clinical chemistry

what can we assess from clinical chemistries

• Organ function

• Endocrine function

• Electrolyte and acid-base status

• Toxicology

what sample type is mostly used

serum

requirements for a patient sample label

Date and time of collection, owners name, patients name and signalment, and clinic ID number

how is a SST used for samples

A serum separator tube is used to separate the serum from the RBCs in a sample.

• inside walls have silica particles to aid in clot activation

• Invert the tube after collection and let it sit for 30 minutes before centrifuging

sample property factors that can affect results

Hemolysis - IMHA 

Icterus - liver disease

 Lipemia - post prandial

iatrogenic factors that can affect chemistry results

Chemical contamination

Improper labelling

Improper  handling

patient factors that can affect results

• Fasted collections

• Post prandial

  • Increased BG

  • Decreased phosphorous

  • Lipemia

  • Increased GFR

• Physiologic 

• Do not need to restrict water intake

pre-analytical factors

• Biological 

  • Age, breed, gender

  • Hemolysis, lipemia, icterus

• Non-biological 

  • Missing labels, transport, req. Forms, incorrect tubes or technique

analytical factors

• Instrument not cleaned

• Equipment not cleaned or sterilized

• plasma/serum 

• Methods

• QC not completed

• Reagents expired

what is a reference range, why is it important

Reference ranges are created by repeatedly assaying samples from a significant number number of clinically normal animals of a given species.

• gives us a baseline when testing sick animals to know how sick they are and how much treatment is required. 

pros and cons of liquid reagents

Pros: available in bulk, least expensive, available in unitized cuvettes to eliminate hazards

Cons: requires storage and handling, and some are flammable and toxic

pros of dry reagents

Pros: only in unitized form, and little to no handling or storage concerns

example of a dedicated-use analyzer

AlphaTrack glucometer

• useful for glucose checks and glucose curves

• more precise and accurate measurements of the element we are testing for. 

proper analyzer instrument care

• maintenance sheet and log

• regular quality control checks

• Manufacturer’s instructions should be followed

• warm-up procedures followed

• Post the toll-free number for help if needed

common protein assays

Albumin, total protein, globulins, and fibrinogen

functions of plasma proteins

• Form structural matrix of all cells, organs, and tissues

• Maintain osmotic pressure

• Serve as enzymes for biochemical reactions

• Buffers in acid-base balance

• Serve as hormones

• Blood coagulation

• Defend against pathogens

• transport/carrier molecules for most constituents of plasma

difference between plasma and serum proteins

plasma = has fibrinogen

serum = no fibrinogen

3 methods for testing TP

1. Refractometer

2. Biuret

3. Protein synthesis electrophoresis

factors that effect TP concentrations

• Altered hepatic synthesis

• Altered protein distribution

• Altered protein breakdown/excretion

• Dehydration = hyperproteinemia

• Overhydration = hypoproteinemia

medical conditions where TP should be monitored

• Edema

• Diarrhea

• Ascites

• Weight loss

• hepatic/liver disease

• Blood clotting disorders

functions of albumin

1. Major binding and transport protein

2. Maintains osmotic pressure of plasma

diseases that can cause hypoalbuminemia

Renal disease - because the nephron filters out albumin, damage to the kidney causes glomerular nephritis, causing protein-losing nephropathy (PLN).

GI disease - decreased nutrient intake and malabsorption cause protein-losing enteritis (PLE).

what conditions can cause altered serum proteins

• hyper/hypoproteinemia

• hyper/hypoalbuminemia

• Hemoconcentration

• Hemodilution

• Blood loss

• Inflammatory disease

• Lymphoma

• Malnutrition 

3 types of globulins

1. alpha

2. beta

3. gamma

alpha globulins function

synthesized in the liver

1. Mainly transport and bind proteins

beta gobulins function

include complement (C3, C4), transferrin and ferritin

1. Aid in iron transport, heme binding, and fibrin formation and lysis

gamma globulin function

synthesized by plasma cells

1. Responsible for antibody production (immunity)

2. IgG, IgA, IgM, IgE, IgD

purpose of the albumin/globulin ratio

help us determine pathological conditions

• first indication of a protein abnormality

• increases or decreases in the A/G concentrations

• if both albumin and globulin concentrations or equally reduced, there is no alteration or disease present. 

[albumin] / [globulin]

normal A/G ratio for cats and dogs

Dogs: A/G >1.0

Cats: A/G <1.0

test used for fibrinogen

Heat precipitation test

functions of the liver

1. Metabolism

2. Synthesis of albumin, cholesterol, PP, and clotting factors (proteins and enzymes)

3. Digestion and absorption

4. Secretion of bile and bilirubin

5. Eliminate and detoxify toxins

6. Catabolism of certain drugs

conditions caused by malfunctions of the liver and gallblader

• Jaundice

• Hypoalbuminemia

• Hemostasis issues

• Hypoglycemia

• Hyperlipoproteinemia

• Hepato-encephalopathy

major hepatobiliary assays

• gallbladder

• bilirubin

• bile acids

• cholesterol

what can affect fibrinogen levels

liver damage because it is synthesized from hepatocytes

• if levels decrease = little to no clotting

• is the precursor to fibrin

what are the enzymes released in liver damaged

• ALT

• AST

• iditol dehydrogenase

• glutamate dehydrogenase

enzymes associated with cholestasis

• alkaline phosphatase (alk phos)

• gamma glutamyltransferase (GGT)

• bilirubin

• bile acids

how and where is bilirubin produced

derived from Hgb breakdown by macrophages in the spleen

bilirubin metabolism

1. Bilirubin is derived from hemoglobin breakdown from heme, by macrophages in the spleen

2. Its insoluble, so it binds to albumin for transport to the liver

3. Liver cells metabolize and conjugate bilirubin to bilirubin glucuronate → gets secreted → part of bile

4. GI tract bacteria remove the glucuronate to → urobilinogen → excreted in urine or feces

how can we measure bilirubin

• unconjugated (free) bilirubin

• conjugated bilirubin

unconjugated (free) bilirubin measurement

an indirect measure

• Increases indicate RBC hemolysis or issues with transport to the liver

conjugated bilirubin measurement

a direct measure

• Increases indicate hepatocellular damage, or bile duct injury/obstruction

function of bile acids

stored in the gallbladder until it contracts in the anticipation or presence of food, from where it releases the salts into bloodstream

• Help digest and absorb fat emulsifications through micelles

• Modulate cholesterol levels

process of bile acids

Reach the duodenum via bile ducts → reaches ileum, transported back to circulation and liver → 90-95% gets reabsorbed in the ileum and remainder is excreted in the feces

how to test for bile acids

Paired serum samples performed after 12 hours of fasting and 2 hours postprandial are needed for the test. The difference in bile acid [ ] is reported

factors that can effect bile acid tests

• Inadequate feeding

• Food aromas

• Prolonged fasting or diarrhea

diseases indicated by elevated bile acids

• PSS

• Chronic hepatitis

• Hepatic cirrhosis

• Cholestasis

• Neoplasms

why is ALT measured

only measured for screening because it is not specific enough for diagnosing

• Is considered a liver-specific test in dogs, cats and primates

why would ALT levels be increased

damage to:

• renal cells

• cardiac muscle

• skeletal muscle

• pancreas

where is ALT found

hepatocytes and free in cytoplasm

where is AST found

hepatocytes, mitochondrial membrane, cytoplasm, RBC, cardiac muscles, skeletal muscles, kidneys and pancrea

why would AST levels be elevated

• Liver disease

• Muscle inflammation or necrosis

• Hemolysis

where is iditol dehydrogenase found

hepatocyytes, kidney, small intestine, skeletal muscle, and RBCs

what is iditol dehydrogenase used to evaluate

liver disease in large animals

where is glutamate dehydrogenase found

mitochondrial bound enzyme

where is alkaline phosphatase found

osteoblasts, chondroblasts, intestines, placenta, bone, kidney, and liver cells

where is gamma glutamyltransferase found

mainly liver

kidney functions

• Produce urine

• Homeostasis 

  • Blood filtering

  • Reabsorption

  • Secretion

• Production of hormones

• BP regulation

• Vitamin D activation

what does BUN test evaluate

kidney function, specifically the ability of the kidney to remove nitrogenous waste or urea from blood

• photometric test (accurate)

• chromatographic (dipstick) → screening only

what does serum creatinine evaluate

GFR and creatinine levels in the blood

• Creatinine if formed from skeletal muscle and diffuses out of the muscle into the blood

what do alterations in serum creatinine indicate

kidney issue

• Any condition that alters the GFR will alter creatinine levels

how much of the kidney must be non-functional before changes can be detected for a serum creatinine test

75%

azotemia

increase in urea nitrogen and/or creatinine in blood

pre-renal issues

decreased blood flow to kidneys

• Dehydration, vomiting, diarrhea, hemorrhage, shock

renal issues

glomerulonephritis 

• Ethylene glycol toxicity

post-renal issues

obstruction of ureters, bladder or urethra

• Bladder rupture (trauma, iatrogenic)

what is the urine protein/creatinine ratio used for

assessing patient status suring treatment

what does an SDMA test evaluate

biomarker for kidney function and reflects the GFR

• Specific for kidneys and less impacted by extrarenal factors (BCS, age, disease)

how does the water deprivation test work

purposely induces dehydration to asses how well the kidneys concentrate urine

how does the vasopressin response test work

injection of ADH to see how kidneys respond to exogenous ADH

1. Patient still has free access to water

pancreas functions

• Exocrine function

  • Produces enzymes for digestion in the small intestine

  • Trypsin, amylase, lipase

• Endocrine function

  • To secrete hormones (insulin and glucagon) to BG regulation

what are the exocrine pancreas tests

• glucose

• lipase

• trypsin

why are serum amylase levels almost always increased

pancreatic disease

increased levels of serum amylase can be due to:

• Acute pancreatitis

• Flare-ups of chronic pancreatitis

• Obstruction of pancreatic ducts

increased levels of serum lipase due to:

Exocrine Pancreatic Insufficiency (EPI) → decreased amylape and decreased lipase

what is a better site for evaluation of pancreatitis

peritoneal fluid

increased levels of serum trypsin due to:

pancreatic mass

• absence in feces = abnormal

endocrine pancreas tests

• glucose

• fructosamine

• glycosylated hemoglobin

what is fructosamine

represents the irreversible reaction of glucose bound to albumin

• increased levels = persistent hyperglycemia

what is glycosylated hemoglobin

represents irreversible reaction of Hgb bound to glucose

• increased levels = persistent hyperglycemia

life-span of RBCs in dogs and cats

dogs: 110 days

cats: 70 days

how should you test glucose levels in a patient

should be fasted

preservative of choice for testing glucose

sodium fluoride

functions of electrolytes

• water balance

• normal muscle and nerve function

• osmotic pressure

• support and activate key enzymes in acid-base regulation

what can changes in electrolyte concentrations indicate

• increase or decrease in fluid intake

• shifts between ECF and ICF

• increase or decrease in renal retention

normal body pH

7.35-7.45

purpose of buffer systems

counteract pH imbalances by altering proton concentrations, intracelllulary and extracellulary

• If pH is out of range, proteins are diminished or destroyed

Respiratory acidosis/alkalosis

acid-base abnormalities in the respiratory system

• Decreased CO2 = increased pH → alkalosis, RR decreases to hold more CO2

• Increased CO2 = decreased pH → acidosis, RR increases to remove CO2

Metabolic acidosis/alkalosis

acid-base abnormalities in the body

• Bicarbonate increases = pH increases

• Bicorbonate decreases = pH decreases

what is the anion gap, how is it calculated

identifies metabolic acidosis

• difference between cations and anions in the body.

• Increases in the gap indicate lactic acidosis, renal failure, or Diabetic ketoasidosis

• decrease in the gap indicates hypoalbuminemia

normal ranges for anion gap

Dogs = 12-24 mEq/L

Cats = 13-27 mEq/L

what elements are assess in electrolyte assays

• sodium

• potassium

• chloride

• bicarbonate

• magnesium

• calcium

• inorganic phosphorous

where is creatine kinase (CK) mostly found

in striated muscle cells

• leaks out of damaged muscles into blood

increased ALT > increased AST, no increase CK

liver injury

increased AST > increased ALT + increased CK

skeletal muscle injury

what can elevated lactate indicate

hypoxia or hypoperfusion

what does the ACTH stimulation test determine

pituitary vs adrenal tumours

is Cushing’s more pituitary or adrenal-dependant

pituitary

how does ACTH stim. test work

Measures adrenal gland response to exogenous ACTH

two types of cortisol tests

1. ACTH stimulation

2. dexamethasone suppression

purpose of thyroid tests

assess the negative feedback loop from the hypothalamus and pituitary for regulating TRF, TSH and thyroxine