AJCP / ORIGINAL ARTICLE
Magnesium Sulfate as an Alternative In Vitro
Anticoagulant for the Measurement of Platelet
Parameters?
Steffen Mannuß, Peter Schuff-Werner, Katrin Dreißiger, and Peter Kohlschein
From the Rostock University Medical Centre, Institute of Clinical Chemistry and Laboratory Medicine, Rostock, Germany.
Key Words: Platelet count; Mean platelet volume; Preanalytics; Cell swelling; Anticoagulation; Magnesium sulfate; EDTA; Citrate
Am J Clin Pathol June 2016;145:806-814
DOI: 10.1093/AJCP/AQW066
ABSTRACT
Objectives: There are conflicting reports on the reliable
measurement of platelet count and mean platelet volume
(MPV) using EDTA or citrate. The anticoagulant properties
of magnesium sulfate (MgSO4) are known from the litera-
ture. The aim of this study was to evaluate MgSO4 as an
in vitro anticoagulant for platelet count, MPV, platelet dis-
tribution width, and platelet activation.
Methods: Whole blood from volunteers was anticoagulated
by EDTA, citrate, or MgSO4. Platelets were counted by the
XE 5000 (Sysmex, Norderstedt, Germany) impedance and
fluorescence optical technique.
Results: The mean impedance platelet counts were 227.7,
197.0, and 201.1  109/L in EDTA-, citrate-, or MgSO4-
anticoagulated blood, respectively. The counts were 4.7%
higher (EDTA) after 3 hours of storage but 4% lower in cit-
rate-anticoagulated blood. The counts in magnesium sam-
ples remained stable. The MPV was 10.4 fL (EDTA), 9.5 fL
(citrate), and 9.3 fL (MgSO4). EDTA samples showed cell
swelling within the first 3 hours. This was lower in citrate
and only marginal in magnesium samples. High activation
of platelets was observed only in EDTA samples.
Conclusions: Magnesium anticoagulation might be advan-
tageous for more reliable MPV measurements. Although
platelet count is underestimated when the impedance
method is used, the platelet count reveals similar results
when measured by the fluorescent optical method.
806 Am J Clin Pathol 2016;145:806-814
DOI: 10.1093/ajcp/aqw066
Downloaded from https://academic.oup.com/ajcp/article/145/6/806/2836709 by guest on 23 March 2024
In the routine hematology laboratory, EDTA and citrate
are the most commonly used anticoagulants for measuring
platelet parameters; for special analyses such as impedance
aggregometry, hirudin is the optimal anticoagulant. The ad-
vantages and disadvantages of these anticoagulants have
been reviewed in detail by Banfi and colleagues.1 Exact
platelet count is of importance in thrombocytopenia, espe-
cially if the necessity for platelet transfusion is discussed.2
The exact and reproducible determination of platelet vol-
ume is of interest as a predictive biomarker, for example, in
cardiovascular disease.3,4 However, there are conflicting re-
ports on the correct anticoagulant and the best method for
platelet counts and volume measurements; therefore, further
standardization is necessary.
EDTA was introduced as an in vitro anticoagulant in
hematology because of its calcium-complexing capacity,
which prevents clotting and therefore stabilizes the fluidity of
whole blood. Blood smears from EDTA-anticoagulated blood
were shown to be optimal for morphologic blood cell differen-
tiation using May-Grünwald-Giemsa (Pappenheim) staining.
The only but relevant disadvantage of EDTA-anticoagulated
blood is the time-dependent swelling of platelets.5
Sodium citrate is mainly used for the study of the plas-
matic coagulation system but is also suitable for performing
a CBC.6
Magnesium sulfate (MgSO4) has been known as an
in vitro anticoagulant since Sahli7 introduced it for conven-
tional platelet count by referring to Bizzozero’s experiences
with the use of MgSO4 for avoiding platelet clumps in glass
tubes. In the late 1980s, this anticoagulant was successfully
used in individuals with EDTA-introduced pseudothrombo-
cytopenia (PTCP)8 and also for hematology tests,9 but it
was never introduced for routine purposes. Our own positive
© American Society for Clinical Pathology, 2016. All rights reserved.
For permissions, please e-mail: journals.permissions@oup.com

experiences with MgSO4 in patients with PTCP led to the
commercial launch of a blood sampling tube prepared with
MgSO4 as an anticoagulant. In a multicenter study10 of anti-
coagulant-induced PTCP, magnesium anticoagulation was
shown to be effective, although platelet counts and platelet
volume seemed to be underestimated compared to when
using EDTA blood.11
The real platelet volume is still a matter of controversy,
especially if one takes into account that EDTA as well as
citrate anticoagulation leads to early platelet swelling.5 This
is a critical issue of preanalytical standardization for meas-
uring the mean platelet volume (MPV) as a possible but
controversially discussed predictive biomarker.12-14
To test MgSO4 as an alternative to EDTA and citrate,
we compared the platelet parameters measured up to
180 minutes from healthy individuals. Finally, we con-
firmed our findings by a comparative study of platelet
counts and MPV in 834 patients’ blood samples.
Materials and Methods
Platelet counts and MPV were measured 20, 40, 60, and
180 minutes after sampling from blood of 21 healthy volun-
teers. The samples were anticoagulated by EDTA, citrate,
and MgSO4. Only apparently healthy individuals without
medication up to 7 days before blood donation and not older
than 65 years or younger than 18 years were included in this
study. The group of volunteers involved 11 men (mean age
33.8 years, ranging from 22-65 years) and 10 women (mean
age 44.3 years, ranging from 24-62 years).
Platelet count and volume of the first consecutive 834
patients of an ongoing PTCP prevalence study (371 women
with a mean age of 59.3 years, ranging from 19-93 years,
and 463 men with a mean age of 60.4 years, ranging from
14-93 years) were analyzed after the exclusion of EDTA- or
citrate-induced PTCP by comparing the platelet counts with
those from MgSO4-anticoagulated blood.
This study was approved by the Ethics Committee of
the University of Rostock (registration no. A 2013-0104).
Written consent is generally given by the patient when
admitted to the hospital but can be withdrawn by the patient
for personal reasons.
Blood Sampling
After releasing the tourniquet, whole blood was collected
from lying or sitting patients and volunteers by venipuncture
of the cubital vein using a 21-gauge butterfly needle
(Sarstedt, Nümbrecht, Germany) with a respective adapter.
Blood was collected in commercial tubes prefilled with
the respective anticoagulant: EDTA (S-Monovette K3-EDTA;
fill volume of blood 2.6 mL), natrium-citrate (S-Monovette
© American Society for Clinical Pathology
807
AJCP / ORIGINAL ARTICLE
9NC; fill volume of blood 2.9 mL), and MgSO4 (S-Monovette
ThromboExact; fill volume of blood 2.7 mL), all manufac-
tured by Sarstedt.
The order of blood sampling was the following: (1)
EDTA, (2) citrate, and (3) MgSO4. Blood from the healthy
volunteers was used for the above-described time kinetics
and measured within 5 to 10 minutes after sampling.
Patients’ blood sampling was performed in the casualty
units of the hospital, located either in the same or in the
nearby buildings. Due to this fact, the time period between
sampling and measurement was between 30 and 45 minutes.
Downloaded from https://academic.oup.com/ajcp/article/145/6/806/2836709 by guest on 23 March 2024
Automated Determination of Platelet Parameters With
the XE 5000
All specimens were routinely analyzed with the auto-
mated hematology analyzer (XE-5000; Sysmex, Norderstedt,
Germany). This analyzer provides two options for platelet
counting: impedance and fluorescence optical technology.
The platelet count correlates with the number of im-
pulses triggered from cells sized between 2 and 30 fL. The
height of the impulse size is calculated and leads to a histo-
gram of platelet volumes. Platelet number and MPV are
estimated on the basis of the platelet histogram surface,
which is limited by the lower and upper volume discrimin-
ator.15 For comparison reasons, we used the fluorescence
method of platelet counting in parallel.
Because of the dilution effect, the platelet counts in cit-
rate-anticoagulated samples were multiplied by a factor of 1.1.
The analyzer was calibrated and maintained according
to the manufacturer’s instructions. Internal quality control
(QC) was performed and samples were assayed only if all
QC criteria were fulfilled.
The imprecision of platelet count, displayed by the co-
efficient of variation, of the impedance technology/fluores-
cence optical technology using differently anticoagulated
sampling devices was 2.07%/2.19% (EDTA), 1.37%/3.71%
(citrate), and 1.23%/2.28% (MgSO4), respectively. The im-
precision of MPV determination was 2.37% (EDTA),
2.46% (citrate), and 1.56% (MgSO4).
Statistical Analysis
The statistical analysis (Wilcoxon signed-rank test and
Spearman rank-order correlation) was performed with the
software SigmaPlot 11. To show the distribution of all quan-
titative values within a group of samples, they are displayed
by box plots. Each box describes the 25th and 75th percent-
iles, including 50% of the measured values. The median
value is depicted by a line inside the box. The whiskers
mark the 10th and 90th percentiles. The black dots mark the
5th and 95th percentiles. Box plots allow the graphical com-
parison of data and their variation.16 Standard error of the
Am J Clin Pathol 2016;145:806-814 807
DOI: 10.1093/ajcp/aqw066
Mannuß et al / MAGNESIUM SULFATE AND PLATELET ANALYSIS
mean was used for plotting the results of comparing the im-
pedance and the fluorescent optical method for platelet
count in differently anticoagulated blood samples.
Results
Time Dependency of the Measurement of Platelet
Parameters in EDTA-, Citrate-, and MgSO4-
Anticoagulated Blood Samples Within 180 Minutes of
Venipuncture
Platelet count and MPV of 21 volunteers were meas-
ured in differently anticoagulated blood samples using the
impedance method of the XE 5000. In nine of the 21 sam-
ples, the optical method was applied in parallel. The first
measurement was done within 10 minutes of venipuncture,
followed by further determinations after 20, 40, 60, and
180 minutes.
Time-Dependent Changes in Platelet Counts
As depicted in Figure 1 , the mean 6 SD platelet count
in EDTA-anticoagulated blood from 21 healthy volunteers
increased within 180 minutes from 228 6 49  109/L (me-
dian, 220) to 240 6 59  109/L (median, 237).
In citrate-anticoagulated blood, the initial platelet count
of 197 6 42  109/L (median, 190) was markedly lower
compared with EDTA-anticoagulated blood. Within the
storage time of 180 minutes, the platelet count decreased to
189  109/L (median, 190).
The platelet count of blood anticoagulated with MgSO4
was determined after blood sampling with 201 6 41  109/
L (median, 193). It remained relatively stable over time and
reached 206 6 42  109/L (median, 194) after 180 minutes.
In nine of the volunteer donors, platelet counts gener-
ated by impedance were measured in parallel by the fluores-
cence optical channel of the XE 5000. The results are shown
in Figure 2 . The time-dependent comparison clearly shows
that both methods measure comparable platelet counts in
EDTA-anticoagulated blood (Figure 2A), although the first
counts were higher when measured by fluorescence
(PLTopt, 228 6 54; PLTimp, 217 6 49  109/L). From
40 minutes onward, both counts were similar, ranging be-
tween 226 6 52 and 229 6 53  109/L for impedance and
between 223 6 54 and 229 6 58  109/L for optical count.
In MgSO4-anticoagulated blood, the mean platelet
count (Figure 2C) measured by impedance was markedly
lower (PLTimp, 190 6 42 109/L) compared with the values
measured in the EDTA-anticoagulated samples. However,
when measured by the fluorescent optical channel, the plate-
let count (PLTopt, 223 6 53  109/L) was similar to that
808 Am J Clin Pathol 2016;145:806-814
808 DOI: 10.1093/ajcp/aqw066
400
350
Platelet Count (× 109/L)
300
250
200
150
100
50
Downloaded from https://academic.oup.com/ajcp/article/145/6/806/2836709 by guest on 23 March 2024
<10 20 40 60 180
Time After Sampling (min)
Figure 1 Platelet count measured by the XE 5000 (imped-
ance method) in samples of 21 volunteers anticoagulated
with EDTA (white), citrate (light gray), and magnesium sul-
fate (dark gray).
measured in EDTA blood by impedance (PLTimp, 217 6 49
 109/L) and by fluorescence (PLTopt, 228 6 54  109/L).
When measured after 180 minutes, the impedance
platelet count increased slightly to a PLTimp of 198 6
44109/L, whereas the mean platelet counts measured by
the fluorescence optical method remained stable over time
within the range of the counts from EDTA-anticoagulated
blood drawn in parallel from the same donors and measured
using both methods.
In contrast to these findings, in citrate-anticoagulated
blood (Figure 2B), platelet counts were lower regardless of
the method used. However, more platelets were counted by
the fluorescence optical method compared with the imped-
ance measurement (PLTopt, 204 6 48  109/L; PLTimp,
184 6 38  109/L). During storage up to 180 minutes, the
platelet count showed a tendency to decrease slightly to
181 6 39  109/L and to 195 6 44  109/L when measured
by impedance or fluorescence, respectively.
Time-Dependent Changes in the MPV
The influence of different anticoagulants and time after
blood sampling is shown in Figure 3 . When the platelet
volume was measured in EDTA-anticoagulated blood, the
initial MPV from 21 healthy donors was 10.40 6 1.18 fL,
which increased continuously to 11.12 6 1.32 fL after
180 minutes of storage at room temperature.
The platelet volume of platelets from MgSO4-
anticoagulated blood was markedly lower than the MPV
measured in EDTA blood. Immediately after blood
© American Society for Clinical Pathology
 AJCP / ORIGINAL ARTICLE

14
A
13
250
Platelet Count (× 109/L)

240
12
230
220

MPV (fL)
11
210
200
190 10
180
170 9
160
0 20 40 60 180 8
Time After Sampling (min)
7

Downloaded from https://academic.oup.com/ajcp/article/145/6/806/2836709 by guest on 23 March 2024

<10 20 40 60 180
B
Time After Sampling (min)

250 Figure 3 Mean platelet volume (MPV) measured by the XE

Platelet Count (× 109/L)

240 5000 (impedance method) in samples of 21 volunteers anti-

230
coagulated with EDTA (white), citrate (light gray), and mag-
220
210 nesium sulfate (dark gray).
200
Different Anticoagulants and Their Influence on Platelet
190
180 Distribution Width and Platelet Large Cell Ratio
170 The influence of EDTA, citrate, and MgSO4 on the plate-
160 let distribution width (PDW) is depicted in Figure 4 by box
0 20 40 60 180
Time After Sampling (min) plots. As already shown, platelets swell with time if stored in
EDTA-anticoagulated blood. This can be confirmed by the ob-
C vious increase of the initial PDW (12.44 6 1.98 fL; median,
12.2 fL) to 14.10 6 2.69 fL (median, 13.8 fL) after
250 180 minutes. This is also expressed by the platelet large cell
Platelet Count (× 109/L)

240 ratio (P-LCR), which increased from 28.2% 6 8.8% (median,

230
220
210
200
190
180
170
160
0 20 40 60 180
Time After Sampling (min)
Figure 2 Comparison of the measured platelet count by im-
pedance (black) and optical fluorescence method (gray) in dif-
ferently anticoagulated samples of nine volunteers. The
whiskers represent the standard error of the mean. A,
EDTA. B, Citrate. C, Magnesium sulfate.
sampling, it was 9.31 6 0.97 fL and increased slightly to
9.66 6 1.06 fL after 180 minutes.
When measured in citrate-anticoagulated blood, the ini-
tial MPV was 9.54 6 1.12 fL (median, 9.45 fL); within
60 minutes, the MPV increased to 9.99 6 1.13 fL (median,
10.0 fL) and then dropped to 9.80 6 0.97 fL, although the
median remained at 10.0 fL.
28.8%) to 34.4% 6 10.0% (median, 33.0%) after 180 minutes.
Corresponding to the MPV changes, the PDW in mag-
nesium-anticoagulated blood is clearly lower than that in
EDTA: at the beginning of the measurement series, the
PDW was 10.35 6 1.50 fL (median, 10.4 fL), while after
180 minutes, it was 11.15 6 2.02 fL (median, 11.0 fL).
The initial PDW in citrate-anticoagulated blood
(10.87 6 2.06 fL; median, 10.6 fL) and that after 180 minutes
(11.26 6 1.96 fL; median, 11.20 fL) were compatible with
the respective MPV values measured at the same time.
The initial P-LCR, determined in magnesium-
anticoagulated blood (19.73% 6 6.93%; median, 21.1%), is
moderately different from that of citrate blood (21.79% 6
8.29%; median, 20.9%). After 180 minutes, the P-LCR of
platelets anticoagulated by magnesium was 22.53% 6
7.83% (median, 22.3%), whereas the P-LCR in citrate blood
was 23.69% 6 7.24% (median, 25.4%).
Platelet Counts in Blood Samples Anticoagulated by
EDTA, Citrate, and MgSO4
Platelets were counted in differently anticoagulated
blood samples from 834 patients using the impedance

© American Society for Clinical Pathology Am J Clin Pathol 2016;145:806-814 809

809 DOI: 10.1093/ajcp/aqw066
Mannuß et al / MAGNESIUM SULFATE AND PLATELET ANALYSIS
20
18
16
PDW (fL)
14
12
10
8
6 0
<10 min 180 min
Figure 4 Platelet distribution width (PDW) measured by the
XE 5000 (impedance method) in samples of 21 volunteers
anticoagulated with EDTA (black), citrate (light gray), and
magnesium sulfate (dark gray).
(PLTimp) and fluorescence optical (PLTopt) method of the
XE 5000 Figure 5 and Table 1 in parallel. The mean
platelet count in EDTA-anticoagulated blood was 243.6
109/L (32-769  109/L) using the impedance method. The
mean platelet count measured by the optical method of the
XE 5000 was 236.5  109/L (39-799  109/L).
The mean PLTimp count for citrate-anticoagulated
blood was 171.2  109/L (24.2-585.2  109/L), although
the dilution effect had already been corrected by a factor of
1.1. The mean value for PLTopt was 195.7  109/L (30.8-
756.8  109/L).
In blood samples anticoagulated with magnesium salt,
the mean PLTimp was 205  109/L (32-642  109/L), and
the respective mean value for PLTopt was 226.5  109/L
(34-787  109/L). The differences between PLTimp and
PLTopt were statistically significant independent from the
anticoagulant used (EDTA, P < .001; citrate, P < .001;
MgSO4, P < .001). These findings are depicted as box plots
in Figure 5.
The platelet counts from EDTA-anticoagulated samples
and those measured from parallel samples anticoagulated by
citrate or MgSO4 were correlated by Spearman rank-order
correlation.
Platelet counts measured by impedance in EDTA blood
(PLTimp) correlated better with platelet counts measured in
MgSO4-anticoagulated blood than platelet counts from cit-
rate blood, r ¼ 0.967 (y ¼ 0.80x þ 9.48) and r ¼ 0.834
(y ¼ 0.64x þ 15.28), respectively.
Using the fluorescence optical method, the platelet
counts (PLTopt) in MgSO4- and EDTA-anticoagulated blood
were correlated with r ¼ 0.977 (y ¼ 0.95x þ 2.46), whereas
810 Am J Clin Pathol 2016;145:806-814
810 DOI: 10.1093/ajcp/aqw066
500
400
Platelet Count (× 109/L)
300
200
100
EDTA Citrate MgSO4
Downloaded from https://academic.oup.com/ajcp/article/145/6/806/2836709 by guest on 23 March 2024
Figure 5 Comparison of platelet counts in 834 blood sam-
ples drawn at the same time but using different anticoagu-
lants and measured by the impedance and the optical
methods using the XE 5000 as a routine analyzer. The results
of the platelet counts are depicted by box plots as described
under statistical methods (PLTimp, blank; PLTopt, gray).
MgSO4, magnesium sulfate.
platelet counts measured in citrate and EDTA showed a
slightly worse correlation (r ¼ 0.932; y ¼ 0.83x þ 0.15).
MPV Measured in Blood Samples Anticoagulated by
EDTA, Citrate, and MgSO4
Platelets anticoagulated with EDTA, citrate, or MgSO4
differ markedly by volume when measured with the Sysmex
XE 5000. The values are depicted as box plots in Figure 6 .
The respective medians and 5th, 25th, 75th, and 95th per-
centiles of the platelet counts are summarized in Table 1.
The MPV in EDTA-anticoagulated samples from 834
patients was 10.4 fL with a minimum of 7.9 fL and a max-
imum of 14.0 fL. In samples from the same patients
anticoagulated with citrate, the mean volume amounted to
9.9 fL, ranging from 7.8 to 13.0 fL, and the mean volume in
magnesium-anticoagulated samples was 9.4 fL with a range
from 7.3 to 12.4 fL. These differences in MPV measured in
differently anticoagulated whole blood are all highly signifi-
cant (P < .001).
The MPVs measured in MgSO4- and citrate-
anticoagulated blood were correlated with the respective
counts in EDTA as a reference. The correlation coefficient r
for MPVs measured in MgSO4- or EDTA-anticoagulated
blood was better than the correlation of the MPVs from
citrated blood and from EDTA blood (r ¼ 0.902 and
r ¼ 0.859, respectively). The corresponding regression lines
are defined by the equations y ¼ 0.78x þ 1.24 and
y ¼ 0.78x þ 1.74.
© American Society for Clinical Pathology
 AJCP / ORIGINAL ARTICLE

Table 1
Summarized Results of Platelet Counts Measured by Impedance (PLTimp) or Fluorescent Optical Technique (PLTopt) and Mean
Platelet Volume (MPV) From Whole-Blood Samples Anticoagulated by EDTA, Citrate, or Magnesium Sulfate (MgSO4) From 834
Individuals
EDTA Citrate MgSO4
PLTimp, PLTopt, PLTimp, PLTopt, PLTimp, PLTopt,
Characteristic  109/L  109/L MPV, fL  109/L  109/L MPV, fL  109/L  109/L MPV, fL
Minimum 32.0 39.0 7.9 24.2 30.8 7.8 32.0 34.0 7.3
5% 139.7 134.7 9.1 86.5 102.3 8.7 115.7 129.3 8.3
25% 193.0 186.0 9.8 132.3 150.7 9.3 164.0 178.0 8.8
Median 229.0 223.5 10.4 165.0 187.0 9.9 194.0 214.0 9.3
75% 284.0 276.0 11.0 201.3 224.4 10.4 238.8 263.0 9.8
95% 380.7 367.4 12.2 267.7 310.2 11.3 319.4 353.1 10.9
Maximum 769.0 799.0 14.0 585.2 756.8 13.0 642.0 787.0 12.4

Downloaded from https://academic.oup.com/ajcp/article/145/6/806/2836709 by guest on 23 March 2024

Mean 243.6 236.5 10.4 171.2 195.7 9.9 205.0 226.5 9.4

14 100

80
12
CD62+ Platelets (%)

60
MPV (fL)

10
40

8
20

6 6
EDTA Citrate MgSO4 7-9.9 10-10.9 ≥11
MPV (fL)
Figure 6 Mean platelet volume (MPV) measured by the im-
Figure 7 Expression of platelet activation markers deter-
pedance method (Sysmex XE 5000) in samples of 834 pa-
mined in whole blood from 21 volunteers and anticoagulated
tients anticoagulated with EDTA, citrate, or magnesium
with different anticoagulants (EDTA, triangle; magnesium
sulfate (MgSO4). The averaged results are depicted by box
sulfate, square; citrate, dot). MPV, mean platelet volume.
plots as described under statistical methods.

MPV and Spontaneous Platelet Activation
It has been reported that the MPV correlates with plate-
let activation. We therefore measured the spontaneous ex-
pression of activation markers on platelets anticoagulated
with all three anticoagulants. In EDTA-anticoagulated
blood, the platelets express CD62 to a variable degree,
whereas in MgSO4- and citrate-anticoagulated blood, only
slight activation is measurable. In Figure 7 , the measured
activity is plotted for three different MPV tertiles (7-9.9 fL/
10-10.9 fL/11 fL). It is obvious that the spontaneous activ-
ity of EDTA-anticoagulated platelets shows a weak but not
significant correlation with the MPV, whereas the otherwise
anticoagulated platelets do not.
Discussion
Today, routine platelet counts and volume measure-
ments are carried out from peripheral venous blood,
anticoagulated with EDTA or citrate, using an automated
hematology analyzer. An important requirement for the cor-
rect determination of platelet count and volume is the stand-
ardization of both, the preanalytical and the analytical
procedure.17 There is a large body of evidence that the
blood-collecting conditions, including the choice of anti-
coagulant, are critical for the reliability of platelet count,
MPV,18 and platelet activation.19
On the basis of our experiences with MgSO4 in individ-
uals with anticoagulant-induced in vitro platelet aggrega-
tion,10 so-called pseudothrombocytopenia, we studied the

© American Society for Clinical Pathology Am J Clin Pathol 2016;145:806-814 811

811 DOI: 10.1093/ajcp/aqw066
Mannuß et al / MAGNESIUM SULFATE AND PLATELET ANALYSIS
suitability of a recently introduced blood-sampling device,
prefilled with MgSO4 (ThromboExact-Monovette; Sarstedt)
for the measurement of platelet count and volume in com-
parison to the established anticoagulants EDTA and citrate.
The rationale and aim of the study was to evaluate the pos-
sible advantages of MgSO4 as an in vitro anticoagulant, es-
pecially under the aspect of some shortcomings of the
established anticoagulants.
The cell-swelling and shape-changing effect of EDTA
within the first hours after venipuncture is known from the
literature20,21 and represents one of the major problems in
standardizing platelet volume measurement.
Therefore, citrate has already been used as an anticoagu-
lant for the measurement of platelet activation, and platelet
function analysis22,23 was also recommended for a more
reliable determination of MPV. On the other hand, whether
morphologic differentiation of WBCs is comparable to that
of EDTA-anticoagulated blood is controversial.24
The anticoagulant potential of MgSO4 has been known
since the beginning of the past century, when Bizzozero
introduced it for platelet count, as cited by Sahli.7 Much
later, magnesium became known as an electrolyte with anti-
coagulant properties and was therefore investigated in clin-
ical medicine as an in vivo platelet aggregation inhibitor,
mainly to prevent cardiovascular complications.25
The antiplatelet activity of magnesium was studied and
reviewed in more detail by Sheu et al.26 Magnesium inter-
feres with the binding of fibrinogen to collagen-stimulated
platelets and obviously has the potential to inhibit intracel-
lular Ca2þ mobilization and thromboxane A2 formation in
stimulated platelets. Furthermore, it induces the formation
of cyclic adenosine monophosphate in platelets and averts
phosphorylation of the protein P47 by inhibiting protein kin-
ase C activity.27
Prior to our study, MgSO4 was used as an in vitro
anticoagulant for blood cell count by a group of researchers
from Japan,8,9 but it was never introduced into the routine
hematology laboratory.
As shown by others28 and now confirmed by our study,
the comparability and reproducibility of platelet count de-
pend on the anticoagulant and on the method used for plate-
let counting. Platelet counts in EDTA-anticoagulated blood
were largely identical regardless of whether they were
counted by impedance or by the fluorescent optical method
of the Sysmex XE 5000.
In citrate-anticoagulated blood, platelet counts were
markedly lower compared with the respective platelet
counts in EDTA blood when measured by the impedance
method. Platelet counts from the same blood sample meas-
ured in parallel by fluorescence were higher but still below
the counts measured in EDTA by both methods.
812 Am J Clin Pathol 2016;145:806-814
812 DOI: 10.1093/ajcp/aqw066
This might be explained by a loss of platelets when they
are in contact with citrate, although there were no signs of
cell destruction. Another explanation might be the fact that
the MPV is markedly lower in citrate-anticoagulated blood,
so very small platelets may bypass the lower platelet dis-
criminator. However, this does not explain the low platelet
counts measured by the fluorescence method.
EDTA is an irreversible calcium chelator, whereas cit-
rate binds calcium in a pH-dependent manner. The release
of calcium might lead to the formation of platelet aggregates
during the storage and repeated agitation of citrate-
anticoagulated blood. Aggregated platelets are then counted
Downloaded from https://academic.oup.com/ajcp/article/145/6/806/2836709 by guest on 23 March 2024
as single large platelets or as leukocytes, thus explaining the
continuous drop of platelets during our time course experi-
ments but not the initial low counts.
Another possible explanation for the lower platelet
counts in citrate-anticoagulated blood was discussed by
Bournazos et al,29 who observed more platelet-leukocyte
conjugates in citrate than in EDTA blood.
Our study clearly shows that in MgSO4-anticoagulated
blood, platelet counts were significantly lower if measured
by impedance and compared with those in EDTA blood.
Using the fluorescent optical method, the platelet count was
largely similar to platelet counts in EDTA blood. This
means that there was obviously no substantial loss of plate-
lets due to magnesium salt anticoagulation11; they seem to
be only partially captured by impedance in contrast to the
fluorescence optical approach.
The fact that higher platelet counts are measured with
the XE 5000 fluorescence optical method refutes the argu-
ment that platelets are wrecked by the anticoagulant. A par-
allel estimation of platelet counts by microscopy using the
Fonio30 approach led to comparable counts in EDTA and
magnesium, thus confirming the fluorescence optical find-
ings (data not shown).
We suppose that the lower platelet count must be dis-
cussed together with the finding that the MPV in citrate-
and MgSO4-anticoagulated blood samples was markedly
lower than the MPV in EDTA blood. We therefore tend to
believe that the impedance platelet count algorithm of the
XE 5000 excludes platelets with a volume below a distinct
volume threshold.
The data analysis of platelet counts in a series of 834 in-
dividuals clearly confirms that the platelet counts in
MgSO4- and citrate-anticoagulated samples are significantly
lower compared with EDTA anticoagulation.
However, the impedance method and the respective
software of the analyzer are supposed to underestimate the
real platelet count in MgSO4-anticoagulated samples, pos-
sibly because the analyzers are adjusted for platelet count in
EDTA-anticoagulated blood only.
© American Society for Clinical Pathology

Lower platelet counts in magnesium-anticoagulated
blood using the routine impedance method underestimate
the EDTA platelet count by 5% to 10%. At first glance, this
might be critical for patients with thrombocytopenia at a
defined critical transfusion threshold of 109/L, for ex-
ample.31 This low threshold was set with the intention to re-
duce the need for and frequency of platelet transfusion
because of economic reasons. In such a situation, platelets
from magnesium-anticoagulated blood have to be measured
by the fluorescent optical method. At least, underestimation
of platelet counts is not disadvantageous for the individual
patient.
Another reason for controversy in platelet analysis is
the absence of standardized MPV measurements. The reli-
ability of this parameter also depends on the kind of anti-
coagulant and the time until measurement because of
platelet swelling, as confirmed in this study.5 Despite these
reservations, MPV is discussed as a useful prognostic bio-
marker, especially in patients with cardiovascular
diseases.14,32
In most of these studies, EDTA was used as an anti-
coagulant, but relatively often the time between blood sam-
pling and MPV measurement was different or not stated.
This lack of standardization of an important preanalytical
criterion is the most critical weak point of MPV biomarker
studies. This is justifiably criticiszed by Lancé et al. 33
One reason to evaluate MgSO4 as an alternative anti-
coagulant to EDTA and citrate was the minimal cell swel-
ling during sample storage.
The initial MPV measured by impedance in 834 un-
selected patients was approximately 1 fL lower in MgSO4-
anticoagulated blood compared with EDTA anticoagulation.
This finding cannot be explained by time differences be-
tween venipuncture and volume analysis, because the sam-
ples were taken and processed at the same time. The mean
MPV of cells anticoagulated with citrate was between the
MPV of EDTA- and MgSO4-anticoagulated samples.
Although the MPV in magnesium-anticoagulated blood in-
creased by around 0.35 fL, it is significantly different from
the MPV in EDTA-anticoagulated blood, which increased
by approximately 0.7 fL within 3 hours.
Both PDW and P-LCR confirm the MPV changes
described for the first 180 minutes of storage at room tem-
perature. There is an increase in PDW because cells
are swelling and the proportion of larger platelets (P-LCR)
increases when platelets are anticoagulated by EDTA.
These parameters increase less if citrate or MgSO4 is used
as an anticoagulant. Although the real clinical relevance of
these new platelet parameters seems to be restricted to the
differentiation of thrombocytopenia,34 it is useful to know
the influence of different anticoagulants on PDW and
P-LCR.
© American Society for Clinical Pathology
813
AJCP / ORIGINAL ARTICLE
There are some indications from other investigators
that the MPV is related to the degree of platelet activation.35
This is in accordance with our findings that the spontaneous
expression of CD62p and CD63 in EDTA-anticoagulated
blood tends to be correlated with the MPV. In contrast, cit-
rate and especially MgSO4 anticoagulation is accompanied
only by marginal spontaneous activation; nevertheless, the
platelets can still be activated by suitable agonists (data not
shown).
We think that the reliability of the MPV is such an im-
portant prerequisite for its use as a predictive and prognostic
biomarker that MgSO4 might be advantageous for this issue;
Downloaded from https://academic.oup.com/ajcp/article/145/6/806/2836709 by guest on 23 March 2024
reliable conclusions from disease-related MPV can only be
made if the study protocol strictly standardizes the preana-
lytical and the analytical conditions, including the kind of
anticoagulation and the method of automated platelet count,
since there is a marked variability of MPV determination by
impedance or light scatter analysis.35-37
Our results presented here suggest that MgSO4 could be
advantageous for studying MPV as a predictive biomarker,
because the values remain more stable after venipuncture
compared with those measured in the presence of citrate or
EDTA. Investigators should be aware of the fact that the
lack of standardizations for measurement techniques and
the use of unsuitable anticoagulants might lead to the misin-
terpretation of MPV as a predictive or prognostic marker or
only as a surrogate marker. The use of MgSO4 as an anti-
coagulant is also suitable to effectively exclude anticoagu-
lant-induced pseudo-thrombocytopenia.
Corresponding author: Peter Schuff-Werner, Rostock University
Medical Centre Institute of Clinical Chemistry and Laboratory
Medicine, Ernst-Heydemann-Straße, 8 D-18057 Rostock
Germany; pschuffw@med.uni-rostock.de.
References
1. Banfi G, Salvagno GL, Lippi G. The role of ethylenediamine
tetraacetic acid (EDTA) as in vitro anticoagulant for diag-
nostic purposes. Clin Chem Lab Med. 2007;45:565-576.
2. Slichter SJ. Evidence-based platelet transfusion guidelines:
Haematology/the education program of the American
Society of Hematology. Am Soc Hematol Educ Program.
2007;172-178.
3. Ki YJ, Park S, Ha SI, et al. Usefulness of mean platelet vol-
ume as a biomarker for long-term clinical outcomes after per-
cutaneous coronary intervention in Korean cohort: a
comparable and additive predictive value to high-sensitivity
cardiac troponin T and N-terminal pro-B type natriuretic
peptide. Platelets. 2014;25:427-432.
4. Lippi G, Filippozzi L, Salvagno GL, et al. Increased mean
platelet volume in patients with acute coronary syndromes.
Arch Pathol Lab Med. 2009;133:1441-1443.
Am J Clin Pathol 2016;145:806-814 813
DOI: 10.1093/ajcp/aqw066
Mannuß et al / MAGNESIUM SULFATE AND PLATELET ANALYSIS
5. Lancé MD, van Oerle R, Henskens YMC, et al. Do we need
time adjusted mean platelet volume measurements? Lab
Hematol. 2010;16:28-31.
6. Perrotta G, Roberts L, Glazier J, Schumacher HR. Use of
sodium citrate anticoagulant for routine haematology ana-
lysis on the CELL-DYNV R 4000: an opportunity to enhance
efficiency in the clinical laboratory. Lab Hematol.
1998;156-162.
7. Sahli H. Lehrbuch der klinischen Untersuchungs-Methoden für
€
Studierende und praktische Arzte mit 291 teilweise farbigen
Holzschnitten im Texte und 5 lithographierten Tafeln/von H.
Sahli. 4th ed. Wien, Leipzig: Deutike; 1905.
8. Nakamoto K, Sugibayashi S, Takahashi A, et al. Platelet
count in EDTA-dependent pseudothrombocytopenia—ap-
plication of MgSO4 as an anticoagulant. Rinsho Byori.
1986;34:167-173.
9. Kondo H, Kobayashi E, Itani T, et al. Haematology tests of
blood anti-coagulated with magnesium sulphate. Southeast
Asian J Trop Med Public Health. 2002;33:6-9.
10. Schuff-Werner P, Steiner M, Fenger S, et al. Effective esti-
mation of correct platelet counts in pseudothrombocytopenia
using an alternative anticoagulant based on magnesium salt.
Br J Haematol. 2013;162:684-692.
11. François D, Masure A, Atallah N, et al. Underestimation of
platelet count on magnesium salt-anti-coagulated samples.
Clin Chem Lab Med. 2014;52:e95-e97.
12. Balta S, Demirkol S, Celik T, et al. Mean platelet volume as
a surrogate marker of long-term mortality in patients
undergoing percutaneous coronary intervention. Am J
Cardiol. 2013;112:142.
13. Shah B, Oberweis B, Tummala L, et al. Mean platelet vol-
ume and long-term mortality in patients undergoing percu-
taneous coronary intervention. Am J Cardiol.
2013;111:185-189.
14. Chu SG, Becker RC, Berger PB, et al. Mean platelet volume
as a predictor of cardiovascular risk: a systematic review and
meta-analysis. J Thromb Haemost. 2010;8:148-156.
15. Briggs C, Harrison P, Machin SJ. Continuing developments
with the automated platelet count. Int J Lab Hematol.
2007;29:77-91.
16. Liu Y. Box plots: use and interpretation. Transfusion.
2008;48:2279-2280.
17. Diaz-Ricart M, Brunso L, Pino M, et al. Preanalytical
treatment of EDTA–anti-coagulated blood to ensure stabil-
ization of the mean platelet volume and component meas-
ured with the ADVIA counters. Thromb Res.
2010;126:e30-e35.
18. Thompson CB, Diaz DD, Quinn PG, et al. The role of anti-
coagulation in the measurement of platelet volumes. Am J
Clin Pathol. 1983;80:327-332.
19. Mody M, Lazarus AH, Semple JW, et al. Pre-analytical
requirements for flow cytometric evaluation of platelet acti-
vation: choice of anticoagulant. Transfus Med.
1999;9:147-154.
20. Vogelaar SA, Posthuma D, Boomsma D, et al. Blood sam-
ple stability at room temperature for counting red and
white blood cells and platelets. Vascul Pharmacol.
2002;39:123-125.
21. White JG. Effects of ethylenediamine tetraacetic acid
(EDTA) on platelet structure. Scand J Haematol.
1968;5:241-254.
814 Am J Clin Pathol 2016;145:806-814
814 DOI: 10.1093/ajcp/aqw066
22. Ahnadi CE, Chapman ES, Lépine M, et al. Assessment of
platelet activation in several different anticoagulants by the
Advia 120 haematology system, fluorescence flow cytometry,
and electron microscopy. Thromb Haemost. 2003;90:940-948.
23. Heilmann EJ, Kundu SK, Sio R, et al. Brief communication
comparison of four commercial citrate blood collection sys-
tems for platelet function analysis by the PFA-100TM system.
Thromb Res. 1997;87:159-164.
24. Narasimha A, Kumar H, Prasad CS. Anticoagulant induced
artefacts in peripheral blood smears. Indian J Hematol Blood
Transfus. 2008;24:43-48.
25. Shechter M, Merz CN, Paul-Labrador M, et al. Oral magne-
sium supplementation inhibits platelet-dependent throm-
bosis in patients with coronary artery disease. Am J Cardiol.
1999;84:152-156.
Downloaded from https://academic.oup.com/ajcp/article/145/6/806/2836709 by guest on 23 March 2024
26. Sheu J, Hsiao G, Shen M, et al. Mechanisms involved in the
antiplatelet activity of magnesium in human platelets. Br J
Haematol. 2002;119:1033-1041.
27. Hwang DL, Yen CF, Nadler JL. Effect of extracellular magne-
sium on platelet activation and intracellular calcium mobil-
isation. Am J Hypertens. 1992;5:700-706.
28. Buttarello M. Quality specification in haematology: the
automated blood cell count. Clin Chim Acta. 2004;346:45-54.
29. Bournazos S, Rennie J, Hart SP, et al. Choice of anticoagu-
lant critically affects measurement of circulating platelet-
leukocyte complexes. Arterioscler Thromb Vasc Biol.
2008;28:E2-E3.
€
30. Fonio A. Uber ein neues Verfahren der
Blutpl€attchenz€ahlung. Deutsch Zeitschr Chirurg.
1912;117:176-194.
31. Segal HC, Briggs C, Kunka S, et al. Accuracy of platelet
counting haematology analysers in severe thrombocytopenia
and potential impact on platelet transfusion. Br J Haematol.
2005;128:520-525.
32. Bath P, Butterworth RJ. Platelet size measurement, physi-
ology and vascular disease. Blood Coagul Fibrinolysis.
1996;7:157-161.
33. Lancé MD, Sloep M, Henskens Yvonne MC, et al. Mean
platelet volume as a diagnostic marker for cardiovascular dis-
ease: drawbacks of pre-analytical conditions and measuring
techniques. Clin Appl ThrombHaemost. 2012;18:561-568.
34. Kaito K, Otsubo H, Usui N, et al. Platelet size deviation
width, platelet large cell ratio, and mean platelet volume
have sufficient sensitivity and specificity in the diagnosis of
immune thrombocytopenia. Br J Haematol.
2005;128:698-702.
35. Shah B, Valdes V, Nardi MA, et al. Mean platelet volume re-
producibility and association with platelet activity and anti-
platelet therapy. Platelets. 2014;25:188-192.
36. Lippi G, Pavesi F, Pipitone S. Evaluation of mean platelet
volume with four haematological analysers: harmonisation is
still an unresolved issue. Blood Coagul Fibrinolysis.
2015;26:235-237.
37. Macey M, Azam U, McCarthy D, et al. Evaluation of the
anticoagulants EDTA and citrate, theophylline, adenosine,
and dipyridamole (CTAD) for assessing platelet activation
on the ADVIA 120 haematology system. Clin Chem.
2002;48:891-899.
© American Society for Clinical Pathology