Lecture 15 - Introduction to biochemical profiles and analyzer methodology Flashcards
List the major testing principles in a Clinical Pathology Lab
- Hematology
- Coagulopathy testing
- clinical biochemistry
- urinalysis/fluid analysis
- cytology
List the major testing principles in a Anatomical Pathology Lab.
- histopathology
- biopsy
- necropsy
List some of the Clinical pathology considerations
- In house vs laboratory- vet
- ‘test’ (assay) selection - vet and/or lab
- sample collection/submission - vet and/or lab
- analysis of sample: laboratory
- interpretation of results: vet and/or lab
Label the sections of the bloodwork pictured below accordingly.
Erythrogram:
Immature reticulocytes or not?
Why is the animal anemic?
Leukogram:
- Tells us if animal has an inflammation happening
Platelets:
- Type of inflammation
- Status of hemostasis in patient.
What are the major goals of a Clinical Biochemistry?
Goal:
* Identify the organ or organs involved
* Create a differential diagnosis list
-top differential would be that single disease that explains all the findings in the case.
ie. explains clinical signs, and abnormal results
- Decide what you want to do next
- re-examine animal
- Better history
- What extra tests are required
The purpose of looking at the serum enzymes is to?
Organ function.
When enzymes are altered, esp when elevated, they are released into the blood and then causes problems.
You can not obtain enzyme results if you do not order a biochem panel. The CBC will not capture this
If you have an elevation in TP, what values do you look at?
Albumin and globulin.
Serum Protein Electrophoresis results show?
Which of the globulins or albumins are elevated.
What type of test is pictured below? What is its purpose?
Cellulose acetate strip and densitometer tracing
List the pros of in-house testing.
- Immediate results – point-
of-care - Customer service
- Additional diagnostics
(when appropriate) - Time sensitive tests – fresh
sample
List the cons of in-house testing.
- Capital equipment
investment $ - Maintenance cost
- Inventory
Define pre-analytical error.
- Pre- Analytical : occurs prior to analysis of sample
- sample : selection, technique, preservation/management
Define analytical error.
- Analytical (at lab): analyzer error
- instrument function
- QC- calibration, internal/external QC Personnel
training and SOPs
Define post-analytical error.
- Post- Analytical : errors in transcription or interpretation of results
Pre-analytical: sample collection and submission
* Aim to collect the ________ quality samples
* Collect an ________ sample for the test sought
* If possible, collect samples _____ treatment
* ______ samples appropriately
* _______ for blood samples whenever possible
* Perform venipuncture with as little tissue _______ as possible
* Use correct sample _______
* Decide if test done ?
* Package _______ – avoid exposure to extremes of _____ or ______
* Avoid shipping _____ cytology together with ______-fixed tissues
* Process/deliver to laboratory ______
highest, appropriate, before, Labeling, Fasting, trauma, containers,
“in house” or external path lab
appropriately, heat, cold, fresh, formalin, promptly
List the different types of blood samples.
Ø blood
* whole
* serum
* plasma
??
Ø bone marrow
* urine
* milk
* feces
List the different bodily fluid samples.
Ø body fluids
pleural, peritoneal, pericardial,
semen,
lymph, CSF
List the different samples you can collect from “washes”.
Ø Washes
tracheal/bronchial
washes
List the different tissue samples you can collect.
Ø tissue samples
* aspirates, impressions,
scrapings,
brushes
What are anticoagulants used for?
- Used to prevent blood from clotting
How soon after you collect a sample, and place it in an anticoag tube, should it be processed?
- Expedite transfer samples to the anticoagulant following collection
Should you mix your sample containing anticoags in it?
- Mix gently but thoroughly
Anticoagulated samples may still clot if ?
- collection is too slow
- tissue fluid is collected with blood.
- the sample is not adequately mixed
- What tube is pictured below?
- This tube is preferably used for?
- What happens to blood samples in this tube when collect from birds and reptiles?
- Other than blood, what other samples are put into this tube?
- How much sample should be put into this tube?
- EDTA (ethylene-diamine-tetra-acetic acid)
- EDTA tube is preferred for hematology/CBC in most species
- Blood will lyse in EDTA in certain species of birds (e.g. cranes, crows, turkeys, hornbills, wood ducks) and reptiles (e.g. tortoises), then in those cases use Heparin tubes
- Also used for cytology of fluids: abdominal/thoracic fluids,
prostatic fluids, tracheal/bronchial washes - Tubes must be filled to the required level to avoid excess EDTA
Half way full is the Minimum
Always submit a _______ made blood smear (preferably _______) to optimize examination of ___ blood cell, _____ blood cell and _____ morphologic features.
freshly, unstained, red, white, platelet
- What tube is pictured below?
- This tube is preferably used for?
- What samples can you put in this tube?
- This tube is not preferred for?
- If you can not analyze these samples within an hour, what should you do?
- Heparin tube
- STAT clinical biochemistry; Whole blood: used for chemistry analysis w/in 1 hr
3. - Not the preferred anticoagulant for hematological analysis
- –> If not possible:
* Centrifuge and transfer plasma to red-top tube, refrigerate
until use
* Warm to room temperature before analysis
Naturally occurring mucopolysaccharide potentiates the action of ___________ __ to inhibit clotting factors
antithrombin III
______________ plasma is used commonly for STAT clinical
biochemistry (other anticoagulants bind _____ and most alter ___________)
Heparinized, Ca++, electrolytes
- Name the blood tube pictured below.
- What is this used for?
- What is its function?
- What is important to note?
- Sodium Citrate
- Used for optimal coagulation assays & platelet function studies
- Reversibly chelates calcium
- Note: at a ratio of 1 ml per 9 ml of blood
* Correct citrate:blood ratio is crucial
* Ensure the tube is filled to the correct level
- Name the blood tube pictured below.
- What is this used for?
- What is its function?
- What is important to note?
“Plain” or serum tubes
* Contains no additive - allows the sample to clot
* Time to clot is variable, usually >20 minutes (thus, heparin used
for STAT analyses)
* Serum is used for many routine biochemistry tests & serology
* Required for specific tests
* Serum bile acids (BA)
* Serum protein electrophoresis (SPE)
* Trysinogen like immunoreactivity (TLI)
- Name the blood tube pictured below.
- What is this used for?
- What is its function?
- What is important to note?
Serum separator tube
* Gel matrix which promotes clotting
* Avoids/decreases alteration of biochemical parameters by
leakage or metabolism from cells
* Serum contains less Ca2+ , has no fibrinogen, no clotting proteins
* Complement proteins may still be active
What is the difference between plasma and serum?
List the syringe size you would use for the following dog sizes:
1. Small breed
2. Medium breed
3. Large breed
4. Giant breed
Do this because sizes of veins are different.
Test question
- One thing that you must remember: chemistry samples contaminated with EDTA
will inaccurate results for _____ and ____. - Samples at room Temp. should be used within ___h, specially for those in ______
heparin (green top) and within ___-__h for EDTA samples - These are recommendation from IDEXX commercial labs.
- Fasting animals at least __h is important to avoid ___
test question
Calcium, Potassium, 1, Lithium, 3-4
4, lipemia
What does a clinical pathology submission form state?
Submission form
* owners details
* animal details
* samples forwarded
* type and number
* Tests required
* Time of sampling
* History
What is important to remember when sending samples to commercial labs?
- USA Department of Transportation and the International Air
Transport Association - Specimen transport guidelines
- Samples are categorized: A or B biological substances
- based on to their potential for causing serious or fatal disease in humans
- If samples are improperly packaged
- shipper may be subjected to fines, jail time, or extra handling fees
Category B, Biological Substances, UN 3373, generally includes?
diagnostic samples from animals
Category A, Infectious Substances, UN 2900, includes ?
samples to be tested for more serious animal diseases, which may cause disability or fatality in humans exposed to the contents
To protect handlers of the package while in transit and personnel at
the destination facility, a?
triple-layer packaging scheme is required
* Contact your lab for transportation/shipping/package requirements
Examples of commercial secondary containers with biohazard label. A pouch on the back holds the paperwork separate
from the sample, and absorbent material is placed in the pouch with the sample. Biohazard labels must not be added to
the outer container.
Things to consider
1. Chemistry Analyzers
2. Clinical relevance of the test
- analytical methods: spectrophotometry
* interfering substances: colored
* accuracy and precision
* Quality assurance
- analytical methods: spectrophotometry
- reference interval ranges and severity of change
* sensitivity/specificity/prevalence
- reference interval ranges and severity of change
Define Photometry?
- Analytical chemistry technique that uses _________ of light to measure concentrations of _____ present in ____
- Most chemistry analyzers used in veterinary medicine use this technology - The photometers break light into many wavelengths (spectrum), and are termed “________”
- Analytical chemistry technique that uses wavelengths of light to measure concentrations of analytes present in serum
- Most chemistry analyzers used in veterinary medicine use this technology
- The photometers break light into many wavelengths (spectrum), and are termed “spectrophotometers”
Describe the common spectrophotometers used:
- Absorbance spectrophotometry – usually veterinary laboratory –wet chemistry
- Reflectance spectrophotometry – usually in house laboratory -dry chemistry
Describe Absorbance spectrophotometry (wet chemistry)
- Light through a solution is ________ + _________
- Wavelengths of light are _______/_______
- _______ dependent
- _______ wavelengths used (optimizes results)
- Light through a solution is absorbed + measured
- Wavelengths of light are absorbed/reflected
- Substrate dependent
- Specific wavelengths used (optimizes results)
Describe Reflectance spectrophotometry (dry chemistry)?
- Blood + carrier are on a slide –> reaction
- Subjected to light –> light reflected is measured
- Intensity is analyte specific
How do Chemistry Analyzers Use Color?
- Uses specific chemicals to react with certain analytes to make a
color change.
Bromocresol Green (BCG) + Albumin —> BCG-ALB - The products of these reactions absorb or reflect different
wavelengths of light in a pattern that are typical of that
substance
The change in light absorption or reflection can be used to
determine the concentration of the analyte
Define Fluorescence analyzer technique.
- A substance exposed to light ________
- _______ that is emitted is measured
- A substance exposed to light fluoresces
- Energy that is emitted is measured
Define Electrochemical analyzer technique.
- Potentiometry
- Conductometry
- Amperometry
What is Beer’s Law?
Beer’s Law shows that absorbance and
concentration have a linear relationship
* A series of samples with a known concentration
of a specific substance is measured by how much
light is absorbed (linear relationship)
* Determines an unknown concentration of an
analyte in a solution
Interfering agents: biochemistry
- Lipemia
- Hemolysis
- Bilirubin
- Drugs
Lipemia
1. Interferes with ?
**2. Avoided by ?
3. Enhances ?
4. INCREASES: ?
- passage of light –Interferes
results with many biochemical tests - at least 12 hour fast prior to
blood collection.
- an increase in lipids in blood can be caused simply by eating a meal.
- ranges from mild, moderate, to severe.
- fasting for 4-6 hours is ok too, but 12 is ideal. - hemolysis (maybe acting as detergent on RBCs).
- Lipemia interferes with enzyme concentrations including:
* Lipase by 10-20%
* ALT (~45%),
* AST (~20%),
* LDH (~50%)
* Glucose, creatinine, bilirubin
What are the causes of Lipemia in vivo?
“PP HEaD”
Lipemia in vivo
* Postprandial
* Pancreatitis
* Diabetes mellitus
* Equine hyperlipidemia
* Hypothyroidism
Hemolysis
1. Usually due to ?
2. Interferes with ?
3. Releases ?
- The RBCs from _____ are rich in K+ (not ?)
- False increased ?
4. Other mild to moderate changes?
- turbulence & erythrocyte trauma at sampling
- how sample was collected
- how you manage tubes post sample collection. - many biochemical tests
- intracellular substances from
RBCs e.g. AST, K+; horses, dogs or cats; Serum K+
- increased hemolysis –> alteration in K in horses more than any other species since their RBC are reach in K –> leading to a false increase in serum K. - ALP, Total Protein, Calcium, Albumin, bilirubin, ALT, CK
Bilirubin
* Interferes with some tests
- Cannot be avoided if the animal is ?
hyperbilirubinemic
What are the causes of Bilirubinemia in vivo?
- Hemolytic disorders
- Fasting
* Horses, ruminants - Cholestasis
* Pre or post hepatic - Hepatic disease
* Decreased functional mass of the liver –> increase in bilirubin pigment.
If fasting ruminants or horses, they will have bilirubinemia which is not necessarily pathological.
Bilirubinemia
Drugs
* Some drugs can interfere with certain tests (biochem results)
* Always state any treatment on
laboratory form
Results of LOW Calcium and HIGH potassium (look at RI)
Does it make sense?
No, likely EDTA contamination
What are the clinical signs of Hypocalcemia?
Hypocalcemia - Clinical Signs (if severe)
* Shivering
* Seizures
* Muscle fasciculations
* Join pain (osteopenia)
* Tachyarrhythmias
What are the DDx of Hypocalcemia?
Differential Diagnosis
* Magnesium deficiency
* Injury to tissues (severe)
* Lactation/pregnancy
* Lab error
* Vitamin D deficiency
* Pancreatitis
* Renal Disease
* Albumin deficiency
* Artifact (EDTA contamination)
* Intake from GI decreased
* Sepsis
* Ethylene glycol
What are the clinical signs of Hyperkalemia?
What are the DDx of Hyperkalemia?
How can the chemistry analysis be contaminated by the EDTA anticoagulant in the purple top tube?
- Assays (reagents and instruments) used for veterinary diagnostics are usually produced for the human market
- Validation of methods is very important
- Some methods are not appropriate for veterinary samples
- e.g. albumin: bromocresol green(V) or bromocresol purple(H)
- ( precludes using human laboratories for many assays)
What is the difference between accuracy and precision?
Accuracy
* ability to give a correct result, the
closeness of a result to the “true” value
Precision
* the ability of an assay to give the same
result when the test is repeated
multiple times
* is a measure of reproducibility and is
usually stated as a coefficient of
variation (CV)
Describe quality assurance at all stages.
