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Effects of common anticoagulants (heparin,

citrate and EDTA) on routine plasma
biochemistry of cattle

Article in Comparative Clinical Pathology · July 2007

DOI: 10.1007/s00580-006-0664-9

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Comp Clin Pathol (2007) 16:207–209
DOI 10.1007/s00580-006-0664-9
ORIGINAL ARTICLE
Effects of common anticoagulants (heparin, citrate
and EDTA) on routine plasma biochemistry of cattle
M. Mohri & H. Shakeri & S. Lotfollah Zadeh
Received: 18 September 2006 / Accepted: 28 November 2006 / Published online: 5 January 2007
# Springer-Verlag London Limited 2007
Abstract The effects of various types of anticoagulants on
plasma biochemistry have been studied in man and various
animals but limited information exists for cattle plasma
biochemistry. Ten clinically healthy Holstein cattle were
blood sampled into different anticoagulants and plain tubes
for harvesting plasma and serum. The concentrations of
glucose, total bilirubin, urea, creatinine, total protein, albumin
and the activity of aspartate aminotransferase (AST) and
creatine kinase (CK) were measured. All of the measured
parameters were significantly lower in citrated plasma than
serum. Significant decreases in the amounts of urea,
creatinine and total protein while significant increases in the
concentration of bilirubin were seen when heparin was used
as anticoagulant. Using EDTA as anticoagulant caused
significant decreases in the amounts of urea, creatinine, total
protein and the activities of AST compared to serum.
Keywords Citrate . EDTA . Heparin . Plasma biochemistry .
Cattle
Introduction
Anticoagulants are additives that inhibit the clotting of
blood and/or plasma, thereby ensuring that the concentra-
M. Mohri (*)
Department of Clinical Sciences, School of Veterinary Medicine,
Ferdowsi University of Mashhad,
P.O. Box 91775-1793, Mashhad, Iran
e-mail: mohri@ferdowsi.um.ac.ir
H. Shakeri : S. Lotfollah Zadeh
Department of Clinical Sciences, School of Veterinary Medicine,
Azad University of Garmsar,
Garmsar, Iran
tion of the substance to be measured is changed as little as
possible before the analytical process (Guder 2001). Anti-
coagulation is achieved either by the binding of calcium
ions (EDTA, citrate and fluoride) or by the inhibition of
thrombin (heparin).
Serum from coagulated blood is the preferred specimen for
clinical chemistry analysis but plasma obtained with an
appropriate anticoagulant may be an equally valid specimen
and in certain conditions preferable to serum. Also, for the
measurement of some trace elements, ammonia, blood pH and
blood gas analysis, whole blood sampled into an appropriate
anticoagulant is required (Young and Bermes 1999).
Heparin is the most widely used anticoagulant for
clinical chemistry analysis. On the other hand, EDTA is
particularly useful for hematological examination and many
blood samples are sent to clinical laboratories anticoagu-
lated with EDTA. Sodium citrate solution is widely used for
coagulation studies because the effect is easily reversible by
the addition of Ca+2.
Because the harvest of serum requires 15–30 min wait
for coagulation completion before centrifugation, the use of
plasma expedites analysis in emergency situations. Further-
more, plasma yield from a given volume of whole blood is
always grater than the yield of serum (Young and Bermes
1999). Also, additional biochemical analyses, not previous-
ly indicated for the initial required hemogram, are often
required. In this situation, it is better to obtain another
sample for serum harvesting but this is not always possible
for all patients especially cattle. Thus, analysis must be
performed on plasma anticoagulated with various types of
anticoagulants, most commonly EDTA.
The effects of various types of anticoagulants on plasma
biochemistry were studied in man and various animals but
there is little information published for cattle plasma
biochemistry (Jones 1985a,b; Young and Bermes 1999;
208 Comp Clin Pathol (2007) 16:207–209

Boyanton and Blick 2002; Stokol et al. 2001; Ceron et al.
2004; Harr et al. 2005). The purpose of the present study
was to determine and compare how the main anticoagulants
may affect the results of routine biochemistry in cattle
plasma specimens.
different anticoagulants. Limits of confidence intervals
were determined as 25th and 75th percentiles. p≤0.05 was
considered as significant.
Results

Materials and methods
Ten clinically healthy Holstein cattle were used in the
present study. Blood samples were collected from the
jugular vein by disposable syringe and 16 G needle. From
each cow, 20 ml of blood were taken and divided into glass
tubes containing appropriate amounts of anticoagulants
(EDTA: 0.1 ml of 10% disodium EDTA solution for 5 ml
of blood, lithium heparin: 100 units for 5 ml of blood,
sodium citrate: 0.5 ml of 3.8% solution per 4.5 ml of blood)
and into plain tubes for serum harvesting.
All samples were transferred, on ice, to the laboratory
and centrifuged at 1,800×g for 10 min providing serum and
plasma, which were refrigerated until measurement (ap-
proximately 90 min after harvesting). No haemolysis was
detected in any of the samples analysed.
The concentrations of glucose (glu, glucose oxidase
method), total bilirubin (bili, dichloroanylin method), urea
(ure, urease/glutamate dehydrogenase method), creatinine
(cre, kinetic Jaffe method), total protein (tp, Biuret method),
albumin (alb, bromcresol green method) and the activity of
aspartate aminotransferase (AST, L-aspartate/2-oxoglutarate
as substrate) and creatine kinase (CK, creatine phosphate as
substrate) were measured by commercial kits (Pars
Azmoon, Tehran, Iran) using an auto analyzer (Biotecnica,
TARGA 3000, Rome, Italy). Control serum (Randox
control sera, Antrim, UK) was used for ensuring measure-
ment accuracy. The within-run CV of all measured
parameters was between 3% and 12%.
The SPSS software, version 9 (SPSS, Chicago) was used
for data analysis. Non-parametric Wilcoxon pair test was
performed to compare the differences between serum and
The median with confidence interval of measured metab-
olites and enzymes activities in serum and different types of
plasma are showed in Table 1.
All of the measured parameters were significantly lower
in citrated plasma than in serum. Significant decreases in
the amounts of urea, creatinine and total protein while
significant increases in the concentration of bilirubin were
seen when heparin was used as anticoagulant. Using EDTA
as anticoagulant caused significant decreases in the
amounts of urea, creatinine, total protein and the activities
of AST compared to serum.
Discussion
In the past, issues concerning serum analyte measurement
and stability were a major concern because serum was the
preferred specimen for most clinical pathology laboratories.
However, some laboratories are switching to plasma to
increase the turnaround time and to avoid the risk of fibrin
clot interference on automated analyzers, especially those
with sample probe without clot detection ability (Boyanton
and Blick 2002).
Sodium citrate solution at a concentration of 3.4–3.8 g/dL
in a ratio of 1 part to 9 parts of blood is generally used for the
analysis of factors related to haemostasis because its effect is
easily reversible by the addition of ionized calcium (Young
and Bermes 1999). In the present study, citrate produced a
decrease in all parameters compared to serum. However,
most of these decreases could be attributed to a dilution (1:9)
effect when the blood was mixed with the liquid anticoag-
ulant. In addition, citrate inhibits aminotransferase activity

Table 1 Median (25th–75th percentiles) of measured metabolites and enzymes in serum and various types of plasma

Metabolites Serum Heparinized plasma EDTA-treated plasma Citrated plasma

Glucose (mg/dl) 52 (49.18–55.35) 50.15 (45.5–56.25) 54.3 (48.48–61.9) 45.35 (42.02–53.43)*

Total bilirubin (mg/dl) 0.24 (0.23–0.27) 0.30 (0.29–0.32)** 0.27 (0.24–0.28) 0.22 (0.21–0.23)**
Urea (mg/dl) 35.45 (32.18–38.38) 33.65 (30.55–37.45)* 32.45 (28.78–33.5)** 31.25 (27.55–33.3)**
Creatinine (mg/dl) 1.32 (1.24–1.43) 1.17 (1.10–1.24)** 1.17 (1.07–1.22)** 1.06 (0.99–1.11)**
Total protein (g/dl) 8.03 (7.8–8.94) 7.91 (7.43–8.61)* 7.42 (7.02–8.19)** 7.09 (6.59–7.55)**
Albumin (g/dl) 2.99 (2.74–3.22) 3.10 (3.02–3.40) 2.99 (2.89–3.09) 2.66 (2.54–2.83)**
AST (IU/L) 68.9 (62.3–80.1) 64.9 (58.85–73.63) 63.15 (58.98–71.53)* 57.75 (50.83–63.48)**
CK (IU/L) 178 (157–220) 178.5(162.5–218.25) 174 (163.5–219) 154.5 (137.5–189.25)**

*p<0.05.
**p<0.01.
Comp Clin Pathol (2007) 16:207–209
and because it complexes molybdate, it decreases the color
yield in phosphate measurements and thus produces low
results (Young and Bermes 1999). In previous studies,
albumin concentration was corrected for the dilution of
blood by liquid citrate anticoagulant; authors expected the
albumin concentration in citrated samples to be lower than
that in heparinized plasma but equivalent to that in serum.
Therefore, the statistically (and clinically) significant lower
mean albumin concentration in citrated plasma compared
with serum was an unexpected finding. They believed that
small variations in the amount of blood collected into citrate
tubes (i.e., a dilution of >1:10) could have contributed to the
lower albumin concentration in citrated plasma. Citrate also
could have inhibited the reaction of albumin with BCG
(Stokol et al. 2001). However, the exact reason for this result
is not clear.
Heparin was generally recommended as the most
suitable anticoagulant for plasma biochemical measure-
ments (Young and Bermes 1999) although in previous
reports significant differences in selected parameters such
as CK, LDH, GGT and potassium were found between
heparinized plasma and serum (Thorensen et al. 1992). In
our study, serum and heparinized plasma yielded different
results for the amounts of urea, creatinine, total bilirubin
and total protein. An artifactual increase in albumin in
heparinized plasma, compared with serum and other anti-
coagulants, using a bromcresol green assay (BCG) was
recently described in canine samples (Stokol et al. 2001;
Ceron et al. 2004). This difference is partly due to the
combination of heparin and fibrinogen (Stokol et al. 2001).
In the study of Stokol et al. (2001), corrected albumin
concentrations in citrated plasma samples (with standard
and modified BCG methods) were not higher than those in
serum and were lower than those in heparinized plasma
samples suggesting that fibrinogen alone was not respon-
sible for the overestimation of albumin concentration in
heparinized plasma. They believed that fibrinogen
explained only 50% of the difference in albumin concen-
tration between heparinized plasma and serum with the
standard BCG method and other unknown causes contrib-
uted to the observed difference. In heparinized plasma of
dog, a sample blank and a reaction time of less than 1 min
for albumin measurements using the BCG method was
recommended to prevent such artifactual increases (Stokol
et al. 2001). The results of the present study demonstrate
that albumin concentration is non-significantly overesti-
mated in heparinized plasma compared with serum samples
using the BCG method. The cause of the differences for the
amounts of urea, creatinine, total bilirubin and total protein
in heparinized plasma compared with serum in cattle is not
clear.
Changes in some metabolites, minerals, electrolytes
and enzymes induced by EDTA were described in man
View publication stats
209
and in dog (Guder 2001; Boyanton and Blick 2002; Ceron
et al. 2004). In the present study, EDTA induced significant
decreases in the detected amounts of urea, creatinine, total
protein and AST activity. In contrast with our result, an
increase in AST activity was described in EDTA-treated
plasma samples from sheep but other studies showed no
variation in AST activity with dog and cattle (Ceron et al.
2004; Jones 1985a,b). Our CK results agree with previous
reports, which found no differences in CK between EDTA
and serum in cattle samples (Jones 1985a) unlike human
EDTA-treated samples, which showed a decrease in CK
activity (Young and Bermes 1999). Distribution of specific
isoenzymes could be responsible for these differences
between species. The significantly lower concentrations of
urea, creatinine and total protein in EDTA-treated plasma
when compared to serum are in contrast with previous
reports in dog and in humans (Ceron et al. 2004; Young and
Bermes 1999). The exact mechanism of these differences
was not clear although the osmotic fluid shift from red cells
to plasma (Dubin and Hunt 1978) and/or differences
between species may be contributory factors.
This study strongly supports the use of serum for the
determination of clinical biochemical profiles of cattle
although more studies into the differences between serum-
derived and plasma-derived results could be helpful.
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