Clinical Chemistry PP
CLINICAL CHEMISTRY
Presented by: Scott Wilson, DVM
Source: Eclinpath.com
AUTOMATED CHEMISTRY ANALYZERS
Chapter 31
Common Types of Analyzers
Spectrophotometers
Electrochemistry
Ion-selective electrode (ISE) technology
Spectrophotometer
Structure and Function
Components:
Light source
Lens
Filter or monochromator
Sample holder
Detector
Readout device
Principles of Spectrophotometry:
Measures the amount of light transmitted through a solution.
Definitions:
Colorimeter: A photometer that uses a filter to select the wavelength of light from the test substance.
Reflectometer: A photometer that detects light reflected off of a test substance (not transmitted light).
Beer’s Law (Beer-Lambert's Law)
Definition: A direct linear relationship exists between the concentration of a solution and light absorption when monochromatic light passes through it.
Key Points:
Transmission: The transmission of monochromatic light through a sample and concentration of an analyte have an inverse exponential relationship.
Degree of Color Change: The degree of color change is proportional to the solution’s concentration.
Electrochemistry Analyzers
Electrodes: Contain biosensor reagent strips or cartridges.
Function: Sample reacts with reagents to create a current, which is measured to determine the ion concentration in the sample.
Common Uses: Evaluation of electrolytes and other ionic components.
ISE Technology
Definition: Ion-selective electrode, also known as potentiometers.
Purpose: Used to evaluate specific ions, most often for electrolytes and ionic components.
End Point vs. Kinetic Assays
End Point Assays: The reaction between the sample and reagent reaches a stable endpoint.
Kinetic Assays:
Do not reach a stable endpoint; reactions are recorded at a specific time while the reaction continues beyond that time.
Factors Affecting Enzyme Activity:
Enzyme activity inhibited by low temperatures.
Enzyme activity accelerated by high temperatures.
Other affecting factors: UV light, salts & heavy metals (copper & mercury), pH extremes, organic solvents.
Most assays performed between (approx. ).
Features & Benefits of Chemistry Analyzers
Reagents:
Liquid or lyophilized (prepared liquid reagents).
Rotor Technology: Utilizes lyophilized reagents.
Bulk Reagents: Most cost-effective but require extra handling & storage.
Pre-measured Cuvettes: Disposable, which limits handling hazards.
Dry reflectance assays: Unitized forms, which eliminate handling hazards, but are more costly compared to bulk.
Dedicated Use Analyzers: Examples include glucosometers.
Analyzer Examples
IDEXX Catalyst DX
VETSCAN VS2
KIDNEY CHEMISTRIES
Chapter 33
Importance of Kidney Function Analysis
Blood and Urine Analysis: Both should be performed to evaluate kidney function.
Functions of the Kidneys
Water and Electrolyte Regulation:
Conserve water & electrolytes in negative balance.
Increase elimination of water & electrolytes in positive balance.
Acid-Base Regulation: Excrete/conserve hydrogen ions to maintain normal blood pH.
Nutrient Conservation:
E.g., glucose & proteins.
End Product Excretion:
Remove nitrogen metabolic byproducts such as urea & creatinine.
Hormonal Production:
Produce renin to control blood pressure and erythropoietin.
Additional Functions of Kidneys
Prostaglandin Production:
Fatty acids used in various physiological functions, including stimulation of smooth muscles and regulation of blood pressure.
Vitamin D Activation: Aids in the active form of the vitamin.
Blood Urea Nitrogen (BUN)
Purpose: Evaluates kidneys’ ability to remove nitrogenous waste (urea) from blood.
Common Influence Factors:
Azotemia: Indicates high BUN levels.
Dehydration.
Hemolysis & Fasting Samples: Can falsely elevate BUN levels due to amino acid breakdown from high protein diets and exercise.
Ruminants: Do not show drastic Urea increases as they recycle it as a protein source.
Creatinine
Significance: Reflects alterations in glomerular filtration rate (GFR).
Sources: Also found in sweat, feces, and vomit.
Influence Factors:
Levels are altered in conditions affecting GFR but are not affected by hemolysis or high protein diets.
Uric Acid
Definition: Important nitrogen end-product in avians & reptiles.
Critical Levels: Concentration increases significantly with greater than 70% renal function compromise.
Additional Serum Chemistries for Renal Disease
Phosphorus: Kidneys are the primary source of excretion in cats and dogs; not the case in horses and cattle.
Potassium: Levels decrease with low urine production and increase with high urine production (polydipsia; hypokalemia).
Calcium:
Hypocalcemia can be found in dogs, cats, and ruminants with chronic renal failure.
Hypercalcemia may be observed in horses with renal failure.
Albumin: Severe kidney failure can lead to substantial protein loss.
SDMA – IDEXX Test
Definition: Symmetric dimethylarginine, a biomarker for kidney function.
Excretion and Benefits:
Excreted by kidneys and more accurately reflects GFR in dogs and cats earlier than creatinine.
Increases as early as 25% loss of kidney function, making it more reliable in acute and chronic kidney disease than creatinine.
Less influenced by extra-renal factors such as body condition, advanced age, and overall disease state.
LIVER CHEMISTRIES
Chapter 32
Importance of Liver Function
Multitude of Functions Regulated by Enzymatic Reactions:
Metabolism: Proteins, carbohydrates, fats.
Synthesis: Albumin, cholesterol, plasma proteins, and clotting factors.
Digestion/Absorption: Nutrient processing related to bile production, which is essential for fat digestion.
Secretion: Bile and bilirubin.
Elimination: Detoxification of toxins and catabolism of specific drugs.
Storage: Vitamins and iron.
Gallbladder Functions
Role: Closely linked to liver functions; primarily stores bile.
Malfunctions of Liver and Gallbladder
Common Clinical Signs:
Jaundice: Yellowing of skin and eyes due to bilirubin accumulation.
Hypoalbuminemia: Low albumin levels often indicating liver disease.
Problems with Hemostasis: Includes clotting issues.
Hypoglycemia: Low blood sugar levels can occur.
Hyperlipoproteinemia: Elevations in lipoproteins.
Hepatoencephalopathy: Neurological syndrome associated with liver dysfunction.
Hepatocytes and Biliary Epithelial Cells
Enzyme Locations: Enzymes are found within cytoplasm and on surface of cell membrane.
Consequences of Damage: Increased enzyme levels in the blood occur when these cells are damaged.
Bilirubin
Definition: A metabolite of the heme portion of hemoglobin.
Formation Process: When RBCs are destroyed in the spleen, bilirubin is bound to albumin and transported to the liver.
Hepatic cells conjugate bilirubin for removal by the kidneys.
Types of Bilirubin
Conjugated Bilirubin (Direct Bilirubin)
Characteristics: Conjugated by hepatocytes to be excreted; water-soluble.
Increased Levels Indicate:
Bile duct obstruction or injury.
Hepatocellular damage.
Unconjugated Bilirubin (Indirect Bilirubin)
Characteristics: Not fully processed by liver and bound to albumin.
Increased Levels Indicate: Hepatic damage or excessive RBC destruction.
Also associated with defects in the transport system allowing bilirubin entry into hepatocytes for conjugation.
Delta Bilirubin
Definition: Conjugated bilirubin bound to albumin.
Measuring Bilirubin
Assays
Total Bilirubin: Measures combined levels of conjugated and unconjugated bilirubin.
Calculation: Total Bilirubin = Direct Bilirubin + Indirect Bilirubin.
Sample Considerations
Effect of Hemolysis: Can produce decreased levels depending on the testing type.
Sample Sensitivity: Avoid lipemic samples, and protect bilirubin from direct sunlight to preserve levels.
Bile Acids Testing
Function: Evaluates hepatocellular function; a non-specific test for liver problems.
Normal Response: Serum bile acid levels elevate postprandial due to gallbladder releasing bile into the duodenum.
Testing Procedure:
Collect fasting blood sample (12-hour fast).
Feed the patient.
Collect a second blood sample 2 hours postprandial.
Result Interpretations
Elevated Levels Indicate Possible:
Portosystemic shunts.
Chronic hepatitis.
Hepatic cirrhosis.
Cholestasis (bile obstruction).
Neoplasms.
Decreased Levels Indicate:
Malabsorptive diseases.
Overview of Bile Acid Tests
Horses: A single sample is sufficient due to continuous bile acid circulation.
Ruminants: Not as sensitive; bile acid testing provides limited diagnostics.
In-house Testing: ELISA tests are increasingly utilized.
Liver Enzymes
Cytosolic Enzymes
Main Enzymes:
ALT (SGPT): Alanine aminotransferase, liver-specific in small animals.
AST (SGOT): Aspartate aminotransferase, not liver-specific and present in muscle.
SD (SDH): Sorbitol dehydrogenase or iditol dehydrogenase; marker for hepatocyte function in large animals.
GLDH: Glutamate dehydrogenase, indicating liver damage, particularly in ruminants and avians.
Leakage: Damage to hepatocytes causes enzymes to leak into the blood, elevating enzyme levels associated with liver disease.
ALT – Alanine Aminotransferase
Definition: Formerly known as serum glutamic pyruvic transaminase (SGPT).
Source: Majorly from hepatocytes in dogs, cats & primates; used to evaluate liver health.
Limitations: Not sufficient in horses, ruminants, and pigs for liver specificity due to low enzyme concentrations.
Other Sources: Found in renal cells, cardiac muscle, skeletal muscle, and pancreas.
Considerations:
Avoid hemolysis and lipemia.
Certain medications (corticosteroids, phenobarbital) can elevate serum levels.
AST – Aspartate Aminotransferase
Definition: Formerly known as serum glutamic oxaloacetic transaminase (SGOT).
Sources: Found in hepatocytes and muscle; indicates non-specific liver damage.
Elevated Levels: Associated with hepatic disease, muscle inflammation or necrosis, and spontaneous hemolysis.
Additional Information:
More liver-damage specific in horses and cattle as opposed to small animals.
Evaluating CK levels can confirm or rule out muscle damage before attributing elevated AST to liver damage.
SD (SDH) – Sorbitol Dehydrogenase (ID – Iditol Dehydrogenase)
Source: Primarily from hepatocytes; offers a better liver-specific diagnostic test in large animals than ALT.
Stability Consideration: Unstable in serum and results must be processed within 12 hours after collection or frozen.
Benefits: Not affected by hemolysis.
GLDH – Glutamate Dehydrogenase
Importance: Found in high concentrations in hepatocytes of large animals.
Significance: Increased levels indicate hepatocyte damage or necrosis in cattle and sheep; potential marker for developing standard ruminant and avian liver function tests.
Other Liver Enzymes
Inducible Enzymes: Located on cell membranes; lysis of hepatocytes does not increase these enzymes' levels.
Key Enzymes:
ALKP (Alkaline Phosphatase): Isoenzymes present in many tissues with varied sources in different ages.
GGT (Gamma Glutamyltransferase): Primary source is the liver, but also found in the kidneys, pancreas & intestine.
ALKP – Alkaline Phosphatase
Source Details: Present in osteoblasts, chondroblasts, and hepatobiliary system.
Distribution: Primarily derived from bones in young animals; in older animals, predominantly from liver.
Clinical Use: Helpful in detecting cholestasis in dogs and cats, but non-reliable in sheep/cattle due to fluctuation in normal levels.
Sample Considerations: Avoid EDTA or oxalate anticoagulants; hemolysis does not affect results significantly.
Cats and ALKP
Variations: Cats show lower liver ALKP than dogs, with a significantly shorter half-life (6 hours in cats versus 72 hours in dogs).
Clinical Note: Any elevation in ALKP levels in cats is noteworthy and requires further investigation.
GGT – Gamma Glutamyltransferase
Source: Primary in liver with additional presence in kidneys, pancreas, intestine, and muscles.
Clinical Indicators: Higher GGT levels are indicative of cholestatic diseases; more reliable than ALKP in horses and cattle.
Colostrum Impact: Neonates ingesting colostrum show elevated GGT levels (in lambs, puppies, and calves).
Considerations: Hemolysis does not affect GGT results.
Pseudo Function Liver Tests
Chemical Testing Outcomes: Some show decreased values when liver function is compromised, particularly with conditions like portosystemic shunts.
Key Indicators: Significant decreases in vital parameters indicate reduced functional hepatocellular mass:
Albumin: >80% liver nonfunctional before change is appreciated.
Urea: Converted from ammonia in the liver for excretion.
Glucose: Produced via gluconeogenesis.
Cholesterol: Synthesized in liver.
Cholesterol
Source: Primarily produced in the liver and ingested through food.
Clinical Applications: Sometimes screened for hypothyroidism, hypercholesterolemia results from various conditions (e.g., diabetes mellitus, hyperadrenocorticism, nephrotic syndrome).
Considerations: Hemolysis can influence results based on testing methods (color) and anticoagulant types may produce false elevations.
More Chemistries & Electrolyte Assays
Chapters 35 & 36
Creatine Kinase (CK)
Analysis Purpose: Evaluated when ALT levels are elevated without signs of liver disease.
Source: Found mainly in brain, skeletal and cardiac muscles; increases indicated cell damage.
Considerations for Testing:
CK is unstable and should be tested as soon as possible after collection.
Susceptible to external factors like UV light exposure; hemolysis does not affect results.
CK Isoenzymes
Evaluation Type and Source:
CK-BB: Brain type, elevated during seizures.
CK-MB: Cardiac type.
CK-MM: Skeletal muscle type, seen with IM injections, surgeries, or strenuous exercises.
Glucose
Function: Indicates carbohydrate metabolism balance; reflects glucose production and utilization rate.
Factors Influencing Levels:
Increased insulin levels correspond to decreased BG levels via enhanced glucose utilization via cells.
Conversely, lower insulin (e.g., diabetes mellitus) results in elevated BG concentrations.
Sample Handling: Centrifuge samples immediately post-collection to preserve glucose integrity; hemolysis does not affect results.
Electrolyte Assays
Common Analytes: Calcium, phosphorus, potassium, sodium, chloride, and magnesium.
Sample Preference: Arterial blood samples are ideal as venous samples yield different reference ranges.
Key Functions of Electrolytes:
Water balance maintenance.
Fluid osmotic pressure regulation.
Muscle and nerve functions.
Activation of enzyme systems and acid-base regulation.
Calcium (Ca++)
Distribution: More than 99% of body calcium is in the bones; remaining plays critical roles in various physiological processes.
Sample Handling: Avoid using EDTA or oxalates as they bind calcium; hemolysis dilutes plasma and affects results.
Phosphorus (P)
Distribution: 80% in bones; primarily energy storage and metabolic function.
Sample Considerations: Separate plasma or serum immediately for accurate results; hemolysis affects reliability of phosphorus measurements.
Sodium (Na+)
Role: Major plasma cation significant for water distribution and osmotic pressure maintenance in the body.
Clinical Implications: Hypernatremia and hyponatremia indicate elevated and decreased sodium blood levels, respectively.
Sample Precaution: Avoid heparin use with samples as it may elevate sodium levels falsely.
Chloride (Cl-)
Function: Acts as the principal extracellular anion for water distribution and cation-anion balance in fluids.
Clinical Considerations: Evaluated through serum or heparinized plasma; hemolysis may dilute sample.
Potassium (K+)
Function: Critical for muscular and cardiac activity, along with other metabolic activities.
Clinical Risks: Can lead to life-threatening cardiac arrhythmias when abruptly elevated (hyperkalemia); hypokalemia associated with inadequate intake or fluid loss.
Sample Handling: Hemolysis can skew results as potassium is more concentrated in RBCs than plasma; refrigeration of samples before separating plasma may cause potassium loss.
Magnesium (Mg)
Distribution and Function: Primarily located in bones; supports many enzymatic reactions.
Clinical Significance: Assess for hypermagnesemia and hypomagnesemia; sample types can influence results.
Bicarbonate (HCO3-)
Function: Major buffer and responsible for CO2 transport; regulates body pH balance.
Testing Practices: Typically measured via detected blood CO2, with arterial blood preferred; lithium heparin recommended as anticoagulant.
PANCREATIC TESTS
Chapter 34
Pancreas Structure and Function
Dual Role:
Exocrine: Secretes digestive enzymes (e.g., trypsin, amylase, lipase).
Endocrine: Regulates carbohydrate metabolism through insulin and glucagon secretion.
Exocrine Pancreatic Assays
Amylase
Function: Digests starches and glycogen but not specific to the pancreas; elevated levels indicate pancreatic conditions such as acute pancreatitis.
Considerations: Results may not directly correlate with the severity of pancreatitis; avoid calcium-binding anticoagulants.
Lipase
Function: Pancreatic lipase breaks down long-chain fatty acids; levels rise during pancreatitis episodes.
Evaluation Practices: Analyze accompanying amylase tests for pancreatic conditions.
Anticoagulant Limitations: Same as amylase; avoid hemolysis and lipemia.
Pancreatic Lipase Immunoreactivity Test
Targets: cPLI (canine) and fPLI (feline).
Sensitivity & Specificity: Particularly sensitive in cats; however, note that elevated levels in dogs may follow other conditions such as GI diseases.
Exocrine Pancreatic Insufficiency
Condition Description: Lack of enzymes leads to poor fat digestion; characterized by large amounts of fatty stool.
Trypsin Testing: Fecal presence of trypsin indicates functionality; if absent, trypsin tests indicate EPI.
Evaluation Techniques: Test tube method and X-ray film to assess trypsin presence.
TOTAL SOLIDS & PLASMA PROTEINS
Chapter 32
Plasma Proteins
Source: Primarily synthesized in the liver and immune system, performing multiple vital functions including structural integrity, osmotic pressure maintenance, enzyme functions, and more.
Common Proteins Assessed:
Albumin
Fibrinogen
Globulins
Total Protein (TP)
Definition: Total protein measurements may include fibrinogen; Total serum protein excludes fibrinogen.
Diagnostic Usefulness: Essential for evaluating hydration status and can be influenced by several factors (dehydration, altered hepatic synthesis, and protein disturbances).
Measuring Total Protein Levels
Methods:
Refractometer: Measures serum or plasma refractive index.
Biuret Method: Measures peptide bonds in serum/plasma.
Electrophoresis: Separates protein fractions based on charge and size.
TP Concentrations and Screenings
Clinical Applications: Used in cases of edema, ascites, diarrhea, and hepatic/renal diseases.
Variations Due to Hemolysis:
Moderate hemolysis does not significantly impact results; marked hemolysis can falsely elevate values.
Albumin
Characteristics: Smallest plasma protein; accounts for 35% to 50% of total plasma protein.
Clinical Consideration: Significant hypoalbuminemia is commonly due to albumin loss; liver disease can directly decrease albumin synthesis.
Globulins
Complex Group of Proteins:
Alpha Globulins: Primarily transport proteins.
Beta Globulins: Iron transport, complement proteins, fibrin formation.
Gamma Globulins: Immunoglobulins produced by plasma cells for immune response.
Measuring Globulins
Methodology: Indirectly measured through electrophoresis, or via TP concentration minus albumin concentration.
Evaluation: Ratio of Albumin to Globulin often indicates protein abnormalities in pathologic conditions.
Immunoglobulins
IgG-Immunoglobulins are produced by plasma cells and are essential for immune response. They are a component of the complex group of proteins known as globulins, which perform various vital functions including structural integrity, osmotic pressure maintenance, and enzyme functions
IgE-an antibody produced by the immune system that is most commonly associated with allergic reactions.
IgA-is primarily found in bodily secretions such as saliva, tears, breast milk, and mucus. It plays a crucial role in protecting the body from pathogens (bacteria, viruses, and other microorganisms) by: Neutralizing toxins, Blocking their entry into cells, and Assisting immune cells in recognizing and eliminating pathogens.
Albumin: Globulin Ratio (A:G Ratio)
Normal Ratios:
>1.00: Normal in dogs, horses, sheep, goats.
<1.00: Normal in cats, cattle, pigs.
Fibrinogen
Source: Synthesized by hepatocytes; serves as precursor to fibrin for blood clotting.
Testing Requirements: EDTA plasma preferred; heparin can yield falsely low levels.
Reference Ranges for Total Solids (g/dL)
Dogs: 5.4-7.5
Cats: 5.7-7.6
Horses: 5.4-7.9
Cows: 6.0-7.5
Evaluation of Protein Fractions
Hyperproteinemia: Typically indicates dehydration.
Hypoproteinemia: Can stem from blood/plasma loss or young animal status.
Albumin Deviations: Hyperalbuminemia is rare; hypoalbuminemia can result from decreased production or increased loss.
Globulin Evaluation
Hypoglobulinemia Causes: Normal in young; blood loss, protein-losing enteropathy may contribute.
Hyperglobulinemia: May be relative (dehydration impacting fractions) or absolute (indicating polyclonal/monoclonal gammopathy linked to immune response or neoplasia).
Protein-Losing Enteropathy (PLE)
Description: Group of diseases characterized by excessive protein loss into the GI tract.
Mechanism: Normal protein leakage can lead to hypoproteinemia via impaired resorption in intestines.
Common Causes include: Lymphosarcoma, enteritis, GI parasites, food allergies, and hemorrhagic gastroenteritis (HGE).
PLE Symptoms & Test Recommendations
Signs to Monitor: Intermittent diarrhea, ascites, LAD (lethargy, anorexia, depression), respiratory distress.
Recommended Tests: CBC for anemia, fecal examinations for parasites, and fractional protein evaluations for albumin, globulins, and fibrinogen.