SPECIAL REPORT

Laboratory Hematology

R e c o m m e n d a t i o n s o f t h e I n t e r n a t i o n a l

C o u n c i l f o r S t a n d a r d i z a t i o n i n H a e m a t o l o g y

f o r E t h y l e n e d i a m i n e t e t r a a c e t i c A c i d

A n t i c o a g u l a t i o n o f B l o o d f o r B l o o d C e l l

C o u n t i n g a n d S i z i n g

INTERNATIONAL COUNCIL FOR STANDARDIZATION IN HAEMATOLOGY:

EXPERT PANEL ON CYTOMETRY*

Of the three ethylenediaminetetraacetic acid (EDTA) salts used for
anticoagulation of blood specimens for hematologic testing, potassium
salts are the most readily soluble. Tripotassium EDTA is dispensed as
a liquid and thus causes a slight dilution of the specimen. This salt also
has been shown to affect the red blood cell size more at increased con-
centrations and on storage than the dipotassium salt. Therefore, dipo-
tassium EDTA is recommended as the anticoagulant of choice in speci-
men collection for blood cell counting and sizing. The amount of dipo-
tassium EDTA used is 1.5-2.2 mg (3.7-5.4 >«mol) per milliliter of
blood. (Key words: Anticoagulant; Blood cell count; EDTA) Am J Clin
Pathol 1993;100:371-372.

Throughout the world, three different salts of the chelating
agent ethylenediaminetetraacetic acid (EDTA; C,0 H16 N2 0 8 )
are being used for hematologic testing: disodium, dipotassium,
and tripotassium EDTA. The potassium salts have an advan-
tage over the sodium salt in that they are more readily soluble
in the blood when present in the dry form in the collection
container; however, all three salts have been shown to affect red
blood cell size, especially after storage of anticoagulated speci-
mens for a number of hours.
Automated blood cell counters are calibrated by reference
methods.' For cell sizing, this requires the measurement of the
packed cell volume (PCV) of fresh blood samples so that values
can be assigned to materials provided by the manufacturer.2
This implies that calibration of automated blood cell counters
will depend on the anticoagulant used. Thus, for harmoniza-
tion between counters, it is essential that the same anticoagu-
lants are used.
Studies have been performed in four laboratories to docu-
ment differences between the three EDTA salts on the apparent
PCV with the microcapillary ("microhematocrit") method and
several hematology analyzers.3 Of the three salts tested,
KjEDTA proved the least suitable because it causes the largest
* Members: J. M. England (Chairman), R. M. Rowan (Secretary),
O. W. van Assendelft, B. S. Bull, W. Coulter, K. Fujimoto, W. Groner, spun immediately after collection (n = 4), K2EDTA appeared
A. Richardson-Jones, G. Klee, J. A. Koepke, S. M. Lewis, C. E.
McLaren, N. K. Shinton, N. Tatsumi, and R. L. Verwilghen.
Prepared for the panel by O. W. van Assendelft and S. M. Lewis.
Revised manuscript accepted for publication December 21,1992.
Address reprint requests to Dr. van Assendelft, NCID 1/2302, MS
D-17, Centers for Disease Control and Prevention, Public Health Ser-
vice, Department of Health and Human Services, 1600 Clifton Road,
Atlanta, GA 30333.
red blood cell shrinkage with increasing EDTA concentration
(11% shrinkage at 7.5 mg/mL blood), the largest change in cell
volume on standing (+1.6% after four hours), and lower mean
cell volume (MCV) values (-0.1 % to — 1.3%, depending on the
type of instrument). In addition, K3EDTA is dispensed as a
liquid, causing all directly measured values (ie, hemoglobin
concentration, red and white blood cell counts, and platelet
count) to be 1-2% lower than the circulating blood values be-
cause of specimen dilution. Finally, a decreased white blood
cell count obtained with an automated blood cell counter has
been reported for blood anticoagulated with K3EDTA in high
concentrations.4
With regard to the choice between the dipotassium and diso-
dium salt of EDTA for standardization purposes, the following
were observed: Compared with PCV measurements on
K2EDTA-anticoagulated specimens, Na2EDTA-anticoagu-
lated specimens consistently gave slightly lower values (n = 40,
PCV range, 0.30-0.46; average, -0.0005; range, -0.0001 to
-0.001); compared with MCV measurements (n = 116),
Na2EDTA-anticoagulated specimens consistently gave slightly
higher values (average, +0.2 fL; range, +0.1 to +0.4 fL).3 Com-
pared with PCV measurements on defibrinated blood (n = 4,
ten individual measurements on each specimen), K2EDTA ap-
peared to cause slight shrinkage of the red blood cells (—0.005)
and Na2EDTA a marginal swelling (+0.001); compared with
PCV measurements on whole blood without anticoagulation,
to cause marginal (-0.003) shrinkage and Na2EDTA slight
(—0.005) shrinkage of the red blood cells.3 On standing for four
hours at ambient temperature, 20-24 °C, K2EDTA and
Na2EDTA appeared to cause approximately the same degree of
swelling of the red blood cells (+0.005 and +0.004, respec-
tively)3; however, when the greater solubility of K2EDTA
(1,650 g/L v.?. 100 g/L for Na2EDTA)5 and the widespread use

371

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 372 SPECIAL REPORT
Laboratory Hematology
of the potassium salt (eg, in Europe, Japan) are taken into ac-
count, the International Council for Standardization in Hae-
matology recommends the dipotassium salt as anticoagulant in
specimen collection for blood cell counting and sizing, espe-
cially when PCV values are required for calibration of auto-
mated blood cell counters.
K2EDTA.2H20, an odorless white crystalline powder, has a
relative molecular mass of 404.4; 1 g (2.4 mmol) will chelate
100 mg (2.4 mmol) of calcium ion.5 The pH of a 1% (weight/
volume) K2EDTA solution is 4.8 ± 1.0.
The amount of dipotassium EDTA, dihydrate, added to
blood should be 1.5-2.2 mg/mL (4.55 ± 0.85 ^mol/mL). This
amount is a compromise between the amount required to pro-
duce anticoagulation and the amount at which cellular changes
are produced.
Tubes containing blood specimens must have an air bubble
constituting at least 20% of the tube volume. The tube must be
inverted completely several times to ensure adequate mixing of
blood and anticoagulant.
With respect to the white blood cell morphologic characteris-
tics of EDTA-anticoagulated blood on storage at ambient (20-
24 °C) temperatures, a slight vacuolization of monocytes was
found after one hour, progressing to moderate after four hours;
a slight vacuolization of neutrophilic granulocytes was found
after three to four hours, progressing to moderate after six
hours.6 Only minimal changes in the white blood cell morpho-
logic characteristics have been reported on storage at 4 °C for as
long as 12 hours.7
A.J.C.P. • October 1993
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Storage of EDTA-anticoagulated blood for as long as 6 hours
is acceptable; extended storage (24 hours) is not recommended
because the stability of the specimens can vary, depending on
the reagents, system, and instrumentation used in testing.5
REFERENCES
1. Lewis SM, England JM, Rowan RM. Current concerns in haema-
tology: 3. Blood count calibration. J Clin Pathol 1991;44:881-
884.
2. International Committee for Standardization in Haematology:
Expert Panel on Cytometry. The assignment of values to fresh
blood used for calibrating automated blood cell counters. Clin
Lab Haematol 1988; 10:203-212.
3. Koepke JA, van Assendelft OW, Bull BS, Richardson-Jones A.
Standardization of EDTA anticoagulation for blood counting
procedures. Labmedica 1988/89,5(6): 15-17.
4. Goossens W, van Duppen V, Verwilghen RL. K2- or K3-EDTA:
The anticoagulant of choice in routine haematology? Clin Lab
Haematol 1991;13:291-295.
5. National Committee for Clinical Laboratory Standards. Additives
to Blood Collection Devices: EDTA. Tentative Standard.
NCCLS document H35-T (ISBN 1-56238-162-8). Villanova,
PA: National Committee for Clinical Laboratory Standards,
1992.
6. van Assendelft OW, Parvin RM. Specimen collection, handling
and storage. In: Lewis SM, Verwilghen RL, eds. Quality Assur-
ance in Haematology. London: Bailliere Tindall, 1988, pp 5-32.
7. Lloyd E. The deterioration of leukocyte morphology with time: Its
effect on the differential count. Lab Perspect 1982; 1(1): 13-16.