SPECIMEN COLLECTION

PHLEBOTOMY the process of collecting blood.

Collection, handling and processing of specimen is essential in
obtaining a reliable result. (Preanalytical QC)
Specimen/ samples analyzed
Substances measured
CATEGORIES OF SUBSTANCES MEASURED IN SERUM:
1.
SUBSTANCES NORMALLY PRESENT W/
FUNCTION IN THE CIRCULATION:
Glucose, Total Protein, Albumin, Individual Proteins,
Electrolytes, Total Albumin Globulin (TAG), Cholesterol,
Hormones, Vitamins, Enzymes
2.
METABOLITES non-functioning waste products in
the process of being cleared.
Urea, Creatinine, Uric Acid, Ammonia, Bilirubin
SAMPLE VARIABLES:
1. PHYSIOLOGICAL CONSIDERATION
2. PROPER PATIENT PREPARATION
3. PROBLEMS WITH COLLECTION
4. TRANSPORT
5. PROCESSING
6. STORAGE
IMPORTANT CONSIDERATIONS IN BLOOD COLLECTION:
1.
PHYSICAL ACTIVITY
a. Transient effects immediate fall and then increase
(Free Fatty Acid or FFA, alanine, lactate, K+)
b. Long term effects increased in alanine, aldolase,
Aapartate Amino Transferase (AAT) or Aspartate
Transaminase, LDH
c. 6 months of physical training increased:
- Plasma testosterone: 21%
- Androstenedione: 25%
- Lh: 25%
2.
FASTING (prolonged)Fasting Blood Sugar: 12hrs
a. after 48 hrs: serum bilirubin increased by 240%
b. after 72 hrs: glucose decrease to 45mg/dl
- Increase: (plasma) triglycerides, glycerol & FFA
- Decrease: transthyretin, albumin, insulin, high
density lipoprotein (HDL) cholesterol, LD cholesterol
- Cholesterol: no change
3.
POSTURE
Supine upright (increased albumin, drug-bound
CHON, cholesterol, enzymes, total CHON,
triglycerides, bilirubin, calcium)
Standing supine/reclining position (decreased
aldosterone, alkaline phosphatase, catecholamines,
calcium, renin, ALT)
4.
ETHANOL INGESTION
- Increase in plasma lactate, urate, triglycerides
- Alcohol abusers increased GGT (Gamma globulin
transferase), urate, MCV

- Long term effects: Increased AST (Aspartate

aminotransferase) & ALT (Alanine amintransferase)
5.
TOBACCO SMOKING
- Increase blood carboxyhemoglobin levels
a. Heavy smokers: inc 8%
b. Non-smokers - <1%
c. Acute tobacco smoking increase plasma
catecholamines, serum cortisol
- Increase glucose, triglycerides, cholesterol, LDL
cholesterol
6.
DEHYDRATION
Hemoconcentration
False elevated: Iron, Calcium, Sodium & Enzymes
Increased hematocrit and protein
7.
LIFESTYLE
High protein diets: increased levels of uric acid, urea
& ammonia compared to vegetarians.
Caffeine: decreased pH, increase ionic calcium &
catecholamines.
GENERAL RULES FOR BLOOD SPECIMEN COLLECTION:
1. PATIENT ID
2. APPROACHING PATIENT
3. COLLECTION SITE
4. TOURNIQUET APPLICATION (not more than 2 minutes)
5. CHECKING PATIENT
6. SPECIMEN HANDLING & TRANSPORT
7. STORING SPECIMEN PRIOR TO TRANSPORT
Serum pH: 8.5 Acid phosphatase (ACP) destroyed
Glycolysis in whole blood decreased serum glucose
Changes in RBC permeability increased serum K, P, Mg
Freezing, subsequent thawing, refreezing, rethawing:
CHON denaturation
Frozen specimen, thawed at RT before analysis increased
Alkaline phosphatase (AKP)
Exposure to light decreased bilirubin
BLOOD SPECIMENS
SOURCE & COMPOSITION OF BLOOD SPECIMENS:
1. VENOUS BLOOD
Composition is affected by metabolic activity of the
tissue it drains & varies by collection site.
Lower oxygen content, dark bluish red color.
2. ARTERIAL BLOOD
Composition is normally uniform throughout the body.
Bright red in color because it is oxygen rich.
Reserved for blood gas evaluation & certain
emergency situations (must put in ice).
3. CAPILLARY BLOOD
Contains arterial & venous blood plus tissue fluid.
Because it enters capillaries under pressure, the
arterial portion is highest.
TYPES OF BLOOD SPECIMENS:
1. SERUM
Normally a clear, pale yellow fluid.

Non fasting sample can be cloudy due to lipids.

Utilized by most chemistry tests, plain tube
2. PLASMA
Normally is clear to slightly hazy, pale yellow fluid.
The fluid portion of an anticoagulated blood contains
fibrinogen.
3. WHOLE BLOOD
Contains both cells & plasma.
Collected in an anticoagulated tube
Mostly for hematology
*Note: regardless of source of blood is approximately 55%
fluid and 45% blood cells.

VENIPUNCTURE
- Deoxygenated blood; contains subs that come from
metabolic activities of diff organs.
- Blood chemistry & immunologic studies.
- More easily collected than arterial blood.
SITES OF VENIPUNCTURE:
ANTECUBITAL FOSSA VEINS median cubital vein (most
preferred because it is large, most stationary and closer
to the surface) located near the center of the antecubital.
MEDIAN CEPHALIC VEIN 2ND vein of choice, fairly well
anchored & often the only vein that can be felt by obese
patients.
MEDIAN BASILIC VEIN last vein of choice, not well
anchored and roll easily.
OTHER VEINS: LEG, ANKLE & FOOT VEINS needs
doctors permission.
OTHER VEINS: HAND & WRIST VEINS but not the
underside of the wrist.

COMPLICATIONS (IMMEDIATE/ DELAYED & LOCAL/

SYSTEMIC):
Hematoma (missed vein)
Collapsed small veins (excessive pull of plunger)
Syncope (loss of consciousness)
Excessive bleeding
Thrombosis of vein
Blood-borne infection: hepatitis b & aids

METHODS OF VENIPUNCTURE:
a.SYRINGE METHOD (most conventional)
- Single sample needles specifically for syringes
- Barrel, plunger, needle, needle holder (5&10), hub,
bevel
b.
EVACUATED TUBE SYSTEM (ETS)
- Multiple-sample needles should be used if more than 1
tube of blood is to be collected during venipuncture
(prevent leakage during tube changes).
- Gel: barrier bet serum & cells
- Marked w/ expiration date (lost vacuum/ ineffective
additive)
- Correct blood-AC ratio
- Needle: 2 way longer for tube; other for vein
c. BUTTERFLY INFUSION SET
- Stainless steel beveled needle with attached plastic
wing to facilitate needle insertion.
- Plastic tubing connects needle to adaptor- screws into
a tube holder = modified evacuated system or a syringe
can be attached to collect blood.
- For pediatrics/ babies & geriatrics
*gauge: bigger number = smaller bore (21: ideal; 26: blood
donation)
VENIPUNCTURE PROCEDURE:
1. Review & assessing of the Test Request
Completeness of patients request & the necessary
preparation (name, test, condition, etc.)
2. Identifying & Positioning the patient
Observe professionalism in approaching the patient.
Position the patients arm in a way comfortable for
both patient & you.
The arm must be supported by a firm surface.
Ask the patient his arm preference.
3. Prepare equipment
Needle & evacuated tubes or syringe.
4. Applying tourniquet to select the vein
- Downward angle position of arm, using force of gravity
to visualize vein.
- Site must be free of skin abrasions, lesions & scar
tissues.
- Select the well anchored vein of the arm.
- Remove the tourniquet to prevent hemoconcentration.
- 3 mins application increases CHON, Fe, AST, Bb & total
lipids
- Repeated fist clinching increases K+ 1-2nm
5. Applying the antiseptic (wet then dry)
- Cleanse with 70% isopropyl alcohol in Circular motion
(inside to out).
- Air drying permits maximum antiseptic action, prevents
contamination, avoids stinging on needle entry and
prevents hemolysis.
6. Reapplying tourniquet
Tight enough to increase pressure not so tight to cut
circulation.
7. Inserting the needle

Bevel up at 15O angle, quickly & smoothly insert

needle.
Avoid probing (fishing), do not attempt a
venipuncture > 2 times
8. Withdrawing blood
- Iv avoided
- Turn off iv for 2-5 mins, discard 1st 5ml of draw
9. Releasing tourniquet
Before the last tube is filled
10. Withdrawing needle
Single, smooth swift motion.
Quickly apply sterile gauze & apply pressure.
11. Removing needle from syringe/ removing evacuated
tube from needle
12. Transferring blood to an appropriate container/tube
13. Checking patients wound
- Betadine falsely high p, uric acid, k+
- Isopropyl (70%) alcohol should not be used for medical
or legal ethanol levels
14. Check label of specimen & transport

5. EDTA
Lavender Top
6. Glycolytic inhibitor (NaF)
Gray Top
*SPS Sodium Polyanethol Sulfonate
*Clot coagulation tests if without the need for
yellow top tube, use a plain/red top tube before the
blue top tube (1st drop is discarded because it can
speed up clotting)
*Gel VS Non-gel: serum or plasma separator tubes
maybe unacceptable for some analytes
(therapeutic drug)

*Note: If all sites of venipuncture have IV, use below IV but

discard the first 5 ml.
VACUTAINER TUBES:

These are tubes for blood collections which are color

coded based upon the anticoagulant present, in various
sizes, 2, 5, 7, & 10 ml.

Needles are sterile, disposable & sized by length &

gauge.

Gauge refers to the diameter or bore of the needle.

The higher the gauge, the smaller the bore.
GREEN G21
BLACK G22
BLUE G23
ADDITIVES INCORPORATED IN VACUTAINER TUBES:
1. ANTICOAGULANTS prevents blood from clotting.
2. ANTIGLYCOLITIC AGENTS prevents glycolysis.
3. CLOT ACTIVATORS coagulation factors such as
Thrombin & substances such as glass (silica) particles &
inert clays like diatomite (Celite) that enhances
clotting by providing more space for platelet activation.
4. THIXOTROPIC GEL SEPARATORS inert substances
contained in or near the bottom of certain tubes.
During centrifugation, the gel lodges between the cell
& fluid, forming a barrier preventing the cells from
metabolizing substances in serum or plasma.
ORDER OF DRAW:

A special sequence of tube collection that reduces

the risk of specimen contamination by microorganisms & additive carry-over, which affects
some tests. (ETS)
1. Sterile tube Blood culture (SPS) Yellow Top
2. Coagulation tube (Citrate)
Blue Top
3. Serum tube with or without clot activator/gel (No
anticoagulant obtains Serum)
Red Top
4. Heparin
Green Top

RATIONALE FOR COLLECTION ORDER:

Yellow Top
minimized microbial contamination
Blue Top
all other additives affects coagulation
Red Top
prevents contamination by additive from
other tubes. *with SST silica particles
activates clotting & affects coagulation
tests.
Green Top
causes the least interference in tests other
than coagulation tests.
Lavender Top
elevates Na & K, chelates & decreases Ca
& Fe levels, elevates Prothrombin Time (PT)
& Partial Thromboplastin Time(PTT).
Gray Top
K oxalate abnormal cell morphology &
interferes enzyme reaction
*EDTA Ethylene Diamine Tetraacetic acid
COLOR
LAVENDER

ACTION
Chelates calcium

USE
Hematologic
assays, lead
assays, CEA
determination &
cell counts

RED

ADDITIVE
EDTA
Versene
(disodium
salt)
Sequestrene
(dipottasium
salt)
none

Allow blood to
clot

RED/ GRAY
OR RED
BLACK

None;
separator
material

ORANGE

thrombin

BLUE

Buffered
citrate

Allows blood to
clot; barrier
between cells &
serum
Accelerated
clot
Binds Ca

Most chemistry,
immunologic &
blood bank tests
Most chemistry
tests

BLACK

Buffered
sodium citrate
NaF/
K2C2O4

GRAY

Binds Ca

Inhibits
glycolytic
enzymes
Iodoacetate
enolase & acts
as AC

STAT serum
test
Coagulation
assays like PT &
APTT
Westergren ESR
Glucose
determination

YELLOW
GREEN

Citrate
dextrose
Heparin (Na+.
Li+ or NH4+)

Inhibits
glyceraldehyde
s 3-phosphate
dehydrogenase
Preserves RBC
Inhibits
thrombin BGA,
ammonia CO-Hb

Blood culture
Active &
methemoglobin

ADVANTAGES OF EVACUATED TUBE SYSTEM OVER

SYRINGE METHOD:
Multiple blood collection can be done.
Requires no prior preparation.
Offers wider range of tube sizes & contained
anticoagulants.
Safer method of blood collection.
ADVANTAGES OF VENIPUNCTURE OVER SKIN PUNCTURE:
Large amount of blood can be obtained for a variety of
tests.
Additional & repeated test can be done.
Fastest method of collecting samples from a number of
patients.
Blood can be transported and stored for future use.
Ideal for blood chemistry determination.

2.
3.
-

pediatrics/ geriatrics: 22; veterinary: 18

needle holder
regular 13, 16mm
pediatric 10mm
tubes/ evacuated tubes
regular 75mm; 100mm

ARTERIAL PUNCTURE
- Oxygenated blood; uniform composition throughout body.
- Used to measure Oxygen tension , Carbon dioxide
tension & blood pH.
- BLOOD GAS ANALYSIS (BGA): critical to patients with
pulmonary problems, oxygen therapy, cardiovascular
problems & those undergoing major operations.
- Sites: RADIAL, BRACHIAL & FEMORAL ARTERIES
- Radial & brachial: preferred sites
- Newborns: umbilical artery catheter
- Brachial: 18-20 gauge, 45-60O
- Radial: 23-25 gauge, 90O
- Heparin is used as anticoagulant.

DISADVANTAGES OF VENIPUNCTURE:
Harm on infants, children & obese individuals.
Requires more time & skill on the part of the operator.
More complications may arise.
COMPLICATIONS IN VENIPUNCTURE:
1.
LOCAL IMMEDIATE COMPLICATIONS
- Hemoconcentration
- Failure of blood to enter syringe
- Circulatory failure
- Fainting or syncope
2.
LOCAL DELAYED COMPLICATIONS
- Hematoma
- Thrombosis of vein
- Thrombophlebitis
3.
GENERAL DELAYED COMPLICATIONS
- Serum hepatitis
- AIDS
- HIV
SST clot activator reduce clotting time
PST green/ gray
- Li Heparin w/ gel
- Electrolytes, routine chem. (invert 505x)
Trace element tube: heparin, EDTA or none
SPS: Soidum/ Polyanithol sulfonate (micro)
ACD acid citrate dextrose (HLA typing)
PARTS:
1. needles
- adults: 20, 21

COMPLICATIONS OF ARTERIAL PUNCTURE:

Hematoma due to increase pressure in artery.
Arterial spasm restriction of blood flow due to reflex
constriction.
Temporary discomfort (aching, throbbing, tenderness,
sharp sensation & cramping).
Thrombosis, hemorrhage & infection.
CONSIDERATIONS:
Intense care must be administered to patients
undergoing arterial puncture.
Sites not to be selected: irritated, edematous, near
wound or area of arteriovenous (AV) shunt or fistula.
Ice water/ coolant (1-5OC): to minimize WBC
consumption of oxygen.
Capillary blood may be a suitable substitute for arterial
blood determination of pH & pCO2 provided the site must
be warmed prior to collection. Warming increases blood
flow through capillaries & arterioles resulting in an
arterial-rich blood.

SKIN PUNCTURE/ CAPILLARY PUNCTURE

- Small quantity blood needed
- Pediatrics, obese w/ thrombotic tendencies & severe
burns
- Geriatric patients: thinner & less elasticity of skin
3 SITES FOR SKIN PUNCTURE:
EARLOBE FREE EDGES, NOT ON SIDES
PALMAR SURFACE OF FINGER
PLANTAR SURFACE OF HEEL & BIG TOE
Infants:
digits of 2nd, 3rd or 4th fingers
lateral plantar heel surface
median plantar heel surface
Children:
plantar surface of big toe
lateral side of finger, adjacent to nose
BLOOD OBTAINED IN SKIN PUNCTURE:
Capillary: BG (prewarmed)
Peripheral (most common term used)
Arteriolar
SITES TO AVOID:
Inflamed area
Congested or edematous site
Cold & cyanotic areas concentrated blood
Scarred & heavily calloused areas

10. Thank patient

11. Transport specimen to lab
THINGS TO REMEMBER IN DOING SKIN PUNCTURE:
Depth: 2.5-3mm
Avoid pressure & squeezing
First drop should be discarded (alcohol & tissue juice)
RBC, hematocrit, hemoglobin & platelets: lower
WBC: higher than in venous blood
Capillary blood from skin puncture is an admixture of
venous, arterial & may contain tissue juice
Puncture depth: 0.85-2mm deep; 1.75-3mm length
Microcapillary tubes used in hema:
Blue ring no AC
Red ring heparinized
Green ring heparinized
MICROTAINER TUBES/BULLETS:

Color coding corresponds to ETS, with markings

minimum & maximum measured in microliters (uL).
CAPILLARY SPECIMEN SOURCES OF ERROR:
1. Abnormal fluid distribution caused by excessive
squeezing.
2. Presence of microclots.
3. Hemolysis.
4. Altered chemistry tests.

ADVANTAGES USING EARLOBE:

Less painful (few nerve endings)
More free flow of blood to thinner skin
Less tissue juice contamination of blood ideal for
searching abnormal cells (histiocytes in bacterial
endocarditis)

WAYS OF INCREASING BLOOD FLOW IN SKIN

PUNCTURE:
1. Dry heat or paper towel w/ warm water (39-42OC).
2. Flicking with index finger until flushing is observed.
3. Chemicals (Trafuril paste Histamine creams).

ADVANTAGES USING THE FINGER:

Accessible to phlebotomist
Easy to manipulate
Ideal for peripheral blood smears
Less intimidating

ARTERIAL BLOOD COLLECTED FROM CAPILLARY

PUNCTURE MAY YIELD UNRELIABLE RESULTS IF:
1. Systolic is less than 95mmHg.
2. Cardiac output is severely restricted.
3. Vasoconstriction is present.

DISADVANTAGES OF SKIN PUNCTURE:

Less amount of blood obtained
Additional & repeated tests cant be done
Blood obtained does hemolyzes easily
Painful especially using the finger

SPECIMEN INTERFERENCE:
A. LYSIS OF CELLS (HEMOLYZED SERUM)
Leakage of intracellular substances
Lysis of RBC = lacking/ hemolysis (in vivo or in vitro)
In vitro hemolysis is due to (common):
1. Use of vacuum tubes
2. Vigorous mixing
3. Use of too narrow/ too wide needle bores
4. Effect of alcohol (have to air dry)
5. Centrifugation & separation steps
Hemolysis is visible only not until a 200mg/L of Hb level.
B. ICTERSIA (ICTERIC SERUM)
Intensely yellow serum sample due to elevated bilirubin
value.
Jaundice: Bilirubin is greater than 430uM (25mg/L).
Bilirubin interferes with test using dyes & turbidity test.

PROCEDURE:
1.
ID patient
2.
Reassure
3.
Assemble equipment
4.
Prepare finger
5.
Puncture finger
6.
Eliminate 1st drop
7.
Produce large rounded drop
8.
Withdraw/collect blood
9.
Stop bleeding

 Interference due to bilirubin may be minimized by sample

blanking or dual wavelength method (ALLEN CORRECTION
METHOD read product at 2 wavelengths; sometimes
higher, to do away with interferences).
Sample blank contains icteric sample & all reagents used
except for the reagent responsible for the color reaction.
When read, the absorbance due to icteric sample os
eliminated.
C. LACTESCENE (LIPEMIC SERUM)
Obtained normally after a meal (elevated exogenous
chylomicrons).
Characterized by milky or highly turbid serum.
Lactescene appears when Triglyceride (TAG) level reaches
4.6mm (4g/L).
Normal Triglyceride level: > up to 100 mg/dL.
Appearance of serum can affect the tests.
Corrected by ultracentrifugation of serum sample
GROUNDS FOR REJECTING SPECIMEN:
Inadequate sample identification
Insufficient volume of the specimen
Inappropriate collection tubeused
Hemolysis
Improper transportation
Interferences
ANTICOAGULANT INTERFERENCE:
Dilution errors especially oxalates which are highly
osmotic.
Inhibition of plasma enzyme activities
- Fluoride which is an enzyme poison.
- EDTA which chelates metallic enzyme activators.
- Oxalates inhibit AMS, LD & ACP.
- Citrate inhibits AMS.
Oxalates, citrate & EDTA lowers plasma Ca levels.
False increase in electrolyte analyses due to
anticoagulant in salt form.
SPECIMEN HANDLING & PROCESSING:
SERUM:
20-30 minutes is the ideal clotting time.
Generally preferred than plasma:
1. Interfering substance are co-precipitate during clotting,
LPL (Low plasma lipoprotein).
2. Optically clearer
3. Free from AC interference
Must ideally reach the laboratory within 45 minutes.
Agitation must be avoided during transport.
Light sensitive/ photo labile substances must be
wrapped with foil or any dark paper or placed in an
amber colored container to prevent light exposure.
(Bilirubin, Vitamin A, Carotene, Erythrocyte,
Protoporphyrin)
CHILLED SPECIMEN (ice at 4OC) for analytes that
needs the metabolic process to be slowed down which may
affect lab results:

ACTH (Adrenocorticotrophic Hormone)

Acetone
Blood ammonia
Catecholamines
FFA
Angiotensin converting enzyme
Lactic acid
Pyruvate
Renin activity

THINGS TO REMEMBER:
All materials should be dry & sterile to avoid hemolysis.
Never puncture or draw blood from vein where IV
medication is running. (false low/decrease)
Site of puncture should be thoroughly clean to avoid
Thrombophlebitis.
Tourniquet should be tied not too tight it may constrict
the arteries and veins.
Remove needle from adaptor of syringe & allow blood to
flow gently down sides of tube.
Containers are to be stoperred & those anticaogulated
tubes are to be inverted several times (6-10).
Needles should be properly disposed into the sharp
container
Always wear your PPE.
Reusable syringes: rinsed w/ tap H2O.
Tourniquet must always be released before withdrawing
needle from vein.
SPECIMEN COLLECTION VARIABLES:
1. TOURNIQUET: >3 minutes increases total protein
(TP), Iron, AST, Bilirubin and total lipids.
2. IV SITE: above IV should be avoided, below IV turn
off 2-5 minutes & discard first 5 mL.
3. CLEANING AGENTS: Betadine (Povidone Iodine)
FALSE HIGH, Phosphorous, Uric acid & Potassium.
4. SOURCE OF SAMPLE:

GLUCOSE CAPILLARY: 1.4% higher than venous

POTASSIUM CAPILLARIES: 0.9% higher than venous

BILIRUBIN, CALCIUM, CHLORIDE & TOTAL

PROTEIN (TP) values are lower in capillaries than
venous

PLASMA POTASSIUM, GLUCOSE & PHOSPHOROUS

lower than serum

PLASMA TOTAL PROTEIN, LACTATE

DEHYDROGENASE (LD) & CALCIUM higher than
serum

EDTA Plasma cholesterol, triglycerides & HIGHDENSITY LIPOPROTEIN(HDL)

CHOLESTEROL are 1.23 times to serum values

5. ANTICOAGULANTS
6. FLUORIDE will interfere with electrolyte studies