Analogues of Ethylenediaminetetraacetic Acid and Sodium Fluoride ...

Osaka City Medical Journal
Vol. 46. No. I 71-87. 2000
Analogues of Ethylenediaminetetraacetic Acid
and Sodium Fluoride as Anticoagulants
MITSUO NARITA, MASAYUKI HINO, TAKAYUKI TAKUBO
and NORIYUKI TATSUMI
Department of Clinical and Laboratory Medicine,
Osaka City University Medical School
Received 29 March 2000; accepted 20 April 2000
Key Words: EDTA, sodium fluoride, laboratory tests, anticoagulant
Summary
Three analogues of ethylenediaminetetraacetic acid (EDTA), ethyleneglycol­
tetraacetic acid (EGTA), ethylendiaminedipropionic acid (EDDP), and hexamethy­
lenediaminetetraacetic acid (HDTA), and sodium fluoride (NaF) were evaluated as
anticoagulants used for rapid and multi-parametric analysis with minimal amounts of
blood. EDDP and HDTA induced rapid blood coagulation «30 min), while NaF was
not useful for coagulation tests because no coagulation was observed by addition of
PT- or APTT-reagents. EGTA could be used for hematology and coagulation tests,
although platelet count was significantly decreased in comparison with EDTA treated
blood due to platelet aggregation, although prothrombin time was prolonged with
EGTA-treated blood. Platelet aggregation could be preven ted by addition of
kanamycin. In chemistry tests, correlations between serum values and EGTA and
kanamycin treated plasma values were good for all items. Based on these findings, we
concluded that EGTA could be used for hematology, coagulation, and chemistry tests,
although with some limitations.
Introduction
Ethylenediaminetetraacetic acid (EDTA), a potent calcium ion chelator, is a stan-
Correspondence: Noriyuki TATSUMl, MD.
Department of Clinical and Laboratory Medicine, Osaka City University
Medical School 1-4-3 Asahimachi, Abeno, Osaka, 545-8585 Japan
Tel: 81-6-6645-3881; Fax: 81-6-6645-3880
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MITSUO NARITA, et a1.
dard anticoagulant for hematological tests 0-3). EDTA enables stable hematological
testing, but EDTA blood can be used for neither chemistry nor coagulation tests (3, 4).
Thus, EDTA is used internationally only for hematological tests. On the other hand,
laboratory automation and systemization has rapidly advanced in the modern medical
laboratory, enabling rapid and multi-parametric analysis with minimal amounts of
blood (5). With current automated analysis systems, large amounts of blood must be
taken from patients for chemistry, hematology, and coagulation tests since specimens
must be collected in different tubes. If blood can be collected in a single tube for differ­
ent tests, this may reduce the volume of blood required for testing. To identify such an
anticoagulant that can be used for different tests, three EDTA analogues and sodium
fluoride were examined for effects on medical testing.
Materials
Materials and Methods
Venous blood was collected from healthy adult volunteers (n=8) who had given
EDTA: Ethylenediamine-N,N,N',N'-tetraacetic acid
ClOH1SNzOs=292.25
HOOCCHz" / CHzCOOH
HOOCCH/ NCHZCHZN'CHzCOOH
EGTA: O,O'-Bis (2-aminoethyl)ethyleneglycol-N,N,N',N'-tetraacetic acid
C'4Hz4NzO,o=380.35
HOOCCHz CH COOH
"NCHzCHzOCHzCHzOCHzCHzN / z
HOOCCHz / " CHzCOOH
EDDP: Ethylenediamine-N,N'-dipropionic acid,dihydrochloride
CSH16Nz04 . 2HCI=277.15
H H
'NCHzCHzN / • 2HCI
HOOCCHzCH/ 'CHzCHzCOOH
HDTA: 1,6-Hexamethylenediamine-N,N,N',N'-tetraacetic acid
C'4Hz4NzOs=348.35
HOOCCHz, / CHzCOOH
/ NCHzCHzCHzCHzCHzCHzN ,
HOOCCH z CHzCOOH
Fig. 1. Chemical formulas of reagents to be studied.
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EDTA analogues and NaF for anticoagulant use
informed consent. Vacuum blood collection tubes containing EDTA-2K for hemato­
logical tests, tubes containing NaF for blood glucose analysis, and tubes for coagula­
tion tests were purchased from Terumo Co. (Tokyo, Japan).
The EDTA analogues used were EGTA, EDDP, and HDTA purchased from Dojin
Kagaku Co. (Kumamoto, Japan). The same way to make a test tube containing EDTA
was used for making a test tube containing EGTA. Following the manufacturer's in­
struction, EGTA was first solubilized with 1 molll NaOH and the mixture was diluted
to make 100 ml with water by adjusting its pH at 7.2. The solution was put into a test
tube and dried up in an incubator. Other chemicals were put into a test tube as a
powder form. Their chemical formulas are shown in Fig. 1. Powder of these reagents
was weighed and placed in polyethylene terephthalate test tubes. Kanamycin was
purchased from Meiji Pharmaceutical Co. (Tokyo, Japan). The product of kanamycin
is a solution, and aliquots of it were placed in test tubes. The values obtained were
corrected by dilution ratios.
Methods
Hematological tests were performed using a fully-automated hematology
analyzer, the NE-8000 (Sysmex Corp., Kobe, Japan). This analyzer can provide
complete blood counts (CBC) and automated white cell differential counts. Morpho­
logical analysis of white cell differentials was performed with traditional Giemsa­
stained film techniques, and stained smears were observed under a light microscope
(6).
Prothrombin time (PT), activated partial prothrombin time (APTT), and plasma
coagulation tests were performed manually in an incubator adjusted to 36°C (7, 8).
Thromborel S (Behring Diagnostics, Lot No. 505410, Marburg, Germany) and Actin
(Behring Diagnostics, Activated Cephaloplastin Reagent, APAC-639c) were used for
PT and APTT, respectively (7, 8). Platelet-poor plasma for PT and APTT was
obtained from anticoagulated blood using sodium citrate, EGTA and NaF in the usual
manner (9). Citrated plasma was used as a control for coagulation tests.
Chemical analysis was performed in our routine laboratory using a fully­
automated chemistry analyzer, the Hitachi 7450 (Tokyo, Japan). Items tested were
total protein (TP) , albumin, total bilirubin (Bil) , triglyceride (TG), total cholesterol
(Chol) , urea nitrogen (UN), creatinine (Cr), uric acid (UA), Na, K, Cl, aspartate
oxoglutarate aminotransferase (AST, GOT), alanine oxoglutarate aminotransferase
(ALT, GPT) , lactate dehydrogenase (LDH), alkaline phosphate (ALP), leucine
aminopeptidase (LAP), r-glutamyl transpeptidase (rGTP), and choline esterase
(ChE) (10).
All data were presented as means (M) ± standard deviation (SD). Statistical
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MlTSUO NARlTA, et al.
analysis of CBC was performed using paired T test. The relationship between variables
was examined in term of Spearman's rank-order correlation coefficient and by simple
regression analysis. Differences were considered to be significant when p-values were
less than 0.05. Analysis was carried out using the Stat View program (Abacus Con­
cepts, CA).
Results
1. Anticoagulative effects of EGTA, EDDP, HDTA, and NaF
In initial trials, inhibition of coagulation by reagents was examined for up to 24
hrs of storage at different temperatures after mixing of the reagents with fresh blood
in polyethylene terephthalate test tubes. Coagulation was checked by eye by tilting the
test tube. EDTA blood was used as a control. EGTA and NaP each blocked whole
blood coagulation, while EDDP and HDTA did not (Table 1). EGTA blood and NaP
Table 1. Whole blood coagulation tests
Reagents Storage temperature Storage period
Cconcentration) COC)

EDTA analogues and N aF for anticoagulant use
Fig. 5. Effects of EGTA or NaF on blood cell morphology after 30 minutes of
storage (1000 x ).
Morphological analysis of each sample was performed with traditional
Giemsa-stained film techniques. Stained smears were observed under a light
microscope. (A: EDTA, B: EGTA, C: EGTA and kanamycin, a: neutrophil,
b: lymphocyte, c: monocyte, d: eosinophil, and e: basophil.)
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c

MITSUO NARITA, et al.
5. CEC and automated white cell differentials using EGTA with kanamycin
The above findings indicated that EGTA is useful for coagulation and hematologi­
cal tests other than platelet counting. The decrease in platelet count during storage
(Fig. 2) was due to platelet aggregation, as confirmed on blood smears. A trial to
prevent platelet aggregation using kanamycin (8) was therefore performed.
Figures 6 and 7 show time-course changes in CBC and white cell differentials in
EGTA blood, compared with EDTA blood. Kanamycin blocked the decrease in platelet
count (18.6 ± 3.4 X 10,,), and this effect lasted for 24 hours to be observed. Changes in
white blood cell count, hematocrit, and mean corpuscular volume were also smaller
with the addition of kanamycin, compared to those with EGTA alone. Leukocyte dif­
ferentials also appeared more stable with the addition of kanamycin, than with EGTA
alone.
6. Effects of kanamycin on blood cell morphology (Fig. 5, series C)
As the photographs show, no clear effects of the addition of kanamycin (100/11)
were observed on blood cell morphology on Giemsa-stained films. With the addition of
200 /11 kanamycin, the cytoplasm of white blood cells was slightly bluer than that in
EDTA blood. However, these morphological changes did not affect visual cell identifi­
cation. No platelet aggregation was observed for blood smears of the sample with ad­
dition of kanamycin.
7. Coagulation tests (Table 2-B)
Kanamycin (100 and 200 /11/ml blood) clearly prolonged PT and APTT, but PT
could be determined when 0.5 mg of EGTA was mixed with 1 ml blood. Thus, 0.5 mg
of EGTA for 1 ml blood was useful for PT tests when kanamycin (100 /11/ ml blood)
was added, although normal reference values of PT should be reestablished for blood
treated with EGTA and kanamycin.
8. Clinical chemistry tests for specimens with kanamycin (Table 3)
In order to determine the usefulness of EGTA-treated plasma for routine chemistry
tests, specimens were prepared within 1 hour after blood withdrawal and all testing
was completed within 6 hours. Serum specimens were used as controls for this study.
Plasma was obtained from blood treated with EGTA with or without kanamycin. For
most items, there was very close agreement between serum and EGTA-treated plasma
values, but for ALP and LAP, values for EGTA-treated plasma were markedly lower
than for serum specimens. For EGTA and kanamycin serum, some items showed lower
values in comparison to serum values. However, significant correlations (p

CIJ
......
Table 3. C.omparison of chemistry data for serum, and plasmas treated with EGTA or EGTA and kanamycin
A. Levels
Item, Method
Total protein (TP), Biuret method
Albumin (Alb), BCG method
Total bilirubin (Bil) , Alkaline azobilirubin method
Triglyceride (TG), Enzymatic method
Total cholesterol (Chol). Enzymatic method
Urea nitrogen (UN), Urease-UV method
Creatinine (Cr). Alkaline picric acid method
Uric acid (UA), Uricase POD method
Sodium (Na), Electrode method
Potassium (K). Electrode method
Chloride (Cl). Electrode method
Aspartame oxoglutarate aminotransferase (AST, GOT), UV method
Alanine oxalo-glutarate aminotransferase (ALT. GPT), UV method
Lactate dehydrogenase (LDH), Wroblewsky-LaDue method
Alkaline phosphates (ALP), PNP substrate method
Leucine aminopeptidase (LAP), L-leu-DBHA substrate method
r glutamyl transferase (r GTP) , L r glutamyl-3-carboxy-4-
nitoroanilide substrate method
Choline esterase (ChE), p-hydroxy benzoyl choline substrate method
Unit Mean±lSD
----------------------------------------------------
Serum EGTA EGT A +kanamycin
g/dl 7.8±O.4 8.2±O.4 7.4±O.3
g/dl 4.9±O.4 4.9±O.4 4.3±O.3
t::J
.-J
mg/dl O.7±O.2 O.7±O.2 O.5±O.2 :J>
mg/dl 112±33 110±33 93±29
mg/dl
mg/dl
220±44
14±2
219±47
14±2
187±39
14±3
0
O'Q
c:
(1)
mg/dl O.9±O.2 O.9±O.1 O.9±O.2
mg/dl 5.9±1.7 5.3±1.6 4.6±1.4 Z
mEq/1
p:>
144±2 145±2 128±3 '"Xj
mEq/1 4.3±O.2 4.0±O.2 4.2±O.2 0' ...,
mEq/1
IU/l/3rC
106±2
31±15
101±1
31±15
97±2
29±13
p:>
::l
'""'" n'
0
p:>
O'Q
IU/l/3rC 26±16 28±16 29±15
IU/l/37°C 353±71 332±65 303±53
IU/l/37°C 162±47 42±12 59±21 c:
rn
(1)
IU/l/37°C 116±9 84±8 78±6
IU/l/3rC 38±24 38±25 32±21
IU/l/37°C 373±87 377±89 330±75
N=8
trj
p:>
::l
p:>
rn
p:>
::l
p,.
c:
Po"
::l
'""'"

MITSUO NARITA, et aI.
Table 3.
B. Correlation coefficients (r) between serum values
and EGTA or EGTA + kanamycin treated
plasma values
Item EGTA
EGTA +
kanamycin
TP 0.976 ** 0.881 **
Alb 0.973 ** 0.960* *
Bil 0.953 ** 0.916* *
TG 0.999* * 0.999 **
Chol 0.997* * 0.988 **
UN 0.992 ** 0.993 **
Cr 0.865 ** 0.745 *
UA 0.999 ** 0.999 **
Na 0.640 0.571
K 0.771 * 0.511
CI 0.887* * 0.818 **
AST(GOT) 0.998* * 0.998 **
ALT(GPT) 0.999* * 0.999* *
LDH 0.969* * 0.972 **
ALP 0.928 ** 0.924 **
LAP 0.999* * 0.999 **
rGTP 1.000* * 1.000* *
ChE 1.000* * 0.996 **
*P

EDTA analogues and NaF for anticoagulant use
Although these systems are able to handle many specimens very rapidly, patients com­
plain that too much blood is required for testing, since blood must be drawn for at
least 2 test tubes: anticoagulant-free tubes for chemical tests, and EDTA tubes for
hematology tests. Therefore, several analyzers for stat test use have recently been
developed for multi-type testing with a single test tube (ll). Several researchers are
attempting to find a new anticoagulant for multiple testing (2). To obtain plasma,
calcium chelation or suppression of thrombin activity is usually performed to prevent
blood coagulation.
EDTA is a potent calcium ion chelator that interferes with the coagulation
pathway. Four EDTA analogues were examined in this study. EDTA is an
internationally-recognized anticoagulant for hematology tests (4), and vacuum test
tubes with EDTA containing 2 ml blood are generally used in clinical laboratories.
EDTA blood enables stable CBC and automated white cell differential determinations
for at least 24 hours after blood collection 03-15). In the blood coagulation pathway,
free calcium ion plays an essential role, and calcium chelators such as EDTA, sodium
citrate and NaF can remove free calcium ion in plasma to block coagulation (4, 16).
EDTA and sodium citrate are used as medical agents, and have low toxicity in human
(17), while EGTA should have low toxicity considering its structural similarity to
EDTA. The chelating activity of EDTA is strong and irreversible enough to provide
lengthy stability on hematological testing, but causes gradual denaturation of blood
cells and thereby changes mean cell volume and plasma constituents. The strong
chelating activity of EDTA sometimes interferes with biochemical reactions requiring
calcium ion such as those involving alkaline phosphatase. Thus, EDTA plasma has not
usually been used for chemical tests (18). In this study, we examined three structurally
different EDTA analogues. Our findings revealed that EGTA is useful for CBC (except
for platelet counting), white cell differential, chemical tests and coagulation tests.
EGTA has been used as a calcium ion buffer for chemical tests which require subtle
control of free calcium ion concentration in mixtures with various biochemical reac­
tions in the presence of proteins (9). EGTA itself is a white powder and water-soluble
at neutral pH. Thus, it can readily be dissolved in blood for laboratory tests as an
an ticoagulant. We assumed that EGTA would be useful as an anticoagulant, and our
results clearly demonstrated that this assumption was correct, although some behav­
iors of EGTA differed from those of EDTA.
On the other hand, NaF is used for blood glucose determination but not for hema­
tology tests and chemical tests. NaF is a potent enolase inhibitor and blocks the
glycolysis pathway, and it is also a calcium ion chelator (8). Based on these charac­
teristics, blood glucose, fructosamine and hemoglobin Al c are determined as part of
routine laboratory testing. However, excess NaF and long term-storage with NaF each
-83-

MITSUO NARITA, et al.
result in partial hemolysis. NaF blood exhibits changes in the chemical profile of
plasma due to platelet aggregation when it is stored for hours. Our study also demon­
strated that NaF produces platelet aggregation and has effects on coagulation studies
different from those of EDTA blood (20). We therefore consider NaF useful as a
multipurpose anticoagulant but not for coagulation tests.
For the four types of chelators investigated, the following conclusions were
obtained. EGTA alone can be llsed for hematology tests other than platelet count, and
it may be useful for coagulation and chemistry tests, and the use of kanamycin can
block the platelet aggregation if the platelet count is needed. Platelet aggregation is
a significant problem for complete blood counting, given the clinical significance of
platelet counting and its requirement in fundamental hematological testing in routine
laboratory practice. The cause of platelet aggregation in test tubes has not been
clearly determined. Pseudothrombocytopenia induced by EDTA is often observed clini­
cally and has been considerod due to activation of IgG-like agglu tinin (21-23). Since
the chemical structure and chelating effect of EGTA are similar to those of EDTA,
similar effects on platelet in vitro aggregation should be observed in EGTA-treated
blood. Various types of reagents such as aspirin (24), prostaglandin E I (25), sodium
citrate mixture (26, 27), and kanamycin (28) have been used to prevent EDTA-induced
pseudothrombocytopenia. In our laboratory, aspirin and prostaglandin El were tested
in EGTA-treated blood. Prostaglandin El was useful, but is too expensive for routine
use. The relatively inexpensive kanamycin was therefore tested in our experiments.
Kanamycin was useful for EGTA-treated blood in preventing aggregation (28),
although it has been thought to reduce activity towards free calcium ion uptaken by the
aminoglycosides incorporated in its molecule (29), and thus to affect the results of
coagulation tests.
Automated white cell differential determination is a new technology, and
enables accurate and precise identification of normal white blood cells. EGTA and NaF
had only small effects on automated white cell differential counts, compared with
EDTA blood. Giemsa-stained cell morphology was markedly affected by the use of
NaF. EGTA blood yielded satisfactory cell morphology, while kanamycin resulted in
slightly bluish cytoplasm. Addition of smaller amounts of kanamycin had lesser
effects. Such effects could also be minimized when these reagents were used immedi­
ately at blood preparation (20), and the effects did not interfere with clinical judgment
of specimen abnormalities.
Total labora tory systemization should include three major elements: chemistry,
hematology and coagulation analysis. Our findings showed that EGTA and NaF
completely inhibited whole blood coagulation, and that only EGTA was useful for PT
and APTT determination. PT and APTT are the most useful markers for screening for
-84-

EDTA analogues and NaF for anticoagulant use
abnormalities of the extrinsic and intrinsic coagulation pathways (30). EGTA pro­
longed PT and APTT, but EGTA blood should be used for such coagulation tests wi th
new normal reference values.
Chemistry testing is performed in conditions in which pH, temperature, metals,
and reagent concentrations have very subtle effects (31). EGTA solution itself is
neutral, but becomes acidic when kanamycin is added due to the sulfuric acid conjugate
of the kanamycin compound. As pH conditions affects on some of chemistry testing,
chemical data obtained from EGTA-treated plasma will therefore be strongly influ­
enced by the addition of kanamycin. Thus, if chemical data close to those of serum
specimens are required, the use of EGTA alone is desirable.
Clinical laboratory tests have in the past decade changed very rapidly from those
of automated instrumentation to those of laboratory systemization, and laboratory
systems are facing very critical situations due to financial difficulties. Efficiency is
now a very important problem for the modern laboratory, and development of a
new anticoagulant is essential for precise and rapid treatment of large numbers of
specimens (16). For these reasons, the results of our trial are very important. In
conclusion, EGTA will be useful as an anticoagulant when hematology tests (other
than platelet counts), coagulation tests, and chemistry tests are to be conducted simul­
taneously.
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EDTA analogues and NaF for anticoagulant use
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