Esophageal dysfunction and gastroesophageal reflux are common and can lead to chronic recurrent pneumonia or pneumonitis, particularly in dogs or cats with laryngeal dysfunction. Laryngeal paralysis in the geriatric Labrador is accompanied by esophageal dysmotility as part of a generalized neuropathy, and swallowing studies can help assess the risk for lower respiratory complications in affected dogs (see Chapter 5). Weak pharyngeal contractions occur in many different breeds of dogs and can predispose to aspiration events, although less is known in cats.

Interestingly, videofluoroscopy has revealed that brachycephalic breeds commonly display pharyngeal collapse (Pollard et al. 2018), likely due to anatomic issues and pressure fluctuations associated with respiratory effort. In addition to perpetuating aspiration injury to the lower respiratory tract, this can result in nasopharyngeal aspiration events that cause nasal discharge and upper respiratory tract inflammation.

Computed Tomography

Imaging the respiratory tract with CT provides superior detail in comparison to standard radiography because of the removal of super‐imposed structures and the ability to reformat images in three dimensions. Assessment of the extent of nasal conditions is improved when CT is performed because of the ability to assess cribriform plate involvement by various disease processes. When a helical CT is available, image acquisition is very fast; however, anesthesia is employed in most instances, particularly to obtain optimal images of the thorax under positive pressure ventilation. Some practices or university facilities perform CT in awake or sedated patients using a plexiglass holding chamber (VetMousetrap™; Oliveira et al. 2011). This might be most useful for nasal imaging as opposed to thoracic studies because respiratory motion artifact can obscure parenchymal details. It is a clinical decision whether the risks of anesthesia outweigh the value of images obtained, but often an intervention is planned after CT (e.g., endoscopy or surgery) which will also require anesthesia. For the thorax, the animal is typically ventilated several times and a breath hold at 15 cm water (H₂

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