SPECIMEN

COLLECTION/SAFETY

DR. C.C. CHEMONGES

SCOPE
INTRODUCTION
SPECIMEN TYPES
BLOOD SPECIMEN
SAFETY PRECAUTIONS
URINE SPECIMEN
STOOL SPECIMEN
AMNIOTIC FLUID
SAMPLE HANDLING
PRE-ANALYTICAL VARIABLES
INTRODUCTION

•Proper sample collection and handling is an integral part of obtaining

valid and timely laboratory test result.
• Specimens must be
 obtained using proper techniques,
 collected in the proper container,
 correctly labeled
 promptly transported to the lab.

• It is the policy of the laboratory to reject samples when there is failure

to follow these guidelines.
• All specimens should be handled with universal precautions, as if they
are hazardous and infectious.
PREPARATION

Prior to each collection review the appropriate test description including:

◦ the specimen type to be collected,
◦ the volume,
◦ the procedure,
◦ the collection materials,
◦ the storage
◦ handling instructions
SPECIMEN TYPES
Biological specimen analyzed in clinical laboratories include;
1. Blood and its components
2. Urine
3. Stool
4. Amniotic fluids
5. Saliva
6. CSF
7. Pleural fluid
8. Ascitic fluid
BLOOD
•Is the most sample used for analytical purposes.
•Three general procedures for blood collection:
• 1. Venipuncture (blood chemistry)
• 2. Arterial puncture (Blood Gas analysis)
• 3. Skin puncture (pediatric client and point of care tests]

•Process of blood collection is called phlebotomy.

•Performed by phlebotomist
•After blood sample is collected may be analysed as:
• Whole blood
• Plasma
• serum
VEIN PUNCTURE
•Refers to all the steps necessary in obtaining an appropriate venous
specimen.
•Involves following steps:
◦ Introduction
◦ Patient identification and preparation
◦ Verifying test requested
◦ Safety precautions
◦ Site selection and preparation
◦ Venous occlusion
◦ Blood collection
◦ Completion of collection
 PATIENT INDENTIFICATION
AND PREPARATION

•Patient Identification should be an active process involving 2 or 3 items

of identification e.g patients name,age,medical records
•Preparation involve:
getting history: surgery, allergy[latex],iv drug use
Explaining the procedure
Positioning[sitting or supine]
SAFETY PRECAUTIONS
•The extent of precaution vary with nature of patient illness and
institution policies.
•They include;
• washing hands
• properly dressing in protective gear including:
 Gown
 gloves
 Mask

This prevents one against infectious material and limits spread of

infection from one patient to another.
SITE SELECTION
•In adults the median cubital vein is preferred because its large and close
to skin surface.
•Other site: Forearm, dorsal aspect of the hand.
•Site to avoid ;
• Arm with Extensive scarring and hematoma
• Arm on side of mastectomy
• Edematous areas
• Arm in which blood is being transfused
• Arms with arterial-venous fistulas or vascular grafts
• Sites above an IV cannula
SITE PREPARATION
•Cleanser commonly used:
• 70% Alcohol swab

•Others
• 70% isopropanolol
• Benzalkonim chloride[specimen for ethanol determination]

•The cleanser usually applied in a circular motion from the site outwards.
•Should be allowed to dry because traces may cause haemolysis.
VEIN OCCLUSION
•Can apply pressure culf [60 mm Hg] or tourniquet.
•Approximately 4 inches above puncture site.
•This obstruct venous return causing venous dilatation.
•Never leave the tourniquet on for over 1 min.
•Change in blood composition is noted slightly after 1 min and markedly
after 3 min of occlusion due to venous stasis.
•Changes include:
• Absorption of water and small molecules eg calcium into cell leading to
concentration of non dissolved molecules eg proteins
VENOUS BLOOD
COLLECTION
There are two methods :
1. Collection with evacuated tubes
2. Collection with a Syringe later transferred to tubes

Evacuated tube method is considered:

cheaper
convenient
 easier to use than syringes

Tubes vary with the additive used and are indentified using stopper color
code.
Commonly used evacuated
tubes
STOPPER COLOR ADDITIVE ACTION
RED None Allows blood to clot
Lavender EDTA Binds calcium
Orange Thrombin Accelerates
clot formation
Blue Na Citrate Binds calcium

Gray a.Na fluoride/K oxalate Inhibits glycolytic

b.Iodoacetate enzyme
Green Heparin Inhibits thrombin
activation
EVACUATED TUBE
METHOD
• A needle is first screwed into the collection tube holder.
• The needle is then gently guided into patient vein
• The tube is then pressed into the holder to puncture the stopper and release
the vacuum.
• Once blood begins to flow in the tube the tourniquet is released.
• When blood flow stops the tube is removed by holding the hub securely
and pulling the tube off the needle.
• Mix gently by inverting the tube
• If more samples needed replace the tube as above.
• Blood collected into a tube should never be transferred to another tube
because additive may interfere with results
EVACUATED METHOD
ORDER OF DRAW
If multiple tubes are needed, the proper order of draw to avoid cross
contamination and erroneous results is as follows:
1.Blood culture[vials or bottles, sterile tubes]
2. Coagulation tube (light blue top)
3. Serum tube with or without clot activator or silica gel (Red or Gold)
4. Heparin tube (Green top)
5. EDTA (Lavender top)
6. Glycoltic inhibitor (Gray top)
BLOOD COLLECTION
USING SYRINGE
•Needle is placed firmly over the nozzle of the syringe.
•Its aligned and pushed into the vein at an angle of 15 degrees
•When the vessle wall resistant is overcomed the forward pressure is
eased and blood withdrawn.
•Then its transferred into tubes followed by gently inverting tubes
containing an additive 5-8 times
•Vigorous withdrawal of blood into syringe or forceful transfer to tubes
may cause haemolysis.
SYRINGE METHOD
COMPLETION OF
COLLECTION
•Place a gauze pad over the puncture site and apply pressure.

•Ask the patient to apply pressure to the gauze for at least 2 minutes.

•When bleeding stops, apply a fresh bandage, gauze or tape.

•Dispose the syringe and needle as a unit into an appropriate sharps

container
 Technique for Venous Puncture

1. Introduction 8. Cleanse site with 70% alcohol

2. Client identification 9. Apply tourniquet

10. Perform the venipuncture(15 degree
3. Inform client of the
angle)
procedure,client reassurance
11. Withdraw needle
4. Verifying Specimen required
12. Release tourniquet
5. Proper positioning of client
13. Place a sterile cotton &apply pressure
6. 6.Preparation of equipment& 14. Transfer specimen
proper labeling
15. Check condition of client
7. Selection of suitable vein for
puncture. 16. Dispose contaminated materials eg
Ideal:antecubitalfossa(median needle and holder in the sharps
container
cubital& cephalic vein)
COMPLICATIONS:

1. Hematoma

2. Excessive pull on syringe plunger collapse of small vein

3. Clients syncope

4. Excessive bleeding

5. Vein thrombosis

6. Infection of the site of venipuncture

ARTERIAL PUNCTURE
The samples are primarily used for blood gas analysis.
Preferred site are:
I. Radial artery at the wrist[commonly used]
II. Branchial artery in the elbow
III. Femoral artery [requires much expertise]

Contraindications of radial artery puncture

A. Cellulitis or other infections over the radial artery
B. Absence of palpable radial artery pulse
C. Negative Allen test
D. Coagulation defects
ALLEN TEST
•Used to assess the patency of Ulnar artery and adequacy of distal arteries
to wrist:
1. The hand is elevated and the patient is asked to make a fist for about 30
seconds.
2. Pressure is applied over the ulnar and the radial arteries so as to occlude
both of them.
3. Still elevated, the hand is then opened. It should appear blanched
(pallor observed at the finger nails).
4. When Ulnar pressure is released the color should return in 7 seconds

•If color does not return or returns after 7–10 seconds, the test is
considered negative
•The ulnar artery supply to the hand is therefore not sufficient. Hence the
radial artery cannot be safely pricked/cannulated
ARTERIAL PUNCTURE
PROCEDURE
[RADIAL]
•Patient is made comfortable by resting his arm on a pillow in front of
him, palm facing up
•Allen test is performed
• Area over the radial artery is cleaned with alcohol wipes. Wear gloves.
•Use pre heparinised syringes but if not available rinse the syringe with
heparin.
• Hyper extend the patient's hand to stretch the radial artery.
• Holding the syringe in one hand placing the finger of the other hand
over the artery at the exact point where the needle should enter the
artery.
ARTERIAL PUNCTURE
CONT
• Puncture the skin with the bevel of the needle up, at an angle of 45 degrees,
towards the direction of blood flow.
• Advance the needle under the skin slowly. When the artery is entered, blood
will enter the flashback chamber spontaneously.
• The blood pressure will push the plunger back and the syringe should fill
without aspiration.
• After the required amount of blood has been obtained, the needle is quickly
withdrawn, while simultaneously placing dry gauze over the puncture site.
• Immediately compress the artery at the site manually and firmly for a
minimum of 5 minutes.
• If the patient is under anticoagulant therapy, hold pressure for a longer period
of time
RADIAL PUNCTURE
ARTERIAL PUNCTURE
COMPLICATIONS
•Infection

•Bleeding

•Vessel trauma

•Air emboli

•Haematoma
SKIN PUNCTURE
Its on open collection technique in which skin is punctured by a lancet.
Used in situations where:
I. Sample volume is limited eg pediatric
II. Severe vein damage eg repeated vein puncture
III. Unavailable site for vein puncture eg severe burns or bandaged
IV. Point of care tests

In adults and older children blood can be obtained from a finger

◦ The recommended site is the distal digit of the third or fourth finger on its
palmar surface, about 3-5 mm lateral from the nail bed

In infants, satisfactory samples can be obtained by a deep puncture of

the plantar surface of the heel
◦ Site outer medial and lateral portions
HEEL SITE
In infants, the
medial and
lateral site
Skin Puncture site
1 Infants:
◦ Lateral or medial plantar surface of the heel.
◦ In almost all infants, the heel bone is not located beneath these areas.
◦ Punctures must not be performed on
a. the posterior curvature of the heel,
b. fingers of an infant less than 6 months old,
c. a swollen site,
d. previous puncture sites,
e. the earlobes.

2. Adults and older children:

◦ The puncture must be on the palmar surface of the distal segment of the
middle or ring finger.
◦ The puncture should occur across the fingerprints, not parallel to them.
SKIN PUNCTURE PROCEDURE
[HEEL]
•Warm the site for three to five minutes[improves blood flow]

•Disinfect the site using a 70% alcohol pad.

•Hold the heel with a moderately firm grip, placing your hand against a
support if possible to prevent movement.

•Then firmly puncture the skin.

• Discard lancet into an appropriate sharps container

 SPECIMEN COLLECTION
[HEEL]
• Wipe away the first drop of blood with a gauze as this drop may contain an
excess of tissue fluid, which could cause erroneous test results.
• Position a collection container beneath the collection site. Blood should
freely flow from the puncture site.
• Avoid touching the puncture site and scraping the collection device against
the skin, as this will produce specimen contamination and hemolysis.
• The order of draw is important because of the tendency of platelets to
accumulate at the site of a wound.
• Therefore, tests for the evaluation of platelets, such as platelet count and
CBC, must be collected first.
• Tubes should be filled to the level indicated on the tube. Work quickly to
minimize the chance of microclots forming
Infant Heel blood collection
COMPLETION OF SAMPLE
COLLECTION
[HEEL]
• Mix each additive collection device by gently tapping and inverting
it.

• It is a good idea to mix anticoagulant tubes by a gentle “thumping”

action after each drop falls into the tube.

•Place clean gauze pad on the puncture site and apply slight pressure.

•Instruct the patient (or guardian) to hold slight pressure on the

gauze pad over the puncture site
 SAFETY PRECAUTION

1. Observe universal (standard) safety precautions.

2. Wash hands in warm, running water with a hand washing product before
and after each patient collection.

3. Gloves are to be worn during all phlebotomies, and changed between

patient collections.

Palpation of phlebotomy site may be performed without gloves providing

the skin is not broken.

4 A lab coat or gown must be worn during blood collection procedures

 SAFETY CONT
5 Needles and hubs are single use and are disposed off in an appropriate 'sharps'
container as one unit
Needles are never recapped, removed, broken, or bent after phlebotomy
procedure.
6 Gloves are to be discarded in the appropriate container immediately after the
phlebotomy procedure.
7 All other items used for the procedure must be disposed off according to proper
biohazardous waste disposal policy.
8 Contaminated surfaces must be cleaned with freshly prepared 10% bleach solution.
9 All surfaces are cleaned daily with bleach.
10 In case of an accidental needle stick,
◦ immediately wash the area,
◦ express blood from the wound
◦ follow guidelines for PEP
FACTORS AFFECTING
BLOOD COLLECTION
Include:
Anticoagulant type

Collection site

Hemolysis
ANTICOAGULANT
Heparin:
commonly used anticoagulant for chemistry, gas analysis, and emergency
tests
Not used in PCR because it inhibit polymerase enzyme
Interferes with binding of EDTA to calcium
Affects the binding of thyroid hormone [T3 and T4] to their carrier protein
leading to high levels of these hormones

EDTA:
o Its a chelating agent binds divalent cation calcium preventing the clotting
mechanism.
o Used in hematological test and Isololation of DNA because it preserves the
cellular details.
o High concentration are hypertonic may shrink the cells
ANTICOAGULANT CONT
ACD:
◦ preserves the form and function of cellular components hence used in samples
for molecular diagnostic

Sodium fluoride:
 is a weak anticogulant
 Used as a glucose preservative
 It inhibits enzymes involved in glycolysis.
 Interferes with urea nitrogen test by inhibiting urease enzyme

Sodium citrate:
Used in the Ratio of 1:9 of blood in coagulation studies.
Its anticaogulation effect is easily reversable by adding calcium
This ratio is critical as osmotic effects and changes in free calcium ion
concentration affect coagulation test results
COLLECTION SITE
•Blood collected from different site differ in composition.

•Venous blood PCO2 is 6 to 7 mm Hg higher than arterial blood.

•Blood from skin puncture is to some extent contaminated with

interstitial fluid
• leading to eg increase in glucose and potasium levels compared to venous
blood
Influence of collection site on
composition of plasma
ARTERIAL CENTRAL PERIPHERY
VENOUS VENOUS

Alanine Aminotransferase U/L 62 61 81

Alkaline Phosphatase U/L 114 113 107

Amylase u/l 149 148 177

Calcium mg/l 81 82 83

Chloride mmol/l 99 97 101

Sodium mmol/l 144 145 144

Urea nitrogen mg/l 320 310 250

Pottasium mmol/l 4.0 3.9 3.8

HAEMOLYSIS
• It’s the disruption of RBC membrane

• Resulting in release of hemoglobin and other cellular component.

• Visual evidence of haemolysis in serum occur when Hb exceed 200 mg/l.

• Severe haemolysis causes :

• slight dilution effect on the constituent present at a lower concentration in RBC
than plasma

• Notable effects in the constituent present at a higher concentration RBC than

plasma e.g potassium, magnesium, LDH
URINE
•The type of urine specimen to be collected depend on the test to be
performed.
•Specimen are:
• Random[few chemical test]
• Timed interval [specific time of day or certain phase of macturation]

•Eg bacteriolology the early phase test for urethritis while mid stream test
for cystitis
•Patients genitalia should be cleaned before specimen collection to
minimise bacteria contamination
TIMED URINE
COLLECTION IN ADULTS
•The bladder is first emptied and this urine discarded.

•There after urine is collected until the scheduled time.

•Incase of bowel movement precaution is taken to prevent contamination

•Urine should be passed into a separate container at each voiding and

emptied into larger container

•This prevents the patient from splashing with preservative eg acid and
storage of container at [4°c]
COLLECTION IN
CHILDREN
•For untimed urine:
• The child’s scrotal/perineum are cleaned
• A plastic bag is placed around the genitalia
• Until urine as been voided

•For timed urine a metabolic bed is used;

• The infant lies on a fine screen
• The screen has a funnel shaped base which drain into a container
• The screen retains fecal matter.
• Therefore the Urine to some extent is contaminated
URINE PRESERVATION
•Most satisfactory form of Preservation is refrigeration
•Other form is adding preservatives whose role include to ;
• Reduce bacteria action
• Reduce chemical decomposition
• Solubilize constituents that may precipitate out of solution

•Acidification to below PH 3 is used in 24 hr specimen for

calcium,VMA,and steroid tests
•Bases NAOH and NA2CO3 are used to preserve spacemen for
porphrins,urobilinogen and uric acid testing
COMMONLY USED URINE
PRESERVATIVES
PRESERVATIVE CONCENTRATION/VOLUME
HCL 6mol/l:30ml per 24-hour collection

Acetic acid 50%: 25 ml per 24 hour collection

NA2CO3 5g per 24 hr collection

HNO3 6mmo/l;15 ml per 24 hr collection

Boric Acid 10g per 24hr collection

STOOL
Tested for :
a) Micro organisms [diarrhea]
b) Heme[bleeding ulcer or gut malignancy]
c) Tryptic activity in children to detect cystic fibrosis
d) Excretion of nitrogen and fat [malabsorption]
e) Occasional porphyria in some types of porphyria

No preservative added.
Should be kept in a refridgerator.
CSF
Spinal fluid is examined incase of:
 CVA
 Meningitis
 Demyelinating disease
 Menigeal involvement in malignancy

Specimen collection:
Initial specimen may be contaminated by tissue debri hence the first tube
used for chemical
Second for microbiology
Third microscopy and cytology examination
SPECIMEN HANDLING
Involves:
◦ Proper labeling where minimal requirements should include: name,
identification number, date and time collected
◦ Separation and storage of specimen

◦ Transport of specimen
SEPARATION AND
STORAGE
Plasma and serum should be separated optimally within 2 hrs.

Premature separation of serum may permit continued formation of fibrin

which may obstruct sample probes in testing equipments.

For labile analytes eg hormones the plasma and serum should be frozen
immediately after separation.
TRANSPORT OF
SPECIMEN
•Primary containers should be constructed to prevent loss of content
when exposed to heat, cold or sunlight.

•Secondary container must prevent primary container from damage.

•For frozen or refrigerated specimen an insulated container is used.

PRE-ANAYLTICAL VARIABLES
AFFECTING SPECIMEN
COMPOSITION
CONTROLLABLE NON-CONTROLLABLE

Posture Biological factors: Age Sex race

Exercise Environmental factors: altitude,
Diet ambient temperature, geographical
Malnutrition location
Time of food ingestion Underlying Medical conditions:
obesity, blindness
Travel
Smoking Pregnancy
Alcohol Menstrual cycle
Drugs
QUESTIONS?