Clinical Chemistry Study Guide: Sample Collection, Assays, and Function Tests

Sample Collection and Handling Procedures

• Samples must be analyzed within $1\,\text{hour}$ after collection to ensure the integrity of the data. If testing cannot occur immediately, freezing is required to preserve the sample's integrity.

• Most clinical chemistry collections should be performed before the animal begins medical treatment and while the patient is in a fasted state.

• Immediate labeling of the collection tube after drawing the sample is mandatory to prevent identification errors.

• For samples being sent to reference laboratories, the accompanying submission forms must be completed in their entirety.

Plasma Characteristics and Handling

• Plasma is defined as the fluid portion of whole blood.

• The composition of plasma is approximately $90\%$ water and $10\%$ dissolved constituents (most of which is proteins).

• If a plasma sample cannot be centrifuged within $1\,\text{hour}$ of collection, it must be refrigerated immediately.

Serum and Serum Separator Tubes (SST)

• Serum is defined as plasma with the fibrinogen component removed (because we let it clot before spinning down).

• Serum Separator Tubes (SST) are specialized collection tools:

  • The inside walls are coated with silica particles that aid in clot activation.

  • The tubes contain a gel that forms a physical barrier between the serum and blood cells during centrifugation.

  • The ones with the separator gel keeps the cells separated the red tops without the gel if you shake them the cells will go back together

• After collection in an SST, the tube should be inverted gently and left to set for $30\,\text{minutes}$ to allow for proper clot formation.

• To facilitate clot contraction, one may "rim" the clot by separating it from the walls of the collection tube using a Pasteur pipette or a wooden applicator stick.

Factors Influencing Clinical Chemistry Results

Hemolysis

• Hemolysis alters the makeup of serum or plasma through the rupture of red blood cells.

• Fluid released from ruptured cells dilutes the sample, which can lead to increased concentrations of Potassium ($K$), Phosphorus ($PO_{4}$), and various enzymes. Potassium main component that will be altered due to hemolysis

General Sample Errors

• Improper labeling of the specimen.

• Improper sample handling, such as failing to analyze within $1\,\text{hour}$ or failing to keep the sample at room temperature or frozen for delayed analysis (greater than $1\,\text{hour}$).

Patient-Specific Influences

• Icterus: Often associated with underlying liver disease.

• Fasting vs. Post-prandial (after eating):

  • Blood glucose levels are typically elevated immediately after a meal.

  • Inorganic phosphorus levels may be decreased post-prandially.

• Lipemia: Results in turbid or cloudy serum, which can affect kidney assays.

• Water Intake: There is no need to restrict water intake when performing kidney assays; an increase in Glomerular Filtration Rate (GFR) can occur.

• Stress: Stress can significantly influence results, especially in feline patients.

Reference Ranges

• Reference ranges (normal values) are established by repeatedly assaying samples from a significant number (typically between $20$ to $100$) of clinically normal animals of a specific species with similar characteristics.

Automated Analyzers and Photometry

Analyzer Principles

• Most chemistry analyzers utilize the principles of photometry to quantify blood constituents.

• Certain in-house analyzers use electrochemical methods:

  • Ion-selective electrode: Features specific electrodes configured to interact with only one ion.

  • Electrochemical: Incorporates electrodes with biosensor reagent cartridges or strips.

Photometry Definitions

• Spectrophotometers: Designed to measure the amount of light transmitted through a solution.

• Reflectometer: Designed to measure the amount of light reflected off a substance.

Beer’s Law

• There is a direct linear relationship between the concentration of an analyte and light absorption when monochromatic light passes through the sample.

• There is an inverse exponential relationship between the transmission of monochromatic light through a sample and the concentration of the analyte.

• The degree of color change produced in a reaction is directly proportional to the solution’s concentration.

Enzyme Activity and Nomenclature

• Enzyme activity can be inhibited by low temperatures and accelerated by high temperatures; the ideal temperature range for measurement is typically $30\text{ to }37^{\circ}\,C$.

• Enzyme activity is measured in International Units.

• Enzymes are named based on the substrate they act upon or the biochemical reaction they participate in:

  • Lipase: Acts on lipids.

  • Lactate dehydrogenase: Acts on lactate.

• Isoenzymes: A group of enzymes with similar catalytic activities but differing physical properties.

Reagent Types

• Liquid reagents.

• Dry reagents: Often found in unitized forms; these require little handling and have fewer storage concerns but are generally more expensive.

Instrument Care and Use

• Analyzers are sensitive instruments

• Must be carefully maintained

• Follow manufacturer’s instructions

• Allow for warm-up periods and leave on throughout the day

• Schedule maintenance sheet and log

• Post the toll-free number for help if needed

Protein Assays

Plasma Protein Functions

• Forming the structural matrix of all tissues, organs, and cells.

• Maintaining osmotic pressure.

• Serving as enzymes for biochemical reactions.

• Acting as buffers in acid-base balance.

• Serving as hormones.

• Functioning in blood coagulation.

• Defending the body against pathogenic microorganisms.

• Serving as carrier or transport molecules for plasma constituents.

Major Protein Components

• Total Protein: Includes fibrinogen when measured in plasma, but excludes it when measured in serum. Total protein levels are vital for determining hydration status: (can be altered due to hydration issues)

  • Hyperproteinemia (elevated): Often indicates dehydration.

  • Hypoproteinemia (decreased): Often indicates overhydration.

  • Primary testing methods include the Refractometer and the Biuret method.

• Albumin: One of the most important proteins, comprising $35\%$ to $50\%$ of total plasma protein. It is synthesized by hepatocytes. Decreases may indicate diffuse liver disease, renal disease, or issues with dietary intake and intestinal absorption. (Patient has to have lost close to 80% liver function to exhibit signs of hypoalbuminemia)

• Globulins: A complex group of proteins.

  • Alpha globulins: Synthesized in the liver; include High-density lipoproteins and Very low-density lipoproteins.

  • Beta globulins: Include complement, transferrin (iron transport), and ferritin (iron storage). Responsible for heme binding and fibrin formation/lysis.

  • Gamma globulins (Immunoglobulins): Synthesized by B lymphocytes (plasma cells) for antibody production.

  • IgG: Fetal and neonatal immunity.

  • IgD: B lymphocyte surface antigen.

  • IgE: Hypersensitivity reactions.

  • IgA: Mucosal immunity.

  • IgM: Activation of complement.

Albumin/Globulin (A/G) Ratio

• Calculated by dividing albumin concentration by globulin concentration.

• In dogs, horses, sheep, and goats: $A/G > 1.0$ (Albumin is greater than globulins).

• In cattle, pigs, and cats: $A/G < 1.0$ (Albumin is equal to or lower than globulins).

Fibrinogen

• Synthesized by hepatocytes (in the liver) and acts as a precursor to fibrin, the insoluble protein forming the blood clot matrix.

• Makes up $3\%$ to $6\%$ of total plasma proteins. It is not found in serum because it is consumed during the clotting process.

• Levels may be elevated by acute inflammation or tissue damage.

• Calculated using the heat precipitation test:

  • TS mg/dL (nonincubated) - TS mg/dL (incubated) = Fibrinogen mg/dL

Hepatobiliary Function Tests

Liver and Gallbladder Functions

• The liver is the largest internal organ, responsible for amino acid, carbohydrate, and lipid metabolism, as well as the synthesis of albumin, cholesterol, and clotting factors.

• It handles bile formation for digestion, bilirubin secretion, and detoxification of toxins and drugs.

• The gallbladder is anatomically and functionally associated with the liver and primarily stores bile.

• Indicators of hepatobiliary malfunction include icterus, hypoalbuminemia, hypoglycemia, hyperlipoproteinemia, and hepatoencephalopathy.

Bilirubin and Bile Acids

• Bilirubin (Liver Specific Tests):

  • Unconjugated (Indirect): Pre-hepatic, bound to albumin. Makes up approximately $2/3$ of total bilirubin. Increases indicate liver uptake problems.

  • Conjugated (Direct): Indicates hepatocellular damage or bile duct obstruction.

• Bile Acids: Synthesized in hepatic cells and aid in fat absorption and cholesterol modulation. Stored in the gallbladder (except in horses). They are largely ($90\%$ to $95\%$) resorbed in the ileum and returned to the liver via portal circulation. The remainder is excreted in feces

  • Testing: Requires paired serum samples: one after $12\,\text{hours}$ of fasting and another $2\,\text{hours}$ after eating (postprandial). Horses require only one sample.

  • Factors for false tests include inadequate fasting, food aromas (like pavlovs dogs), or diarrhea.

  • Post prandial serum BA concentrations are higher than fasting concentrations

Cholesterol: We do not pay much attention to in vet med if high check thyroid

• Plasma lipoprotein produced primarily in the liver but also ingested in food

• Cholestasis causes increased cholesterol in some species

• Not well characterized in veterinary species

• Sometimes used to screen for hypothyroidism

• Does not cause grossly lipemic plasma after eating

Liver Enzymes: As hepatocytes are damaged, enzymes leak into the blood

• Alanine Transaminase (ALT): Liver-specific in dogs, cats, and primates. Horses, ruminants, pigs, and birds lack sufficient levels for it to be useful. Can be increased by corticosteroids or anticonvulsants.

• Aspartate Transaminase (AST): Not organ specific. Found in hepatocytes, RBCs, cardiac muscle, skeletal muscle, kidney, and pancreas. Elevations may mean liver disease, muscle necrosis, hemolysis, or strenuous exercise.

• Alkaline Phosphate (AP/ALP): Used to detect cholestasis (bile duct obstruction) in small animals. Also present in osteoblasts, chondroblasts, intestines, placenta, and liver cells.

• Gamma glutamyltransferase (GGT): Found in many tissues - Primary source is the liver. Elevated in obstructive liver disease. Cattle, horses, sheep, goats, and birds have higher baseline levels than dogs and cats.

Kidney Function Tests

• The kidneys maintain homeostasis by controlling water/electrolyte levels, maintaining blood pH, conserving nutrients, and producing renin, erythropoietin, and prostaglandins, aid in vitamin D activation.

• Blood Urea Nitrogen (BUN): Measures the ability of the kidney to remove urea, a nitrogenous waste product. Since urea is insoluble, it requires high water volume for excretion. Increases (azotemia) can be caused by dehydration, high-protein diets, or strenuous activity. (Lepto/urinary obstruction)

• Serum Creatinine: Formed from creatine in skeletal muscle. Diffues out of the muscle and into the blood. It is used to evaluate renal function but is not a sensitive early indicator; nearly $75\%$ of kidney tissue must be nonfunctional before levels rise.

• Uric Acid: A metabolic by-product of nitrogen catabolism.

  • In Dalmatians, a defect in uric acid uptake into hepatocytes leads to excretion (uric acid) in the urine.

  • In birds, uric acid is the major end product of nitrogen metabolism ($60\%$ to $80\%$) and serves as the primary index of renal function. $70\%$ of function is lost before levels rise.

Pancreatic Function Tests

Exocrine Pancreas

• Produces enzyme-rich juice (trypsin, amylase, lipase) for digestion.

• Amylase: Breaks down starches and glycogen in sugars. Elevated in acute pancreatitis or duct obstruction.

• Lipase: Breaks down lipids. As pancreatic disease progresses and cells are replaced by connective tissue, lipase and amylase levels gradually decrease.

• Serum Trypsinlike Immunoreactivity (TLI): Radioimmunoassay specific to dogs and cats; detects trypsinogen (produced only in the pancreas).

• Serum Pancreatic Lipase Immunoreactivity (PLI): Standard for diagnosing pancreatitis (fPLI for cats, cPLI for dogs); recommended over TLI for pancreatitis specifically.

Endocrine Pancreas

• Islets of Langerhans contain $80\%$ Beta cells (secretion of insulin) and $20\%$ Alpha cells (secretion of glucagon).

• Glucose: Insulin is the only hormone that lowers blood glucose.

• Factors that increase glucose (hyperglycemia) include glucagon, growth hormone, epinephrine, and glucocorticoids.

• Hypofunction - diabetes melitus

• Hyperfunction - hyperinsulinism (Insulinoma in ferrets)

• Testing: Use fasted animals. Glucose drops $10\%\,\text{per hour}$ if left on RBCs at room temperature. Sodium fluoride is the preferred anticoagulant.

• Fructosamine: Represents the irreversible reaction of glucose bound to albumin. Reflects persistent hyperglycemia over the previous $1\text{ to }2\,\text{weeks}$.

• Glycosylated Hemoglobin: Reflects glucose concentrations over the lifespan of an RBC ($3\text{ to }4\,\text{months}$ in dogs; $2\text{ to }3\,\text{months}$ in cats). Persisent hyperglycemia

• $\beta$-Hydroxybutyrate: The primary ketone produced in diabetic ketoacidosis (DKA).

  • Ketone bodies can be detected in plasma

Electrolytes and Acid-Base Status

Electrolytes

• Negative ions or anions

• Positive ions or cations

• Are found in all body fluids

• Function in maintenance of water balance, fluid osmotic pressure, and normal muscular and nervous functions

• Also maintain and activate several enzyme systems in the acid-base regulations

Acid-Base Balance

• Normal blood pH range is $7.35\text{ to }7.45$.

• Acidosis: pH below $7.35$ (excess $H^{+}$).

• Alkalosis: pH above $7.45$ (low $H^{+}$).

• Buffers:

  • Bicarbonate: $\text{HCO}_{3} + H^{+} \rightarrow H_{2}\text{CO}_{3} \rightarrow H_{2}O + \text{CO}_{2}$. The respiratory system removes $\text{CO}_{2}$ within minutes; kidneys work over days.

  • Potassium: $K^{+}$ and $H^{+}$ exchange between intracellular (ICF) and extracellular (ECF) fluids.

    • Decreases in plasm potassium cause potassium to move from cells to ECF and H+ to move from ECF into the cells, vice versa.

  • Protein: Hemoglobin binds $\text{CO}_{2}$ and $H^{+}$.

• Both respiratory and renal systems work to regulate the pH in acid-base imbalance

  • Respiratory works within minutes

  • Renal works for days to restore balance

Acidosis and Alkalosis

• Categorized by the cause

• Respiratory acidosis or alkalosis

  • Abnormalities in the respiratory system

• Metabolic acidosis or alkalosis

  • All causes other than respiratory system

Respiratory Acidosis/Alkalosis

• If respiratory rate decreases, less CO2 is eliminated

  • Hypercapnia

  • Increase in partial pressure CO2 (PCO2)

• If respiratory rate increases:

  • Hypocapnia

  • Decrease in PCO2

Metabolic Acidosis/Alkalosis

• Any metabolic condition that results in the buildup of acids in the body

• Ketones – produced when glucose metabolism is abnormal

• Decrease in blood bicarbonate levels

• Vomiting can cause alkalosis

• Increase in blood bicarbonate levels

Specific Electrolytes

• Sodium ($Na^{+}$): Major ECF cation. Hypernatremia (elevated) vs. Hyponatremia (decreased). Do not use heparin for collection.

• Potassium ($K^{+}$): Major intracellular cation. Important for muscle and cardiac function. Avoid hemolysis to prevent false elevations. Important in normal muscular function, respiration, cardiac function, nerve impulse transmission, and carbohydrate metabolism

  • Hyperkalemia – K+ leaves ICF and elevates plasma heparin K+ levels

  • Hypokalemia – decreased K+ levels due to inadequate K+ intake, alkalosis, or fluid loss from vomiting or diarrhea

  • Plasma preferred – K+ may be released during clotting

  • Avoid hemolysis – releases K+

• Chloride ($Cl^{-}$): Predominant ECF anion. Involved in water distribution

  • Predominant extracellular anion

  • Plays an important role in maintenance of water distribution, osmotic pressure, and the normal anion/cation ratio

  • Hyperchloremia – elevated chloride levels

  • Hypochloremia – decreased chloride levels

  • Interference in results

  • Hemolysis

  • Prolonged storage

• Bicarbonate ($\text{HCO}_{3}^{-}$): Regulated by kidneys. Estimated as $95\%$ of total measured blood $\text{CO}_{2}$.

  • Second most common anion of plasma

  • Kidney helps regulate bicarbonate levels

  • Frequently estimated from blood CO2 levels

  • The bicarbonate level is approximately 95% of the total CO2 measured

  • Arterial blood preferred

Anion Gap

• Calculated as: $(Na^{+} + K^{+}) - (Cl^{-} + HCO_{3}^{-})$

• Normal gaps: $12\text{ to }24\,\text{mEq/L}$ in dogs; $13\text{ to }27\,\text{mEq/L}$ in cats.

• Increases are seen in renal failure, lactic acidosis, and DKA.

Miscellaneous Tests

• Creatine Kinase (CK): Organ-specific enzyme for striated muscle damage and to some extent in the brain. It leaks out of damaged muscles into the blood.

• Lactate: Produced by anaerobic metabolism; indicates hypoxia or hypoperfusion.

• Adrenocortical Function:

  • Cushing disease: Hyperadrenocorticism (excess cortisol).

  • Addison disease: Hypoadrenocorticism (decreased cortisol).

  • ACTH Stimulation Test: Evaluates adrenal response to exogenous ACTH ($80\%$ accurate in dogs, $50\%$ in cats). Used in animals with suspected Addison or Cushings disease)

  • Dexamethasone Suppression Test: Low-dose confirms Cushing; high-dose differentiates pituitary from adrenal causes.

• Thyroid Assays: Thyroid hormone affects metabolic rate and growth. Typically hypofunction in dogs/horses/swine and hyperfunction in cats. TSH response tests are used primarily in dogs to diagnose hypothyroidism.

Chemical tests for GI function

• Fecal occult blood

  • Dramatic signs

    • Melena – black feces, heavy blood loss

    • Hematochezia – frank fecal blood

  • Subtle signs

    • GI ulcers, neoplasia, parasitism

    • Low-level bleeding may lead to iron deficiency anemia

• Toxicology:

  • Require accurate history

    • Environmental factors

    • Through clinical examination

    • Diagnosis can be difficult

  • Toxicology specimens

    • Handling, packaging and transport

    • Must be free of contamination by environmental compounds and debris

    • LABEL!!

      • Lead Poisoning (Plumbism): Causes basophilic stippling of RBCs. Clinical signs vary from GI to CNS. No simple, reliable in house test.

      • Nitrate/Nitrite: Found in heavily fertilized grains or well water from heavily fertilized areas. Nitrites degrade hemoglobin to methemoglobin, turning blood dark red to brown.

• Anticoagulants and rodenticides:

• Warfarin, diphacinone, and pindone act by inhibiting the metabolism of vitamin K in the body

  • Vitamin K is required for factors II, VII, IX, and X in the liver

  • ↑PT↑PTT↑ACT

  • Diagnosis based on these screening tests and response to vitamin K therapy

• Ethylene Glycol (Antifreeze): Minimal lethal dose is $1/8\,\text{teaspoon per pound}$ for cats and $1/2\,\text{teaspoon per pound}$ for dogs. Antidote (fomepizole) must be administered within $3\,\text{hours}$ for cats and $8\text{ to }12\,\text{hours}$ for dogs.

  • Strongly suggested in urine sediments containing large amounts of calcium oxalate monohydrate crystals

• Chemicals That Denature Hemoglobin

  • Alter hemoglobin in RBCs and form Heinz bodies

  • Cats – acetaminophen and methylene blue

  • Dogs – onions, garlic

  • Horses – red maple leaves

  • Ruminants – onions and brassicas