Blood collection and handling

Question 1

Whole blood is and comprised of

Answer 1

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

Question 2

Plasma is

Answer 2

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

Question 3

Serum is

Answer 3

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

Question 4

Anticoagulants are

Answer 4

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

Question 5

Serum collection tube is

Answer 5

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

Question 6

Sample collection is

Answer 6

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

Question 7

What to do before collecting blood

Answer 7

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

Question 8

Needle and syringe for collecting blood

Answer 8

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

Question 9

Vacutainer system for blood collection

Answer 9

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

Question 10

Collection tube is

Answer 10

Can be empty, sterile tubes or they may contain anticoagulants
Vary in size from a few microliters to 15ml

Question 11

Heparin tube is

Answer 11

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

Question 12

EDTA tube is

Answer 12

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

Question 13

Oxalate and citrate tubes is

Answer 13

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

Question 14

Sodium fluoride tubes are

Answer 14

(Grey top)
Contains sodium fluoride
Used for preserving blood glucose in a blood sample- effectiveness is questionable
Interferes with many enzyme tests

Question 15

Sure-Sep tube or serum separator tube is

Answer 15

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

Question 16

Sample volume that should be taken

Answer 16

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

Question 17

How much blood should you collect and why

Answer 17

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

Question 18

Collection procedure

Answer 18

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

Question 19

General handling procedure

Answer 19

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

Question 20

Tips for filling vacutainers

Answer 20

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

Question 21

How to fill a vacutainer properly

Answer 21

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

Question 22

Order of blood in the tube

Answer 22

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

Question 23

Current recommendation for fill order

Answer 23

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)

Question 24

Record keeping for blood is important for

Answer 24

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)

Question 25

Lab submission forms should contain all of the following info

Answer 25

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)

Question 26

Chemistry analysis is

Answer 26

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

Question 27

What do you use for a chemistry analysis

Answer 27

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

Question 28

How quick should a chem panal be preformed after blood is drawn

Answer 28

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

Question 29

What to do once the chemistry analysis blood is drawn

Answer 29

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

Question 30

Serum sample prep

Answer 30

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

Question 31

Plasma sample prep

Answer 31

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

Question 32

Factors that influence chemistry analysis

Answer 32

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

Question 33

Hemolysis will occur because

Answer 33

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

Question 34

How does hemolysis impact results

Answer 34

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

Question 35

Chemical contamination is

Answer 35

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

Question 36

Improper labeling

Answer 36

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

Question 37

Improper sample handling is

Answer 37

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

Question 38

Patient influences

Answer 38

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

Question 39

Reference ranges are

Answer 39

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