— EDTA can be used for hemograms, ESR (Wintrobe’s
method), platelet count, DLC and peripheral smear
FIG. 9.2: Sites for obtaining blood by venipuncture from forearm
¾ Cellular morphology is preserved better, even 2–3 hours
¾ As platelet clumping is prevented, EDTA is a better
anticoagulant for platelet counts
¾ EDTA 2K salt is recommended for CBC, is more water
soluble (1.5 + 0.25 mg/mL of blood).
¾ When in excess, EDTA shrinks RBCs and leukocytes. If
• PCV is significantly reduced
• MCHC is proportionately increased
• Platelets swell and disintegrate, therefore, a fallaciously
high platelet count may be obtained
• It cannot be used for coagulometry applications.
Four (4) grams of disodium or dipotassium salt is added to
100 mL of deionized water. About 0.2 mL of this solution
is added to chemically clean vials, the vials are later kept
in an incubator or hot air oven till complete liquid dries
3–4 mL of blood, as it contains 8 mg of EDTA per vial.
Oxalates act by chelating calcium, and calcium oxalate is
formed as insoluble precipitate. These are used for blood
¾ Potassium oxalate (2–3 mg/mL of blood) but it may
cause shrinkage of cells. Not used anymore.
¾ Double oxalate used for ESR and hematocrit. Potassium
oxalate and ammonium oxalate are used together in a
ratio of 2:3, this is done to counter the swelling effect of
ammonium oxalate and shrinking effect of potassium
• Double oxalates can be used for
– Hemoglobin, TLC, RBC count, ESR by Wintrobe’s method and PCV estimation.
– Leukocytic morphology is not well preserved
and hence not suitable for peripheral smear
– The calcium chelated is precipitated in calcium
oxalate, which is a toxic substance, it is never to
be used for blood banking applications.
Prepare double oxalate solution as follows:
3. Deionized water 100 mL. Mix well, 0.2 mL of the
solution will contain 8 mg of the oxalates, which
prevent clotting of about 3 to 4 mL of blood.
Trisodium citrate is used for ESR and some coagulation
studies. This too acts by chelating calcium. For ESR, ratio is
1:4; while for coagulation studies ratio is 1:9. 1 part of 3.8%
trisodium citrate and 4 or 9 parts of blood respectively.
Heparin (powder or liquid) acts by inhibiting thrombin
and other stages of clotting factor activation.
Special anticoagulants include ACD (acid-citrate
dextrose) used in blood banking and fluoride and oxalate
for sugar estimations. Other blood banking anticoagulants
are also used. Wherever possible, the necessary tests,
investigations and preparation of blood films should be
If this is not possible, refrigerate the sample at 4°C.
Before taking blood from the venous blood containers,
invert them gently several times (60) or else unacceptable
deterioration in precision may ensue.
Anticoagulated Blood Storage and
Peripheral smears (anticoagulated or direct blood used
and stored at 25 + 5°C). Unfixed smears.
¾ Up to 60 minutes: No worthwhile notable change
¾ Up to 3 hours, few changes may be visible
¾ Up to 12–18 hours: Neutrophils are affected
¾ Cytoplasmic borders may appear ragged with small
nuclear disintegration or budding
¾ RBCs do not change for up to 6 hours at room
temperature (25 + 5°C) but longer periods may cause
progressive crenation and sphering.
¾ On storing EDTA blood, the following changes may
• Sedimentation rate gradually decreases
• TLC and platelet counts decrease
• Reticulocyte count decreases within 6 hours
• Hemoglobin remains unchanged if the sample does
¾ Perform all investigations as soon as the blood sample
¾ Never freeze the sample. On storing the sample at 4°C,
the deterioration rate slows down
¾ Perform all counts within 2 hours of blood collection
¾ Excessive EDTA in the sample will significantly lower
¾ Leukocytic degenerative changes will affect automated
¾ A refrigerated sample must always be brought to room
temperature before being used. All samples must
be mixed gently, preferably by rotation, for at least 2
¾ Whatever be the reason for obtaining blood, in the
interest of the patient and your own interest, it is ideal
and necessary to use sterile disposable blood collection
systems, viz. disposable syringes or the Vacutainers.
These are meant for single use and are to be discarded
(never to be used again). Relatively new in our country,
but established all over the world and being used for
decades, is the Vacutainer blood collection system
manufactured by Becton Dickinson (BD). Other makes/
¾ The Vacutainer system consists of a needle, a needle
holder and a glass/plastic vacuum tube instead of the
syringe barrel and plunger. Once the vein is punctured,
the Vacutainer tube appropriately in contact with
the needle, the requisite quantity of blood flows
automatically into the Vacutainer tube so that the need
to pull the plunger out is obviated. Vacutainer is simple
to use, quicker, cleaner and safer. It offers— leakproof
tubes, standardization of specimen quality at high
level, opportunity to rationalize laboratory procedures
and innovative, high technology tubes. Appropriate
anticoagulants, and other additives are preadded
in appropriate quantities so that all that is required
is clean venipuncture. Containers also available for
collecting blood from infants with the help of a skin
puncture, these are called microtainers. The blood
so collected is adequate for micro or dry chemistries.
From a single venipuncture, blood can be collected in
separate vacutainers (for different purposes—EDTA
or oxalate-fluoride or citrate vacutainers) meant
for different purposes and very easily identified by
the color of their caps. The vacutainer system is a
cleaner system, as blood does not come in contact
with atmosphere as it flows straight from the vein
through the sterile needle into the sterile tube. The
process of transferring blood from syringe to different
bulbs is eliminated. Contamination from fallen blood
is entirely removed. The incidence of hemolysis is
significantly reduced because the major cause of it—
the transfer of blood from the syringe to container—
is eliminated. In hematology, because of the instant
contact between blood and anticoagulant minimizes
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