Hematology Lab-1
Hematology Lab-1
Hematology Lab-1
Phlebotomy
Introduction
Blood collection for hematological studies can be performed via venipuncture (blood collected
from Vein) or by capillary puncture (blood collected from the finger, heel, earlobe, or toe).
There are many preanalytical steps to follow in the process of blood collection such as patient
identification, requisition verification of laboratory orders and biohazard safety, to name a few.
these items as well as venipuncture of laboratory order capillary puncture methodology will be
discussed in this sheet.
Collection of blood sample by Venipuncture: It is one of the most critical phases of obtaining
accurate laboratory test result.
Equipment
1. Syringes:
In the past syringes were the only practical way blood could be collected. They were made of:
A. Glass: and after each use, they were placed in paper bags or cloth wraps and sterilized (by
boiling) for reuse. These have been replaced by:
B. Plastic: disposable syringes
C. Evacuated System: tubes and needle holders
These are best for patients with small veins, veins that easily collapse (when exposed to the
pressure in the evacuated tubes)
They may also be the best choice when other collecting devices are used, such as Butterfly
infusion sets and intravenous infusion sets.
Two advantages for syringes use:
1. Blood usually appear in the hub of the syringe as a vein is penetrated, which is helpful for the
phlebotomist and when collecting blood from arms with fragile, deep or mobile veins.
2.Blood –flow rate and withdrawal pressure is easily controlled by the phlebotomist, so that
collapse of thin walled veins can be avoided. Large amounts of blood (up to 60 mL) can be
withdrawn using a small diameter needle and syringe.
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II. Evacuated System
The most common blood collecting system use:
1.A disposable needle. 2.A needle holder .3. An evacuated tube
The needle screw into the holder and each end of the needle is pointed;
one to enter the vein; the other is to puncture a collecting tube. The vein
is entered first, and then any number of collecting tubes can be added to
the other end. Each tube is punctured through the rubber cap and blood
flows into it due to the partial vacuum. (gradually loss of vacuum from
the tubes that have been stored on shelf a few years).
Collecting tubes are available in many sizes and the tube walls are made of soda lime glass or
borosilicate glass. Some tubes also employ a special material that separates serum from the blood
clot. Many tubes are coated with silicon to minimize clotting and hemolysis. The collecting tubes
may contain additives such as anticoagulant and preservatives ( will mention in the next section
), which are usually denoted by a stopper color code. Draw blood collection tubes in the correct
order, to avoid cross-contamination of additives between tubes The Clinical and Laboratory
Standards Institute recommends the following sequence of collection of evacuated tubes in a
multi-draw should be in this order:
1. Sterile/Blood cultures
2. Light blue-top tube (sodium
citrate): Tube contains 3.2% sodium
citrate as an anticoagulant. This tube
is used for preparing citrated plasma
for coagulation studies (1:9 ratio).
Other tubes contain 3.8% sodium
citrate (1.4 ratio) with black top tube
is used for ESR test.
Note: Tube should be inverted several
times immediately after blood
collection to prevent coagulation. It is
also important to completely fill the
tube with blood, since the ratio of
anticoagulant to blood is critical for
certain coagulation studies.
3. Red-top tube: Tube does not
contain an anticoagulant. This tube is
used for collecting serum or clotted
whole blood specimens.
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4. Other additives
a. Green: Green-top tube (sodium heparin): Tube contains sodium heparin as an anticoagulant.
This tube is used for preparing heparinized plasma, whole blood, and bone marrow specimens.
Note: Tube should be inverted several times immediately after blood collection to prevent
coagulation.
b. Lavender-top tube (EDTA): Tube contains EDTA as an anticoagulant. This tube is used for
preparing EDTA plasma, whole blood, and bone marrow specimens.
Note: Tube should be inverted several times immediately after blood collection to prevent
coagulation
c. Gray: Sodium fluoride and potassium oxalate, inhibits Red cell glycolytic pathway and used
for Glucose test.
Correct order of tube draw is important in order to obtain the best possible specimen. The blue
tube must be drawn BEFORE the green heparinized tube. If the green tube is drawn or filled
prior to the blue tube, there may be a chance of heparin contamination in the blue tube. More
details about Recommended order of draw for plastic vacuum tubes are attached in Annex 1
Advantages of the Evacuated System:
1. One needle holder can replace many bulky syringes saving space on Phlebotomy trays.
2. A number of collecting tubes with various additives can be used sequentially (some patients
experience anxiety at the sight of a 30 mL syringe being filled with their blood whereas 5-6 tubes
may not seem as threatening).
3. Blood enters each tube and mixes rapidly with a specific anticoagulant, whereas blood in a
syringe must either forgo anticoagulant until the phlebotomy is complete, or the syringe must be
coated with anticoagulant, thus limiting the variety of additives used.
4. Blood collection is faster with the evacuated tubes than with the syringes especially if several
tubes are to be filled.
III. Anticoagulants
Coagulation of blood requires about 15 clotting factors, most of them are proteins which are
activated into enzymes when injury occurs: the last steps for clot formation:
Prothrombin + Prothrombinase Thrombin
(Ca + Plasma thromboplastin)
Fibrinogen + Thrombin Fibrin (Clot)
Most procedures used in hematology require whole blood or plasma. Anticoagulants prevent
blood clotting by enhancing the action of natural inhibitors (as antithrombin, by Heparin) or by
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removing (chelating) Calcium (most other anticoagulants). Selection of the anticoagulant
depends on the requirements of the procedure. Some used in hematology includes the following:
1.Heparin: inactivates FX and FII (thrombin).
Available as Sodium, Potassium and Ammonium salts, it inhibits Thrombin action. About 20
units of heparin are required to anticoagulated 1 mL of blood.
Advantages :1. minimum interference in most chemical tests .2. Available as a liquid or a
powder. 3. can be used in Phosphorous test.
Disadvantages: 1. Relatively expensive. 2. Produces blue background on Wright –Stained smear
.3. Inhibits acid Phosphatase activity .4. Causes clumping of WBCs & Platelets.
2. EDTA (ethylene diaminetetra acetic acid) (sequestrene, Versene):
It is used as a disodium or dipotassium salt (Na2and K2 EDTA) that prevent coagulation by
chelating or binding Calcium in the plasma. Calcium is required in many steps of the coagulation
process. About 1-2 mg of EDTA is needed for each mL of blood, it could be powder or solution.
EDTA is the most common anticoagulant used in routine hematological studies.
Advantages: a. Prevents Platelet clumping.
b. Preserves cellular morphology.
c. Prevents artifacts in preparing blood films.
d. Little effect on chemistry tests
e. EDTA blood stored at 4c for 24hrs. If tested again show no changes in results compared to the
previous results.
Disadvantages: a. Inhibits Alkaline phosphatase
b. Not useful for Calcium and Iron testing
c. If in excess: shrink RBCs, so decrease Hematocrit, False ESR results and MCHC.,
Degenerates WBCs & platelets so increase platelet count.
3.Sodium Citrate: this is the anticoagulant of choice for the coagulation studies. A
concentration of 3.2% is used, mixing 1 part of Na citrate with 9 parts of blood for coagulation
tests and3.8% is used, mixing 1 part of Na citrate with 4 parts of blood for ESR test. Calcium is
chelated, which is easily reversed by the addition of ionized Ca since it is required to clot the
plasma in coagulation tests.
Advantages: Maintain stability of coagulation factors (V&VIII & platelets)
Disadvantages: a. Not suitable for many chemistry tests. b. Inhibits Alkaline Phosphatase. c.
Cause distortion of cells morphology.
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Anticoagulants are already in the tubes in the precise amount needed to mix with the amount of
blood that will fill the tube. The color of the stopper on each tube indicates what, if any,
anticoagulant the tube contains as we mentioned before. It is important to completely fill each
tube so that the proportion of blood to chemical additive is correct, otherwise, the test results
may not be accurate or the specimen will be rejected and will need to be recollected.
1. Lavender, Green or Gray (EDTA, Heparin or Potassium Oxalate): Manufacturer recommends
that tubes be filled to 1/2 the maximum capacity. Test results may be skewed if tubes are over or
underfilled.
*Underfilling: Decreased test results may occur due to the dilution effect of the anticoagulant.
*Overfilling: May yield a sample which contains small microclots. This may decrease test
results or cause damage to instrumentation, if the clot is aspirated into the machine.
2. Blue (Sodium Citrate): Manufacturer’s recommend that tubes must be filled to capacity
because test methodology requires a strict 1:9 anticoagulant blood ratio in order to achieve
accurate test results.
*Underfilling: Prolonged coagulation results due to a decrease in the anticoagulant: blood ratio.
The excess unbound anticoagulant spills over into the patient plasma prolonging coagulation
results. This produces a falsely elevated result.
*Overfilling: Small microclots may occur in the sample, indicating that coagulation has begun to
occur in tube. This also leads to prolonged coagulation results because the coagulation factors
being tested were already being “used up” in the tube. (The fewer coagulation factors present in
the plasma- the longer the coagulation time.)
IV. Needles
The Gauge (diameter) and Length of the needle used on a
syringe or evacuated system depends on:
1.Amount of blood to be drawn.
2.Type of analysis to be performed
3.Condition of potential phlebotomy sites
Its gauge number gives the bore size, or diameter of a
needle. The smaller the number, the greater the diameter, For ex:
G24&G26 indicates small needle Lumen(diameter) while, G16&G18
indicates needles with large lumen.
Most blood samples are collected with needles of 19-23G , .Patients
with very small veins or collapsible veins may require a small needle
used with syringes . On the other hand , blood donors providing a
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unit (450mL )of blood or plasma are usually drawn with a 16 G needle . Collection of large
single sample volume can be facilitated with a butterfly set.
This consists of a needle and a long flexible tube attached
to a syringe. The tube can be taped to the patient ‘s arm to
withdraw a large sample over a larger interval of time
than would occur with an evacuated system or syringe
alone.
This may be necessary in elderly or critical ill patients
with delicate or badly scarred veins. Anticoagulants are
sometimes used in the tubing or syringe to ensure
adequate blood flow and prevent clotting if the sample
collection is prolonged.
The length of the needle is usually either 1inch →11/2
inches. The choice of length depends on the characteristics of the phlebotomy site. (depth of
veins, scarred tissues) and the phlebotomy’s preference. All needles in routine use today are
disposable and sterile, so that blunt, barbed or contaminated needles should not be a factor of
error. Visual examination of the tip prior to venipuncture is a good practice.
When an evacuated system is used, tubes for collection should be in the order: 1. Sterile blood
culture.2. Tubes without additives (serum) .3. Na citrate or Heparinized tubes. 4.Tubes with
other additives (EDTA, Fluoride)
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Phlebotomy Protocol:
Venipuncture
Guidelines that form the basis for an acceptable blood collection
1.Assemble the required supplies
All items needed during the procedure, including a few spares should be close at hand.
For a routine venipuncture, supplies include:
a. Tourniquet: several types are available; a blood pressure cuff may also be used.
b. Alcohol preps or swabs.
c. Dry gauze pads, to dry alcohol and place over the site after the needle is removed.
d. Collection tubes, as needed, with appropriate additives for requested tests.
e. Needles
f. Adhesive bandages
g. Puncture-resistant sharps container for used needles
h. On top of these, there should be laboratory forms, specimen labels and pens that you
will need.
i.
2. Identify the patient: Now that you have the tools you need, it’s time to deal with the patient.
Greet the patient with a friendly, professional manner. Patient cooperation is essential for
successful results. The tone of the patient encounter is set within the first 30 seconds of
communication.
The process differs when you are dealing with an adult (conscious) and a child.
When patient is an adult:
Make sure to introduce yourself and have the patient state their full name.
Assure that correct samples are obtained from the intended individual. Test results posted to the
wrong records could have adverse effects on the diagnosis, treatment, and medical condition of a
patient.
• A. If possible: the patient should state his or her name when asked by the phlebotomist.
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• B. The phlebotomist should compare identification data on the test request slip with that
provided by the patient. (bed or door labels and wrist identification bands for in patients).
• C. Compare request slip data with data of labels to be placed on the collecting tubes.
When patient is a child or a baby:
When patient is child
When a parent or guardian is present, ask the person for the first and last names of the child.
Match the identity of the patient with that of the name, date of birth, and file number written
on the laboratory form.
Before carrying out phlebotomy, technicians must wash their hands with soap and water and then
dry with a single-use towel. An alternative would be to cleanse the hands with 3 ml of alcohol
rub, starting with the palm of the hand, rubbing it into the fingertips and all over the hands until
the alcohol dries out. Put on well-fitting latex.
5. Position the patient: A successful venipuncture is much more probable if the patient is
immobilized and the phlebotomist is comfortable. Seated patients should sit comfortably with
arm extended, below shoulder level on an armrest or supporting surfaces. Placement of their
other hand beneath their elbow may provide added support. A reclining patient should lie on his
or her back with an arm extended either outward (distally) from the body or alongside the body.
A rolled towel or pillow can provide additional support near the elbow.
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6. Select venipuncture site:
• In most cases, blood will be drawn from an arm veins, since these vessels are usually:
Large, close to skin surface, and easy to penetrate.
• On occasion an alternate site must be selected, because of burns, amputation, presence of
a cast or other medical consideration. Alternate sites include the back of the hand, the
ankle, or foot or (jugular &femoral).
• A few special considerations should be noted:
• a. avoids using skin areas marked by excessive scarring or containing large hematomas.
• b. Do not draw from an arm in which an intravenous line is inserted, since dilution of the
blood sample may occur. If the I.V. needle is inserted high in the arm, it is possible to
draw blood a lower site of the same arm. If I.V. therapy is occurring in both arms, a nurse
can usually discontinue fluid administration for few minutes. If Blood is drawn from the
I.V. connector, the 1st 5 mL is discarded to avoid I.V. fluid contamination and the sample
is drawn as usual. The phlebotomist should note this in case discrepancies arise when
sequential test results are compared.
• C. When a patient has had a mastectomy, don’t obtain blood from the arm that is on the
same side as the breast that has been surgically removed.
8. Select a vein:
Most venipunctures are made in an arm vein at, or just
below the bend of the elbow. The Medial Cubital vein
is most often used since it is large, superficial, and lies
in a site that is relatively insensitive to pain. The
Cephalic and Basilic veins may also be used, but they
are more likely to roll and bruise.
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During this inspection, the choices of syringes versus evacuated system and needle size are
made.
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• If Syringes are used: One hand grasps the barrel and the other hand is free to pull back
the plunger. If the needle is properly located in the vein, blood should enter the hub of the
syringe. Pull back slowly on the plunger until the desired volume of blood has been
obtained.
lf there is no blood flow:
a.) Needle is incorrectly positioned; reposition by withdrawing slightly and trying another
area. Index finger may be used to feel for position of needle relative to vein.
b.) Tube has no vacuum; replace with another.
If draw is successful, allow tube to fill completely. All tubes with additives must be at
least half-full to ensure accurate test results.
If multiple tubes are being drawn, carefully remove first tube after blood flow stops and
replace with second tube according to Order of Draw. Perform in such a manner so
needle remains in correct position. If tube has an additive, remove and gently mix
immediately.
If draw is unsuccessful, another vein may be palpated and used. Do not attempt to draw
patient more than twice. Arrange for a different staff member to attempt third draw with
patient's consent or arrange for a next day draw after oral hydration. Inform the ordering
clinician if the phlebotomy was not successful.
11. Draw samples in the correct order
Draw blood collection tubes in the correct order, to avoid cross-contamination of additives
between tubes. As color coding and tube additives may vary, verify recommendations with local
laboratories. For illustration purposes, Table 2(Annex2 ) shows the revised, simplified
recommended order of draw for vacuum tubes or syringe and needle, based on United States
National Committee Clinical Laboratory Standards consensus in 2003
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• Sample tube processing:
• While the patient applies pressure to the puncture site, the phlebotomist should tilt tubes
containing anticoagulants to ensure mixing, label all sample tubes with information
appropriate to the collecting and processing facility. In most hospitals a label might
consist of:
• Patient’s full name, hospital case or record number, room or ward number, date & time
sample was drawn, initials or code number of the phlebotomist.
• Assess patient's state. Make sure that s/he is all right before allowing patient to stand and
leave. THANK THE PAIENT.
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Important Note: Never shake the blood vigorously, but invert them so that additives will be
mixed. For most additives, inverting 4 times will suffice, while others require 8 to 10 times, or as
specified by the laboratory. Afterwards, prepare the samples for testing or transportation, and
clean up any spills of blood or other body fluids.
Capillary Puncture
• A skin puncture of the finger, heel, ear lobe or toe(>6months) may be used to provide a
small amount of blood when required or when a venipuncture is too difficult or
impractical. Can be applied on children finger stick or newborn infants (heel stick <1yr).
Devices used include:
• 1. Sterile disposable lancet.
• 2. Spring activated devices, which have a lancet held in place by a cocking lever. When
the blade is released, it penetrates the skin to depth of 2 mm.
• (N.B.: jugular, femoral and umbilical cord blood collection is performed by a physician.)
• Procedure:
• 1. Select a puncture site: 2nd, 3rd, or 4th finger. Finger stick. Avoid the tip of the finger
near the nail (less thickness in tissue than in the center. Do not use old puncture sites or
areas with obvious calluses.
• 2. If necessary warm the puncture site > If exposed to cold or impaired circulation, poor
capillary blood flow, hold or rub the patient’s hand.
• 3.Cleanse the puncture site area with isopropanol and dry it with gauze.
• 4.Grasp the finger firmly but not too tightly with one hand and use the other hand to hold
the lancet and perform the venipuncture. Position facing or parallel to the patient.
• 5. Wipe the 1st drop that wells up from the wound with clean gauze, this drop contains a
large amount of tissue fluid that may dilute the blood and interfere with some coagulation
studies.
• 6. Collect subsequent blood drops into the appropriate containers. Blood should flow
freely from the puncture with a minimum amount of pressure on the finger. Gentle
massage of the finger. The site can be wiped from time to time to remove platelet clumps
and facilitate additional blood flow.
• 7. Apply pressure to the puncture site with a clean gauze or cotton ball until bleeding has
stopped.
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Annex 1:
Clot activator (red Clot activator Blood clots, and the Chemistries, immunology
3 top) serum is separated by and serology, blood bank
No anticoagulant
centrifugation (cross-match)
present
No additives
present
6 Sodium heparin Sodium heparin Inactivates thrombin For lithium level use
(dark green top) or lithium and thromboplastin sodium heparin, for
heparin ammonia level use either,
Blood gases , special
hematology tests ,
biochemisyrt electrlytes
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Order Type of Additivec Mode of action Uses
of usea tube/usual colourb
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Annex 2
Do Do not
DO carry out hand hygiene (use soap and water or alcohol DO NOT forget to clean your hands
rub), and wash carefully, including wrists and spaces
between the fingers for at least 30 seconds (follow WHO's
‘My 5 moments for hand hygiene’a)
DO use one pair of non-sterile gloves per procedure or DO NOT use the same pair of
patient gloves for more than one patient
DO NOT wash gloves for reuse
DO use a single-use device for blood sampling and drawing DO NOT use a syringe, needle
or lancet for more than one patient
DO disinfect the skin at the venepuncture site DO NOT touch the puncture site
after disinfecting it
DO discard the used device (a needle and syringe is a single DO NOT leave an unprotected
unit) immediately into a robust sharps container needle lying outside the sharps
container
Where recapping of a needle is unavoidable, DO use the DO NOT recap a needle using both
one-hand scoop technique (see Annex G) hands
DO seal the sharps container with a tamper-proof lid DO NOT overfill or decant a sharps
container
DO place laboratory sample tubes in a sturdy rack before DO NOT inject into a laboratory
injecting into the rubber stopper tube while holding it with the other
hand
DO immediately report any incident or accident linked to a DO NOT delay PEP after exposure
needle or sharp injury, and seek assistance; start PEP as to potentially contaminated
soon as possible, following protocols material; beyond 72 hours, PEP is
NOT effective
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References:
Pendergraph, G.E., Handbook of Phlebotomy, Third Edition, Lea and Febiger, Philadelphia,
1992.
Cilinical hematology and fundamentals of hemostasis ,Third Edition , Denise M .Harmening,
1997,
WHO Guidelines on Drawing Blood: Best Practices in Phlebotomy.
Geneva: World Health Organization; 2010.
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