Transcript Chemical Assay of Serum, Plasma, or Whole Blood

Chemical Assay of Serum,
Plasma, or Whole Blood
Clinical Pathology
Chemical Assay
• Most vet clinics use equipment to perform
routine chemical assays in-house.
• Important aid in accurate diagnosis,
treatment, and response to medications.
• Measure levels of dissolved substances
normally in blood.
Sample Collection
• Whole Blood: Made up of fluid and
cells. (sample is mixed with
anticoagulant and can not be frozen)
• Fluid=plasma
• Cells= RBC’s, WBC’s, Platelets
• Plasma: 90% water and 10%
dissolved components such as
proteins, carbohydrates, vitamins,
hormones, enzymes, lipids,
electrolytes, wastes, etc.
• Can be refrigerated or frozen
• Collected by centrifuging unclotted
blood.
Sample Collection Continued
• Serum: blood is allowed to clot then
centrifuged. Serum is plasma from
which fibrinogen has been
removed.
• Sometimes may freeze sample
• Red top or clot activator tubes
Anticoagulants
• Heparin: interferes the least
with chemical assays.
• Available in different salt
forms.
• Should be used at least 20
units/ml of blood
• Green top tube
• EDTA: Choice for hematologic
studies because it does not
alter cell morphology.
• Should be used at 1-2
mg/ml blood collected.
• Lavender top tube
Anticoagulants Continued
• Oxalates: sodium, potassium, ammonium,
or lithium salts.
• Potassium is the most common form
used.
• May interfere with enzymatic activity
• Sodium Fluoride: glucose preservative
• Sodium citrate: coagulation test.
Factors Affecting Results
• Hemolysis: syringe collection,
forcing blood through the needle.
• Chemical contamination: tubes
must be chemically pure.
• Improper labeling
• Improper handling: use sample
within one hour.
• Patient influences: e.g. fasting
animal may decrease glucose,
lipemia post meal ingestion.
Enzymology
• Enzymes are proteins produced in cells.
• Catalysts for chemical reactions.
• Most enzymes are formed and function
intracellularly.
• Blood levels should be low in a healthy animal.
• The blood level can increase if the enzyme has
leaked out of damaged cells.
• Substrates are substances enzymes help to form
into specific products.
• Assays performed determine the enzyme
concentration by measuring the enzymatic reaction.
• An uncatalyzed reaction requires a higher
activation energy than does a catalyzed reaction.
• There is no difference in free energy between
catalyzed and uncatalyzed reactions.
Endpoint Assays
• Product formed from enzymatic action
interacts with a reagent to produce a color
complex.
• The amount of light absorbed by the color
complex present indirectly reflects the
amount of enzyme present in the sample.
Factors Influencing Enzyme
Activity
• Inhibited: Low temperatures, dehydration,
UV light, and heavy metals.
• Accelerated: High temperatures
• Can be denatured by temperature and pH
extremes.
• A small change in the polypeptide chain
can cause the enzyme to lose activity.
• Each enzyme has an optimum
temperature in which it works effectively.
Components of Enzymes
• Substrate: substance acted on by the
enzyme.
• Buffer: keeps the pH of the system
constant to ensure maximal enzyme
activity.
• Cofactor/coenzyme: Acclerate a reaction.
Temperature
• If refrigerated serum is used, it must be
prewarmed before added to the test
system.
• If serum needs to prewarmed, then the
test needs to be prewarmed also.
• Anticoagulants may nterfere with the
activity of enzymes.
Terms
• Named for the substrate they act on or the
biochemical reaction which they
participate.
• Most enzymes end in –ase.
• Isoenzymes: group of enzymes with
similar catalytic activities but different
physical properties.
• Ex. Serum alkaline phosphatase is
found in many tissues such as
osteoblasts and hepatocytes.
Hyperlipidemia
• Increased concentration of lipids,
specifically cholesterol an triglycerides.
• Can find gross lipemia in the plasma
sample.
• May be postprandial or indicate disease.
• Pancreatitis.
• Hyperhthyroidism
• Diabetes mellitus
Amylase
• Digestive enzyme stored in pancreas.
• Leakage of amylase may be measured in
blood during inflammation (pancreatitis).
• Levels may also be high due to renal
dysfunction.
• Why does amylase not go up in cats?
Lipase
• Secreted by the pancreas in active form.
• Should be measured with amylase.
• Kidneys play a role in the degradation of
lipase.
• Found in animals with a high fat diet.
Trypsin-like Immunoreactivity
(TLI)
• Test for exocrine pancreatic insufficiency
(TLI).
• Useful in cases of malabsorption.
• Common in German Shepards over 8
months of age.
• When animals do not secrete amylase and
lipase properly.
Glucose
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Formed by digestion of carbohydrates and the
conversion of glycogen by the liver.
Primary source of energy for most cells.
Sources of blood glucose:
• Dietary
• Increases2-4 hours postprandial except in
ruminants
• Malabsorption and starving may cause
hypoglycemia.
• Glycogenolysis
• Primary source of glucose while fasting
• May be caused by catecholamine release (>300
mg/dl in the cat).
• Hyperglycemial in cattle with neurologic signs
• Corticosteroids
• Growth Hormone
• Certain drugs
Fate of Glucose
• Insulin stimulates cellular uptake (muscle,
fat, liver).
• Blood glucose levels \>30 mg/dl stimulates
insulin release.
Hypoglycemia
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Insulin hypersecretion
Insulin overdose
Sepsis
Extreme leukocytosis
Pregnancy
Prolonged Starvation
Hyperglycemia
• Increased peripheral resistance to insulin
• Diabetes mellitus and decreased insulin
production
• Stress (cat)
• Postprandial
• Pancreatitis