Biochemistry Profiles & Analyzer Methodologies Flashcards
What ate the 3 major veterinary laboratory disciplines?
- clinical pathology - hematology, coagulopathy testing, clinical biochemistry, urinalysis, fluid analysis, cytology
- anatomical pathology - histopathology, biopsy, necropsy
- other - bacteriology, virology, mycology, immunology, parasitology
What are 3 goals of clinical biochemistry?
- identify the organs involved
- create a differential diagnosis list with a top differential that explains all findings in the case
- guides to a decision of what to do next, like re-examine, obtain more history, extra testing, etc.
Serum enzymes:
What is used as a control for serum protein electrophoresis?
cellulose acetate strip and densitometer tracing
What are 4 pros and 3 cons of in-house testing?
PROS
1. immediate results for point-of-care testing
2. customer service
3. additional diagnostics
4. time-sensitive results with fresh samples
CONS
1. investment for equipment ($$$)
2. maintenance cost
3. inventory availability
What are the 3 sources of error with in-house testing?
- PRE-ANALYTICAL - prior to analysis; sample selection, collection technique, preservation/management
- ANALYTICAL - analyzer error; miscalibration, internal/external QC personnel training, following SOPs
- POST-ANALYTICAL - errors in transcription or interpretation of results
When is it most beneficial to collect samples?
- before treatment
- with a fasted patient
What should be avoided when sending out samples?
shipping fresh cytology with formalin-fixed tissues
What are 6 common samples collected for clinical pathology?
- blood - whole, serum, plasma
- bone marrow
- body fluids - pleural, peritoneal, pericardial, semen, lymph, CSF
- washes - tracheal, bronchial
- tissue samples - aspirates, impressions, scraping, brushes
- urine, feces, milk
How are anticoagulants used for clinical pathology samples? In what 3 situations do they tend to not work as directed?
used to prevent blood from clotting and expedites transfer samples into anticoagulants following collections
- collection is too slow
- tissue fluid is collected with blood
- sample not adequately mixed
What is the purple top tube? What is it most commonly used for? What can it also be used for?
EDTA - anticoagulant
hematology/CBC
cytology of fluids - keeps high protein fluids, like abdominal/thoracic fluids, prostatic fluids, and tracheal/bronchial fluids, from coagulating
What tube is preferred for bird and reptile hematology/CBC? Why?
green top heparin tubes
EDTA will lyse their RBCs
What is recommended to also submit with EDTA purple top tubes? How much should these tubes be filled?
freshly made (unstained) blood smear to optimize examination of RBC, WBC, and platelet morphology
at minimum halfway, since excess EDTA can alter results
What is the green top tube? When is it most commonly used?
heparin tube containing the naturally occurring mucopolysaccharide that potentiates action of antithrombin III to inhibit clotting factors
STAT clinical biochemistry or whole blood, since other anticoagulants bind calcium and would alter electrolytes
- NOT prefered anticoagulant for hematological analysis, unless in bird and reptile patients
What is another option if STAT biochemistry is needed, but a heparin tube is not available?
- centrifuge and transfer plasma to red top tube
- refrigerate until use
- warm to room temperature before analysis
What is the blue top tube? What is it most commonly used for?
contains sodium citrate, which reversibly chelates calcium
coagulation assays and platelet function studies
What is the preferred ratio of blood to be collected in a sodium citrate tube?
1 mL of sodium citrate per 9 mL of blood
- 1 tube requires 9 mL of blood
What is the red top, or plain, tube? What is it most commonly used for?
contains no additive, allowing the sample to clot and getting serum upon centrifugation
biochemistry tests and serology
Why are the red top tubes not used for STAT biochemistry?
time to clot is variable and it typically requires more than 20 mins to clot
What 3 specific tests require the use of a red top tube?
- serum bile acids
- serum protein electrophoresis
- trypsinogen-like immunoreactivity
What is the tiger top tube? What is achieved upon centrifugation?
tube containing a gel matrix that promotes efficient clotting and avoids altering biochemical parameters by leakage/metabolism from cells
serum containing less calcium, no fibrinogen, no clotting proteins, and some active complement proteins
What is the difference between plasma and serum?
PLASMA has anticoagulants, allowing it to contains clotting proteins and fibrinogen
SERUM is the result following coagulation, so it has no clotting proteins or fibrinogen
How does the concentration of ions compare in plasma and serum?
plasma has slightly greater calcium and slightly less potassium compared to serum
Why is EDTA inappropriate for biochemistry?
alters samples, falsely increasing calcium and potassium and decreasing magnesium
Why is serum preferred for electrophoresis and biochemistry?
does not contain fibrinogen that can interfere with results
EDTA vs Serum tube:
What are the preferred gauges of needles used on the different sizes of patients? Why is it important to use the correct needle size?
- SMALL BREED up to 10 kg = 25g
- MEDIUM BREED 10-30 kg = 22g
- LARGE BREED 30-60 kg = 20g
- GIANT BREED over 60 kg = 18 g
avoids hemolysis and compliments blood vessel sizes
How should blood samples be transferred into sample tubes?
- closed system (butterfly) and vacutainer = directly dispense through needle
- otherwise, remove the needle and dispense slowly into the sample tube
avoids hemolysis
What is the correct order of filling samples for blood panels?
- blue top (sodium citrate)
- red top (serum)
- greed top (heparin)
- purple top (EDTA)
How should samples contained with EDTA and heparin be handled to ensure even disbursement of the anticoagulant?
gentle invert 10-15 times and used within 3-4 hours or 1 hour
What are 3 common problems with test results due to sample quality? How can they be avoided?
- HEMOLYSIS - broken RBCs distort analyte values, making plasma/serum pink
- LIPEMIA - milky plasma/serum filled with lipids interferes with chemical reactions and light absorption
- ICTERUS - excessive bilirubin pigments makes plasma/serum yellow
have pets fasted for 4-6 hours prior to appointment
What are the 2 classifications of samples sent to commercial labs? What packaging scheme is required for transit?
- Category B, biological substances - includes diagnostic samples from animals
- Category A, infectious substances - includes samples tested for more serious animal diseases that may cause disability or fatality in humans exposed to the contents
triple layer
What are 2 important characteristics of commercial secondary containers for veterinary samples?
- pouch in the back for paperwork separate from sample
- absorbent material placed in the pouch
biohazard labels must not be added to the outer container
What analytical method do most chemistry analyzers use? What commonly interferes with sample detection?
spectrophotometry
colored substances alter light absorption and reflection
What is spectrophotometry?
analytical chemistry technique that uses light broken down into many wavelengths (spectrum) of light to measure concentrations of analytes present in the serum
What are the 2 most common spectrophotometers used?
- ABSORBANCE - wet chemistry in veterinary laboratory
- REFLECTANCE - dry chemistry in house
How does absorbance spectrophotometry work?
WET CHEMISTRY
- light moves through a solution and is absorbed and measured
- wavelength of light are absorbed and reflected, depending on the substrate
- specific wavelengths are used to optimize results
How does reflectance spectrophotometry work?
DRY CHEMISTRY
- blood and a carrier is placed on a slide
- sample is subjected to light, which is reflected and measured
- intensity of reflection is analyte specific
What concept is absorbance spectrophotometry dependent on?
Beer’s Law - absorbance and concentration have a linear relationship (higher absorbance = higher concentration)
Icterus, hemolysis, lipemia:
In what 2 ways can lipemia affect samples? How can this be avoided?
- interferes with passage of light, altering many biochemical tests
- acts as a detergent on RBC membrane, enhancing hmolysis
have patients fasted for 12 hours prior to blood collection
What 5 specific effects does lipemia have on biochemistry results?
increased…
- lipase (10-20%)
- ALT (45%)
- AST (20%)
- LDH (50%)
- glucose, creatinine, bilirubin
What naturally causes lipemia in vivo? 4 pathological causes?
posprandal
- pancreatitis
- diabetes mellitus
- equine hyperlipidemia
- hypothyroidism (decreased metabolism)
What causes hemolysis of samples? What effect does this have on biochemical tests?
turbulence and RBC trauma at sampling
releases intracellular substances from RBCs, like AST and potassium, causing falsely increased serum potassium levels
- also moderately changes ALP, TP, calcium, albumin, bilirubin, ALT, and CK
In what 2 ways does bilirubin affect biochemistry results? When can this not be avoided?
- decreased creatinine
- increased bilirubin
if the animal is hyperbilirubinemic
What are 4 common causes of bilirubinemia in vivo?
- hemolytic disorders
- fasting in horses and ruminants
- cholestasis (pre or post-hepatic)
- hepatic diseased causing decreased functional mass
Why is it important to state any treatment an animal is receiving on a laboratory form?
some drugs can interfere with certain tests
CASE: 3 y/o Scottish Terrier presents for annual PE and wellness testing. Apparently healthy at home, good energy and appetite, and no current meds. PE shows BAR, TPR WNL, and overall unremarkable. CBC also unremarkable.
What is likely the cause of the significant findings in the biochemistry profile?
likely blood was collected in the improper tube for biochemistry serum —> EDTA will bind to Ca+ and make it unable to participate in the clotting process and increase K+
at these levels, hypocalcemia would present with shivering, seizures, muscle fasciculations, joint pain, and tachyarrhythmia, while hyperkalemia would present with bradyarrhythmia and generalized muscle weakness
How would collection of blood from a purple-top or blue-top tube alter biochemistry results?
EDTA –> chelates Ca+ = decreased Ca+ and increased K+
citrate —> decrease Ca+, no effect on K+
CASE: 5 y/o CM Boxer presents for annual PE and wellness testing. Apparently healthy at home, good energy and appetite, no current meds. PE shows BAR, TPRR WNL, and is overall unremarkable. CBC is WNL.
Upon first collection, there are some discrepancies with the biochemistry panel. A second test was done and all analytes were WNL. What likely caused the odd results?
hemolysis of the sample caused the contents of the RBC, like ALT and K+ to release, increasing their values, and falsely increased ALP
What is method validation?
assays (reagents and instruments) used for veterinary diagnostics are usually produced for the human market, so they must be validated to ensure they are accurate for animals
- for example, bromocresol green (V) or bromocresol purple (H) albumin assays are not accurate for animal diagnostics
What is the difference between accuracy and precision?
ACCURACY = ability to give correct results; closeness of the result to the true value
PRECISION = ability of an assay to give the same result when the test is repeated multiple times, independent of accuracy, commonly states as a coefficient of variation
What quality assurance can be done at the pre-analytical, analytical, and post-analytical stages?
correct identification, appropriate samples, appropriate quality
valid method, calibration, control samples, external samples
interpretation, transcription
How is quality assurance done?
- known control samples are used daily (internal QA)
- unknown samples are provided by QA groups to compare results to a group mean
- results are plotted to follow trends
(question all odd results!)
