3. Sample Collection and Handling

Question 1

What is whole blood and what is it comprised of?

Answer 1

Circulates thru blood vessels
composed of RBC, WBC, platelets, plasma (fluid that contains albumin, globulins and fibrinogen, waste products and ions)

Question 2

What is the purpose of whole blood and its components? Why is examination of it important?

Answer 2

blood transports O, CO, nutrients,w aste products and hormones thru the body
WBC and Ab’s big immunity role
Exam of blood gives trained professionals a clue on patients health. Whole blood must be mixed with anticoagulant to look t it

Question 3

What is plasma

Answer 3

The FLUID portion of blood containing an anticoagulant which RBC’s, WBCs and platelets are removed after centrifuging
Contains fibrinogen, other clotting factors and proteins
in a healthy patient, clear or straw colored
plasma sample preparation will be performed in a clin path lab

Question 4

What is serum?

Answer 4

The clear, straw, liquid portion of blood that does NOT contain fibrinogen, or formed elements
liquid that remains after the sample has clotted
nothing has been added to the sample, it does not contain an anticoagulant

Question 5

What must you do before sample collection?

Answer 5

must know what tests to run! Will determine appropriate vein, V, blood tubes

Should collect b4 tx and if already started, make a note of it

Question 6

What collection equipment do you need?

Answer 6

Needle and syringe
Vacutainer system
Collection tubes

Question 7

What are anticoagulants

Answer 7

they are chemicals that will prevent or delay blood from clotting
there are a # of diff anticoagulants that are used for blood analysis, know which one you need

Question 8

What is a serum collection tube

Answer 8

Red top tube!
No anticoagulant
once blood clot has formed, serum is harvested
used for lg # of biochemical blood tests
can also be used for storage and shipment of histology samples, hair samples, urine
NEVER used for hematology tests

Question 9

What are heparin tubes?

Answer 9

Green top tubes
Contains heparin as anticoagulant to react with Na, K, lithium or ammonium salt
used for biochem 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 sm sample, you can coat the inside of the syringe w/ heparin b4 collecting the blood

Question 10

What is EDTA tubes?

Answer 10

lavender/purple top
contains EDTA or ethylenediaminetetraacetic acid, it can be in power or preferably liquid form which comes as either a sodium or potassium salt
anticoagulant of choice for hematology tests
Never used for chem analysis

Question 11

Why do we use EDTA tubes to look at cells?

Answer 11

gives the most consisten preservation of cell volume and morphological features of the cells
prevents clotting of the blood
tube must be mixed after the blood has been added
^^ done gently with a figure 8 motion, a gentle back and forth rocking motion or by inverting the sample 8-10 times
Excess EDTA in the blood sample will nullify automatic machine analysis

Question 12

What are oxalate and citrate tubes

Answer 12

blue top
used for coagulation tests (they interfere with clot formation by binding calcium
interfere w/ chemistry test results
oxalates are available in na, k, ammonium or lithium salts
citrates are available in na or lithium salts also used for blood transfusions, some need to be refrigerated

Question 13

What are sodium fluoride tubes?

Answer 13

grey tops
contain sodium fluoride
used for preserving blood glucose in a blood sample -effectiveness is questionable
interferes with many enzyme tests

Question 14

What are sure-sep tubes or serum separator tubes

Answer 14

Tiger top
a variation of red top, no 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 15

How much sample volume do we need for collection?

Answer 15

Depends on tests and patients dehydration status
in a well hydrated patient, approx 1/2 V of whole blood collected should be serum or plasma
dehydrated patients will yield 1/2 of that volume ^^
ideal to collect enough serum, plasma or whole blood to run the test 3 times. Allows for technical error, instrument failure, dilution of sample and forearding to a reference lab

Question 16

You need 1.2 mL of serum tor un a chem panel. What is the min V of blood that you need in a well-hydrated this sample?

Answer 16

3mL

Question 17

You need 1.2 mL of serum tor un a chem panel. What is the min V of blood that you need in a dehydrated this sample?

Answer 17

6mL

1.2 needed, 3ml for well hydrated. 2x3 = 6ml in dehydrated

Question 18

Before collecting, what should you do before you get your patient out?

Answer 18

Gather all equipment
Label the tube with patients name, date and time, sample type
Prep the area for venipuncture with alcohol swab, allow area to dry b4 collecting the sample
Use less is more if possible to reduce hemolysis and contam of tissue fluid
Mix anticoag containing vacutainers gently by inverting 8-10 times or by moving it in figure 8

Question 19

What are some general sample handling procedures to keep in mind

Answer 19

Blood collected for CBC analyzed <1hr (if not possible, blood smear prepared and tube refrigerated)
DO NOT refrigerate blood smears
Blood analyzed @ room temp - remove from fridge about 30 mins b4 running sample
Mix sample b4 removing sample for testing
inadequate mixing =erroneous data
Never freeze blood samples 4 hematology testing
Fill tubes completely to prevent artificial reduction in PCV - due to sample dilution w/ anticoag
If sm veins use heat or lots of alcohol, can pump foot to stim blood flow

Question 20

What are some sick tips for filling vacutainers?

Answer 20

1. select an appropriate site to give enough blood/is tolerable 4 anim
2. Appropriate vacutainer size
3. Make sure tube not expired
4. Use minimum force req (gentle pulsing force of syringe will prevent vein collapse)
5. Think about order to fill vacutainer tubes
6. fill tube w/ required blood amount to mix w/ anticoag

Question 21

Why should we make sure that the tube is filled with the required amount of blood?

Answer 21

To prevent cell distortion, hemolysis and clotting which would all affect results
The vacuum in tube should be automatically fill the tube w/ the correct volume if a vacutainer system is used to collect the sample
need to know what a properly filled tube looks like so we know what normal is

Question 22

What is the order of draw?

Answer 22

the order if filling is determined based on minimizing risk of cross contamination of tubes w/ anticoag
Cross contam can result in inaccurate lab results
reflective study showing risk of cross contam of a blood sample is higher when a needle and syringe are used than if a vacutainer system is used - tip of syringe never touches inside of vacutainer

Question 23

What is the current recommendations for fill order?

Answer 23

Culture tubes/bottles
Sodium citrate tubes
serum tubes
serum separator tube
heparin tube
EDTA tube
Sodium fluoride/potassium oxalate
In terms of color
Sterile (yellow), light blue, red, SST (pale orange), green, lavender, gray

Question 24

Why is record keeping important

Answer 24

vital part of business
provides continuity of care
have a consistent protocol to avoid human error
double check math - most common errors
report units (conventional vs SI units - very diff numbers)

Question 25

What should a lab submission form contain?

Answer 25

patients name, owners name, signalment, short physical description of patient (weight, color, microchip, or tattoo), clinical hx, clinicians name (clinic name, address and phone # if acting as a reference lab)
tests required, space to report results, RVT’s initials, remarks area, case ID # if used

Question 26

What is a chem analysis?

Answer 26

A chemical analysis of blood to evaluate electrolytes, proteins, enzyme lvls and other analytes within the liquid portion of the blood
a chem analysis may be performed for routine health testing, as an aid to Id why an anime is sick, to eval the progression of dz or to monitor for organ toxicity when a patient is being medicated for a chronic condition

Question 27

How does chem analysis work?

Answer 27

Usually performed on serum or plasma in which machines use whole blood with anticoag (like heparin). Know which tube are req b4 sample collected
Analysis performed <1hr of collection, if delayed, sample can be refrigerated but serum or plasma MUST be harvested first. Do not freeze whole blood
Samples that have been thawed cannot be refrozen
the quality of the results obtained are determined by your sample collection and handling technique

Question 28

What happens if the animal is sick?

Answer 28

sample should be collect b4 treatment is initiated
fasted samples are preferred - glucose, urea, lipase and cholesterol lvls may be affected if animal has eaten recently (12hr fast req for tests like vitamin b12 and PTH)
tube should be labelled w/ patients info, sample type, date and time b4 blood collected
if blood submitted to reference lab, a request formmust be filled out and submitted with the sample

Question 29

How do we prepare a serum sample?

Answer 29

blood collected and transferred to a red top tube (no anticoag)
let sit for 20-30 @ room temp to clot
gentle run a wooden applicator btw cloth and the inside of wall of blood tube to loosen clot from tube
replace cover and centrifuge at 2000-3000 rpm for 10 min
remove serum from tube w a capillary pipette and transfer sample to a labelled red top tube
process immediately

Question 30

How is plasma samples prepared?

Answer 30

collect sample, transfer to blood tube w/ anticoag
mix sample
centrifuge <1hr of collection
remove fluid layer w/ capillary pipette and transfer the plasma to labelled red top tube
process sample immediately

Question 31

What are some factors that influence results

Answer 31

A pathological condition in anim
hemolysis
icterus
lipemia
certain medications
diets
improper sample handling

Question 32

What is hemolysis?

Answer 32

rupture of the cell. occurs bc of aspiration of alcohol into the syringe
if the syringe is damp
if a needle with a sm diameter is used
too much suction on the syringe or by the vacutainer will increase risk of hemolysis
transferring the blood from the syringe thru the needle into 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 33

How does hemolysis impact results?

Answer 33

cell rupture will cause the sample to be artificially dilute due to fluid leakage from the cells - will dec the conc of some analytes within sample
cell rupture will cause conc of some metabolites to be inc - how is this possible
hemolysis interferes with lipase activity
hemolysis will alter bilirubin determinatio

Question 34

What is chemical contamination?

Answer 34

if the blood tubes are contaminated with detergents or disinfectants will affected chem results - can be avoided by not re-using tube sor strings
take care to avoid contamination of the red top tube with anticoagulant
vacutainer system - fill tube w/ appropriate order
needle and syringe - avoid touching tip of syringe to EDTA tube when filling to prevent transfer of EDTA to red top

Question 35

How can we ensure proper labelling?

Answer 35

Ensure tubes are labelled w/ patient ID, date and time of collection, ensure sample type is on red top tubes

urine, serum and plasma look similar

Question 36

How can we prevent improper sample handling

Answer 36

same run within 1hr of collection, otherwise sample must be processed and stored
if sample frozen, can be mixed after thawing but cannot be refrozen
if sample becomes too warm, the lab results may be affected

Question 37

How might patient influence results?

Answer 37

ideally fast for 12 hrs b4 blood collection bc inorganic phosphorus lvls may be decreased immediately after a meal
Glucose lvls, cholesterol and lipids will be increased in a non-fasted patient
lipemia - inc turbidity of the sample altering lab results

Question 38

What are reference ranges?

Answer 38

AKA normal range
ref ranges are specific to analyzer and test method being used
used to determine based on running samples on multiple normal anims
species specific