‫نقابة المهن العلمية بالمنوفية‬ [1]

lood ampling
The vascular system

The vascular system is like a road system from aorta to the vena cava

Artery structure

The arteries walls are thicker than veins as they need to be able to
expand as the blood is pumped through them.

✓ All arteries except for the pulmonary artery carry oxygenated blood.

✓ Normal systemic arterial blood is bright red because it is oxygen rich.

Vein structure

Blood is kept moving through veins by skeletal muscle movement and the opening and closing of valves that line their inner
walls

✓ All veins except for the pulmonary veins contain deoxygenated blood

✓ Normal systemic venous blood is dark bluish red because it is oxygen poor

Capillaries structure

Capillary walls are one cell thick

✓ This enables exchanges of oxygen, carbon dioxide, and nutrients between blood and the surrounding body tissues.

✓ Capillary blood contains arterial and venous blood plus tissue fluid but because it enters capillaries under pressure,
the arterial portion is highest.
 ‫نقابة المهن العلمية بالمنوفية‬ [2]

Blood specimens

Whole blood

Contain both cells and plasma, like blood in the body. It must be collected in an anticoagulant tube to keep it from clotting

Anticoagulant is the substances that prevent coagulation of blood and it is used to prepare a plasma sample in laboratory.

Complete mixing of the blood immediately following venipuncture is necessary to ensure the anticoagulant can adequately
inhibit the blood’s clotting factors.

Plasma sample

It is normally a clear, pale yellow fluid that separates from the cells when blood in an anticoagulant tube is centrifuged

For plasma samples, blood should be centrifuged as soon as possible (within 1 hour after collection) and separated immediately
after centrifuging then plasma transferred to a clean tube

Serum sample

It is normally a clear, pale yellow fluid separated from clotted blood by centrifugation

The blood samples are allowed to completely clot at 37 °C for about 20 minutes (to prevent latent fibrin formation, which may
cause undesirable clogging of automated chemistry analyzers) then blood samples centrifuged and serum should be separated
from clot and transferred to a clean tube

Stability studies have shown that clinically significant analyte changes occur if serum or plasma remains in prolonged contac t
with blood cells.

Loosening the clot by “trimming” or“ringing” the tube may cause some hemolysis and should be avoided.

Serum Vs Plasma

▪ In clinical chemistry, serum is most commonly the specimen of choice, owing to its simplicity in collection and handling.
Additionally, interference from anticoagulants is obviated.
▪ The major difference between plasma and serum is that serum does not contain fibrinogen (used in clot formation)
▪ Usually, a greater volume of plasma than serum is obtained from a given volume of whole blood owing to the clot formation
process.
▪ After separation from blood cells, analytes have the same stability in plasma and serum when stored under the same conditions .
 ‫نقابة المهن العلمية بالمنوفية‬ [3]

Blood coagulation

The intrinsic pathway responds to spontaneous, internal damage of the vascular endothelium whereas the extrinsic pathway beco mes
activated secondary to external trauma. Both intrinsic and extrinsic pathways meet at a shared point to continue coagulation, the
common pathway.

▪ Clotting factors involved in the intrinsic pathway include factors XII, XI, IX, and VIII.
▪ Clotting factors involved in the extrinsic pathway include factors VII, and III.
▪ The common pathway includes clotting factors X, V, II, I, and XIII.

Clotting factor IV is a calcium ion that plays an important role in all 3 pathways.
 ‫نقابة المهن العلمية بالمنوفية‬ [4]

Laboratory anticoagulants

EDTA (Ethylene diamine tetraacetic acid)

use 1.8 mg EDTA per milliliter of blood or use 25-30 l for each 1ml
blood.

EDTA is a chelating agent which can sequester metal ions

including ferric ions and calcium ions in a mixture, diminishing their reactivity.
Therefore, the main function of K2 EDTA in the blood specimen is to chelate calcium
ions from the mixture. Furthermore, the process of blood coagulation requires calcium
ions. But, the addition of EDTA to the specimen makes the calcium ions unavailable for this
process, arresting coagulation.

Must be used in tests that need whole blood such as CBC, blood group, and
Rh

It is the preferred anticoagulant for CBC because cellular elements are preserved well
and it prevent platelets clumping

Of course, EDTA cannot be used for coagulation tests or serum electrolytes

K2EDTA
Dipotassium EDTA, is spray-dried to the walls of the tube
After collection the tube should be inverted several times to mix
the blood and anticoagulant
K2EDTA will not dilute the sample
K3EDTA
Tripotassium EDTA, is in a liquid form
This enable easier mixing of blood and anticoagulant
K3EDTA will dilute the sample ~ 1-2%.

K2EDTA is recommended by the CLSI (Clinical & Laboratory Standards Institute) and the ICSH (International Council for
Standardization in Hematology) as the anticoagulant due to these reasons:

▪ The increasing EDTA concentration in K3 EDTA results in increasing the shrinkage of red blood cells (11% shrinkage with
7.5 mg/ml blood). and
▪ Upon standing, K3 EDTA increases the cell volume (1.6% increase after 4 hours).
▪ Another drawback of K3 EDTA is that it results in the dilution of the blood specimen. It lowers most of the measurements
including RBC, WBC, platelet, and Hgb counts by 1-2%.
 ‫نقابة المهن العلمية بالمنوفية‬ [5]

Sodium citrate

Dissolve 3.2 gram in 100 ml DW (3.2% solution)

It converts ionized calcium into unionized soluble

complex but not as strongly as EDTA.

Its action is reversible so it can be used in coagulation studies (calcium ion is

added to the test during PT determination)

The most popular tests use citrate is:

▪ ESR test (400 l citrate + 1.6 ml blood)

▪ PT & PTT test (200 l citrate + 1.8 ml blood)

Sodium fluoride

It can be used as a powder 2.5 mg/ml blood

▪ Anticoagulant action: Sodium fluoride prevents coagulation through the formation of a weakly dissociated Ca complex. It is often
combined with potassium oxalate anticoagulant (NaF/KO).
▪ Antiglycolytic action: Sodium fluoride also prevents glycolysis, which can decrease glucose concentration by up to 10 mg/dl per
hour. Sodium fluoride preserves glucose for up to 3 days by forming an ionic complex with Mg++ , thereby inhibiting the Mg + +
dependent enzyme, enolase of the glycolytic pathway.

Mainly used for glucose determination

High concentrations of sodium citrate and sodium fluoride must be avoided; citrate and fluoride inhibit urease so urea
determination cannot be done

Oxalate anticoagulants may shrink red cells; thus, blood anticoagulated with oxalate cannot be used to measure hematocrit.

Heparin

Heparin is available as lithium heparin (LiHep) and sodium heparin

(NaHep) and can be used in concentration of 10-50 U/ml /5ml blood

It facilitates the action of anti-thrombin III which is a

protease that inhibit active factors (IX, X, XI, XII) so inhibit conversion of
fibrinogen to fibrin and Inhibits thrombin formation.
 ‫نقابة المهن العلمية بالمنوفية‬ [6]

▪ Some samples must be collected on heparin such as blood gases.

▪ Heparinized plasma is preferred for potassium measurements to

avoid an elevation due to the release of potassium from platelets as
the blood clots

Heparin is not recommended for CBC as it cause platelets

aggregation, WBCs clumping and bluish background of blood film

Vein puncture (phlebotomy)

Equipment preparation

▪ Read the patient request.

▪ Estimate the blood volume to take.
▪ Prepare equipment for blood sampling (Tourniquet, Alcohol, Cotton, Adhesive
strip, Sterile disposable syringes)
▪ Prepare the suitable tubes for each test.

Tube stopper Anticoagulant Used for

Grey tube Potassium oxalate/Sodium fluoride Blood glucose
Black tube Sodium citrate ESR test
Blue tube Sodium citrate PT and PTT test
Red tube Plain containing no anticoagulant Serum samples
Lavender tube EDTA for hematological procedures
Green tube Sodium heparin heparinized plasma as blood gas
Contains a clot activator (Such as silica that activates factor XII) and serum gel separator
Yellow tube (Thixotropic gel), an inert substance that forms a barrier between the blood cells and the
serum that prevents the cells from metabolizing substances in the serum

Never collect blood sample before the preparation of equipment's and tubes
 ‫نقابة المهن العلمية بالمنوفية‬ [7]

Vein selection

▪ The patient should sit comfortable in a chair in a lying down position.

▪ Patient arm extends from shoulder to wrist
▪ Arm should not bend at elbow.
▪ The vein selected should be large, straight that does not roll, and sufficiently
close to the surface to be seen and palpated.

The preferred collection site is the median cubital vein (it has the lowest risk of
damaging nerves in the arm), then the cephalic vein and basilic vein

Dorsal hand veins can be used but it is not suitable in patients with poor
circulation

Avoid arm with: Burn area, edema, hematoma, scaring, recently injected or withdrawn syringe (they do not allow the blood to
flow freely and may make it difficult to obtain an acceptable specimen)

Drawing blood from the arm on the same side as a mastectomy should be avoided, mastectomy inveloves lymph node removal
and can cause lymphostasis, thus making the arm vulnerable for swelling and infection.
 ‫نقابة المهن العلمية بالمنوفية‬ [8]

Blood collection

▪ Check the syringe to be sure that the syringe works smoothly by pressing
the piston.
▪ Ask the patient to make fist this helps make the vein more prominent.

Never allow pumping fists while tourniquet is in place (this increase

potassium and phosphorus)

▪ Apply tourniquet to distend the vein (tourniquet obstructs the venous

return so it helps to distend the vein).

The tourniquet should be applied 3 to 4 inches above the

venipuncture site

The tourniquet should not be placed too tightly (as to stop arterial flow) or left on the patient for more than 2 min.

Prolonged application of the tourniquet results in partial stasis of blood

which leads to hemoconcentration that increase concentration of serum
enzymes, potassium, proteins, and protein-bound substances as calcium.

If it necessary to apply a tourniquet, more than 2 minutes, release it and

reapply again directly before drawing blood

▪ Palpate the vein by left hand. It should rebound (It is important to notice
size of vein, depth of the vein and direction of the vein)
▪ Use 70% alcohol as disinfectant in concentric circle and let it to dry for
30–60 sec to avoid hemolysis and burning sensation.

Never touch the cleaned site with finger again

If it necessary to palpate the site before the venipuncture, disinfect the finger

▪ Fix the vein by drawing skin tight over the vein to prevent it from rolling
▪ Enter by the needle with an angle less than 30 degrees between the needle
and the skin (Bevel Up)

The most common needle size for adult venipuncture is 21 gauge (bore or
opening size) with a length of 1 inch. The advantage of using a 1 -inch
needle is that it provides better control during venipuncture.
 ‫نقابة المهن العلمية بالمنوفية‬ [9]

Insert the needle smoothly and rapidly to minimize discomfort

When the needle enters the vein there is sudden loss of resistance and blood come in the head of needle

▪ Withdraw blood gradually by gently pulling upon the syringe plunger

Vigorous pulling on the plunger of the syringe can collapse the vein
and produce hemolysis of the blood specimen

▪ Remove the tourniquet once the needle has been inserted in the lumen
of the vein before removing the syringe
▪ Place a sterile cotton piece over the point where the needle entered the
skin.
▪ Remove the syringe quickly and use the single-handed technique to
recap it.
▪ Remove the needle from the syringe
▪ Pour the blood gently from the syringe at the wall of the tube
according to the following order:
o Sterile tubes (blood cultures)
o Coagulation tube
o Plain tubes
o Heparin tube
o EDTA tube
o Fluoride tube

Sodium citrate for (ESR) may be after PT tube or after

EDTA tube

▪ Mix well blood with anticoagulant if found gently inverts the tube 8–
10 times to mix the additive with the blood.
▪ Dispose of contaminated materials and needles in special disposal
containers.
▪ Patient press cotton for 2 min to stop bleeding with the arm extended.

Do not forget to label the specimens at the sampling room before sending it to the laboratory
‫نقابة المهن العلمية بالمنوفية‬ ‫]‪[10‬‬
 ‫نقابة المهن العلمية بالمنوفية‬ [11]

Complications of blood sampling

Ecchymosis (Bruise)

Bruising is the most common complication encountered in obtaining a

blood specimen. It is caused by leakage of a small amount of blood in the
tissue around the puncture site.

The phlebotomist can prevent bruising by applying direct

pressure to the venipuncture site with a gauze pad.

Hematoma

A hematoma results when leakage of a large amount of blood around the

puncture site causes the area to rapidly swell. This may be avoided by:

▪ Use the major superficial veins

▪ Do not penetrate the vein from the side
▪ Remove tourniquet before removing the needle
▪ Ask the patient NOT to bend the arm after sampling
▪ Apply pressure by cotton to the venipuncture site

Hematoma will go away, but patient can apply ice and use the
affected arm as little as possible.
 ‫نقابة المهن العلمية بالمنوفية‬ [12]

Hemolysis

It is the destruction of red blood cells which leads to the release of

hemoglobin from within the red blood cells into the blood plasma. The
following may cause blood hemolysis and must be avoided:

▪ Collecting blood from a narrow vein

▪ Using excess EDTA or moisten EDTA
▪ Slow drawing blood from the vein
▪ Forcing blood through the needle into tubes
▪ Using wet tube or stopper
▪ Vigorous shaking of tubes after collection
▪ Holding blood in too warm area

Fainting (Syncope)

Fainting is also a common complication encountered. Before drawing blood, the phlebotomist should always ask the patient whether he
or she has had any prior episodes of fainting during or after blood collection.

The CLSI does not recommend the use of ammonia inhalants to revive the patients because they may trigger an adverse
response that could lead to patient injury.

If the patient begins to faint, the phlebotomist should remove and discard the needle immediately, apply pressure to the site with a
gauze pad, lower the patient’s head, and loosen any constrictive clothing.
 ‫نقابة المهن العلمية بالمنوفية‬ [13]

Nerve damage

The phlebotomist must select the appropriate veins for venipuncture and should not blindly probe the arm with the needle or try to
laterally relocate the needle. If a nerve has been affected, the patient may complain about shooting or sharp pain, tingling, or numbne ss
in the arm.

The phlebotomist should immediately remove and discard the needle, apply pressure with a gauze pad, and collect the
blood from the other arm.

Neonatal heel prick

Purpose and indications

The neonatal heel prick is a common procedure for taking a blood sample from the heel of newborn which is used for a variety of tests
such as bilirubin testing and for newborn screening tests for inherited metabolic disorders.

Blood obtained by skin puncture is also called as capillary blood. It is a mixture of blood from, venules, arterioles and also
contains some tissue fluid

Site of sampling
 ‫نقابة المهن العلمية بالمنوفية‬ [14]

The recommended location for blood collection from a newborn baby is the heel (distal edge of the calcaneal protuberance)

Only lateral parts are used not central part to avoid medial calcanous nerve injury

Heel prick procedure

1. Make sure baby is lying in a safe position with the foot lower than the torso so
gravity can assist blood flow

2. Clean the site to be punctured with 70% isopropanol sponge (recommended

by CLSI)

3. Hold the baby's foot firmly to avoid sudden movement and to apply slight
tension to the skin.

4. Using a sterile blood Heel stick lancet, puncture the heel

5. Wipe away the first drop of blood with a piece of cotton to prevent excess
tissue fluid from contaminating the specimen and rid the site of alcohol
residue that could prevent formation of well-rounded drops and hemolyze
blood.

6. Collect blood sample on a card, using test tube, capillary tube, or automatic
pipette (collect as quickly as possible).

7. When finished place a piece of clean, dry cotton on the puncture site, and hold
it in place until the bleeding has stopped.

• Pre-warming the infant's heel (with a warm, moist towel no hotter than 42°C for 3 to 5 minutes) is important to increases the flow
of blood for collection of specimens
• Letting the site dry naturally permits maximum anti-septic action, prevents contamination caused by wiping, and avoids stinging on
puncture and specimen hemolysis from residual alcohol (which can decrease bilirubin results).
• Heel punctures in infants should not be made more than 2 mm deep because of the risk of bone injury and possible infection
(osteomyelitis)
• Use gentle pressure to to encourage blood flow, however avoid squeezing as tissue fluid dilu tes the collection, and haemolysis may
occur.
• Do not apply a bandage to an infant as it can become a choking hazard and can also tear the skin when removed
‫نقابة المهن العلمية بالمنوفية‬ ‫]‪[15‬‬