Blood collection and handling Flashcards
Whole blood is and comprised of
Circulates within the blood vessels
Whole blood is comprised of
RBC
WBC
Platelets
Plasma- fluid which contains proteins( such as albumin, globulins and fibrinogen) waste products and ions
Blood transports oxygen, CO2, nutrients, waste products and hormones b/w the lungs and tissues
Its components also play a vital role in our immune system
WBC and globulins (antibodies)
Examination of blood gives a trained person many clues to a patient’s state of health
To examine a sample, whole blood must be mixed with an appropriate anticoagulant
Plasma is
The fluid portion containing anticoagulant in which formed elements (RBC, WBC and platelets) are removed
Also the fluid portion of whole blood is circulating in a live animal
Contains fibrinogen, other clotting factors and proteins
In a healthy patient it is clear and straw coloured
Plasma sample preparation will be performed in the lab
Serum is
The clear, straw-coloured, liquid portion of blood that does not contain fibrinogen, or formed elements (RBCs, WBCs or platelets)
It is the liquid that remains after the sample has clotted
Nothing has been added to the sample–it does not contain an anticoagulant
Serum sample preparation will be performed in the clinical pathology lab
Anticoagulants are
Anticoagulants are chemical that will prevent or delay blood from clotting
There are a number of different anticoagulants that are used for blood analysis
It is essential that you know which anticoagulant is required for the test that is ordered as they can interfere with test results
Serum collection tube is
Red top
Does not contain an anticoagulant
Once a blood clot has formed, the serum is harvested
Used for a large number of biochemical blood tests
Can also be used for storage and shipment of histology samples, hair samples, urine, etc
NEVER used for hematology test
Sample collection is
Before collecting a blood sample, you need to know what tests have been requested
This will help you to decide which vein to collect the sample from
The volume of blood required
The blood tubes that you will need to have ready
Samples should be collected before any treatments are started
If treatments have been initiated, make note of it in the medical record
The preferred blood source is venous blood
What to do before collecting blood
The collection site must be cleaned and swabbed with alcohol before collection
Allow the alcohol to dry before proceeding
The animal should be restrained using minimal manual restraint if at all possible
This minimises stress, which can compromise the sample
To generate reliable results, you MUST collect and handle the sample properly
Sample collection, processing, testing, and interpretation must be performed as a complete, sequential chain of events
Develop a routine and stick to it
Needle and syringe for collecting blood
Thai system is what is usually used in small animals and exotic species
Use the largest gauge needle that is practical
The syringe size should be as close to the volume required as possible
Pulse plunger of the syringe to let the vein refill
When transferring the blood from the syringe to the vacutainer, remove the needle from the syringe and the cap from the vacutainer before rejecting the blood into the vacutainer
DO NOT stick the needle into the stopper top and let it drown in, it will create artefacts
Try to allow the blood to flow down the side of the vacutainer
Vacutainer system for blood collection
Made up of a needle, needle holder and collection tubes
Use the tube size that is required for the volume of your sample
It will reduce artefacts
Prevent collapsing of the vein
Collection tube is
Can be empty, sterile tubes or they may contain anticoagulants
Vary in size from a few microliters to 15ml
Heparin tube is
Green top
Contains heparin as an anticoagulant
Sodium, potassium, lithium or ammonium salt
Used for biochemistry tests, particularly when whole blood is required
Check your machines
Some use serum others use lithium heparin samples, others use both
NEVER used for hematology tests
For a small sample, you can coat the inside of the syringe with heparin before collecting the blood
EDTA tube is
Purple top
Contains EDTA- ethylenediaminetetraacetic acid, it can be in powder or preferably liquid form
Comes as either a sodium or potassium salt
Anticoagulant of choice for hematology tests
NEVER for a chemistry analysis
Most consistent preservation of cell volume and morphological features of the cells
Prevents clotting of the blood
The tube must be mixed after the blood has been added
This is done gently with a figure 8 motion, a gentle back and forth rocking motion, or by inverting the sample a minimum of 10 times
Excess EDTA in the blood sample will nullify automatic machine analysis
Oxalate and citrate tubes is
Blue top
Used for coagulation test
They interfere with clot formation by binding calcium
Interfere with chemistry test results
Oxalates are available in sodium, potassium, ammonium or lithium salts
Citrates are available in sodium or lithium salts
Also used for blood transfusions
Some need to be refrigerated
Sodium fluoride tubes are
(Grey top)
Contains sodium fluoride
Used for preserving blood glucose in a blood sample- effectiveness is questionable
Interferes with many enzyme tests
Sure-Sep tube or serum separator tube is
Tigger top
A variation of a red top tube
Does not contain an anticoagulant
Contains a gel in the bottom of the tube to separate the cells from the serum after the sample has been centrifuged
Prevents cells from metabolizing analytes
Sample volume that should be taken
The amount of blood we collect depends on the volume required for the tests and the patient’s hydration status
In a well hydrated patience, approx ½ of the volume of whole blood collection should be serum or plasma
Dehydrated patient will yield about ½ of that volume
How much blood should you collect and why
It is ideal the collect enough serum, plasma or whole blood to run the required tests 3 times
This allows for
Technical error
Instrument failure
Dilution of sample
Forwarding to a reference lab
Collection procedure
Gather all required equipment together
Needle, vacutainers, alcohol, etc.
Label the tube
Patients name, date and time, sample type
Prep the area for venipuncture
Alcohol swab, allow the are to dry before collecting the sample
Perform venipuncture with as little tissue injury as possible
Reduces risk of hemolysis and contamination within tissue fluid
Apply pressure to venipuncture site after needle is withdrawn- done by the restrainer
Mix anticoagulant containing vacutainers gently by inversion (minimum 10 times) or by moving it in a figure 8 pattern
General handling procedure
Proper handling prevents technical artefacts*
Blood collected for a CBC (complete blood count) should be within 1 hour
If this is not possible, a blood smear should be prepared and the tube refrigerated
Do not refrigerate blood smears
Blood should be analyzed at room temp
Remove from the ridge about 30 min before running sample
Mix the sample gently immediately before removing the sample for testing
Inadequate mixing results in erroneous data
Never freeze blood samples for hematology testing
Fill tubes completely to prevent artificial reduction in packed cell volume (PCV)- due to sample dilution with the anticoagulant
If a patient’s veins are small (dehydration, shock, size of the patient, blood loss, etc) use heat therapy (warm towel) or use lots of alcohol
You can pump the foot to stimulate blood flow
Tips for filling vacutainers
Select a venipuncture site that
Can provide the appropriate volume of blood with one poke
Is tolerated by the animal
Select the correct vacutainer size
They come in various sizes- 250μL, 500μL, 1.8 mL, 2 ml, 2.7 ml, 3 mL, 3.5 ml, 4 ml, 5 ml, 6 ml, 7 mL, 10 mL, 15mL
Make sure the tube is not expired
Use minimum negative force
Use a gentle or pulsing force on the syringe when drawing the sample- this will help to prevent the vein from collapsing
Use as large a bore needle as possible to reduce hemolysis and force required to draw the sample
How to fill a vacutainer properly
Fill the tube with required amount of blood properly mix with the anticoagulant- this will prevent cell distortion, hemolysis and clotting which will all affect results
The vacuum in the tube should automatically fill the tube with the correct volume if a vacutainer system is used to collect the sample
You need to know what a properly filled tube looks like so if it doesn’t fill you are aware
This may occur due to improper vacuum, a clot in needle, moving the needle outside of the vein, loss of suction, etc
In exotics and in small animals, a needle and syringe are used, blood is then transferred to a vacutainer
The stopper and needle are removed and then the vacutainer is filled –MAKE SURE to fill it properly
Order of blood in the tube
The order if filling is determined based on minimizing risk of cross contamination of tubes with anticoagulant
Cross contamination can result in inaccurate lab results
Reflexive study showing that the risk of cross contamination of a blood sample is higher when a needle and syringe are used than if a vacutainer system is used
When using a needle and syringe- ensure that the tip of your syringe never touches the inside of the vacutainers
Current recommendation for fill order
Culture tubes/bottles
Sodium citrate tubes (light blue)
Serum tube (red)
Serum separator tube (tigger)
Heparin tube (green)
EDTA tube (purple)
Sodium fluoride/Potassium oxalate (grey)
Record keeping for blood is important for
A vital part of the business
Provides continuity of care
Have a consistent protocol to avoid human error
Double check math- one of the most common errors
Report units (conventional vs SI units- very different numbers)
Lab submission forms should contain all of the following info
Patients name
Owners name
Signalment
Short physical description of patient (weight, color, microchip or tattoo information)
Clinical history
Clinicians name (clinic name, address and phone # if acting as a reference lab)
Tests required
Space to report results
RVTs initials
Remarks area
Case identification number (if used)
Chemistry analysis is
Chemical analysis of the blood is performed to evaluate electrolytes, proteins, enzymes levels and other electrolytes within the liquid portion of the blood
A chemistry analysis may be performed for routine health testing, as an aid to identify why an animal is sick, to evaluate progression of disease or to monitor for an organ toxicity when a patient is being medicated for a chronic condition
What do you use for a chemistry analysis
The chemical analysis of blood is usually performed on serum or plasma
Some machines will use whole blood with an anticoagulant added to it (usually heparin)
Ensure that you know what type of blood tubes are required before the sample is collected
How quick should a chem panal be preformed after blood is drawn
The analysis should be performed within 1 hour of sample collection if possible
If sampling will be delayed, the sample may be refrigerated or frozen
If you are freezing the sample, the serum or plasma MUST be harvested first, do NOT freeze whole blood
Sample that have been thawed can not be refrozen
What to do once the chemistry analysis blood is drawn
The quality of the results that you obtain are largely determined by your sample collection and handling techniques
In the case of sick animals, the sample should be collected before treatment is initiated
Fasted samples are preferred- glucose, urea, lipase, and cholesterol levels may be affect if the animal has eaten recently
A 12 hour fast is required for some tests (Vitamin B12, PTH)
The blood tube should be labelled with the patients info, sample type, dat and time before the blood is collected
If the blood is to be submitted to a reference lab, a request form must be filled out and submitted with the sample
Serum sample prep
Blood should be collected and transferred to a red top tube (no anticoagulant)
Let the sample sit for 20-30 minutes at room temperature to allow a blood clot to form
Gently run a wooden applicator between the clot and the inside wall of the blood tube to loosen the clot from the tube
Replace the cover on the blood tube and centrifuge at 2000-3000 rpm for 10 minutes
Remove the serum from the tube with a capillary pipette and transfer the sample to a labelled red top tube
Process the sample immediately
If you are unable to process it immediately, refrigerate or freeze the sample
Plasma sample prep
Collect the blood sample and transfer it to a blood tube with the appropriate anticoagulant
Gently mix the sample
Centrifuge within 1 hour of collection
2000-3000 rpm for 10 minutes
Remove the fluid (plasma) layer with a capillary pipette and transfer the plasma to labelled red top tube
Process the sample immediately
If you are unable to process it immediately, refrigerate or freeze the sample
Factors that influence chemistry analysis
Abnormal chem results may occur due to a variety of reasons
A pathological condition in the animal
Hemolysis
Icterus
Lipemia
Certain med
Diet
Importer sample handling
Hemolysis will occur because
Hemolysis will occur for a number of reasons
Aspiration of alcohol into the syringe
If the syringe is damp
If a needle with a small diameter is used
Too much suction on the syringe or by the vacutainer will increase risk of hemolysis
Transferring the blood from they syringe through the needle to the vacutainer
Transferring the blood from the syringe into the vacutainer too quickly
Rough agitation of the sample after collection
Over centrifuging- time and speed
Freezing whole blood
How does hemolysis impact results
Cell rupture will cause the sample to be artificially dilute due to fluid leakage from the cells
This will decrease the concentration of some analytes within the sample
Cell rupture will cause concentrations of some metabolites to be increased
How is this possible?
Hemolysis interferes with lipase activity
Hemolysis will alter bilirubin determination
Chemical contamination is
If the blood tubes are contaminated with detergents or disinfectants, this will affect chemistry results
This can be avoided by not re-using tubes or syringes
Take care to avoid contamination of the red top tube with anticoagulant
Vacutainer system-fill the tubes in the appropriate order
Needle and syringe-avoid touching the tip of the syringe to the EDTA tube when filling it to prevent transfer of EDTA to the red top tube
Improper labeling
An easily avoidable error
Ensure that the tubes are labelled with the patient identification, date and time of collection
Ensure that the sample type is indicated on red top tubes
Urine, serum and plasma look remarkably similar
Improper sample handling is
A sample should be run within an hour of collection
If this is not feasible, the sample must be processed and stored properly to ensure accurate results
If a sample has been frozen, it must be mixed gently after thawing
Samples that have been thawed should not be re -frozen
If the sample becomes too warm, the lab results may be affected
Patient influences for chem panels
Ideally the patient should be fasted for 12 hours prior to blood collection
Inorganic phosphorus levels may be decreased immediately after a meal
Glucose levels, cholesterol and lipids will be increased in a non-fasted patient
Lipemia–increases turbidity of the sample altering lab results
Reference ranges are
Also known as the normal ranges
Reference ranges are specific to the analyzer and test method that being used
Reference ranges are determined based on running samples on multiple “normal” animals
Species specific
May also be impacted by breed and age of the animal
