ORIGINAL ARTICLE INTERNATIONAL JOURNAL OF LABORATO RY HEMATO LOGY

Erroneous automated optical platelet counts in 1-hour

post-transfusion blood samples
E. MAURER-SPUREJ* , , C. PITTENDREIGH*, J. YAKIMEC , M. HUDOBA DE BADYN , , K. CHIPPERFIELD ,

*Canadian Blood Services, SUMMARY

University of British Columbia,
Vancouver, BC, Canada Thrombocytopenic patients with acute leukemia may show high post-

Department of Pathology &
Laboratory Medicine and Centre
for Blood Research, University of
British Columbia, Vancouver, BC,
Canada
transfusion count increments that significantly exceed the number of
transfused platelets. This study demonstrates that the automated
hematology analyzer Sysmex XE-2100 reports erroneously high optical
platelet counts when the blood sample contains particles in the size

Vancouver General Hospital, range of platelets or smaller. Thrombocytopenic or low-normal whole

Vancouver, BC, Canada

Division of Hematopathology,
Vancouver Acute, Vancouver, BC,
Canada
Correspondence:
Dr Elisabeth Maurer-Spurej,
University of British Columbia,
Centre for Blood Research, 2350
Health Sciences Mall, Vancouver,
BC, Canada V6T 1Z3.
Tel.: +1 604 827 5993;
Fax: +1 604 822 7135;
E-mail: emaurer@interchange.
ubc.ca
doi:10.1111/j.1751-553X.2008.01097.x
Received 31 January 2008;
accepted for publication 7 July
2008
blood samples were spiked with 1 lm latex beads (n = 14) to mimic
contaminants under controlled conditions. Optical and impedance
measurements of spiked and control samples with the Sysmex XE-2100
were compared with the Advia 120 and the manual counts. The added
beads unexpectedly increased the automated optical platelet counts in
the Sysmex XE-2100 and, to a lesser extent, the Advia 120 (Wilcoxon
signed ranks test, P < 0.05), while the beads were not included in the
impedance or the manual microscopic platelet counts. Differential
interference contrast microscopy was used to investigate samples from
platelet concentrates for transfusion. Platelet concentrates (32/128)
were identified as possible sources for particles that were microscop-
ically distinct from platelets but would be included in the automated
optical count. Transfusion of platelet concentrates containing contam-
inating particles can lead to unexpectedly high post-transfusion platelet
counts and misdiagnosis of thrombocytopenic patients.

Keywords
Platelets, platelet count, thrombo-
cytopenia, hematology analyzer,
platelet transfusion

detection methods the accuracy of automated cell

INTRODUCTION
The clinical use of automated hematology analyzers
started in the early 1960s and has since dramatically
progressed. With the combination of different
 2008 The Authors
Journal compilation  2008 Blackwell Publishing Ltd, Int. Jnl. Lab. Hem.
counts was vastly enhanced. For example, the combi-
nation of impedance and optical platelet counts in the
Sysmex XE-2100 has particularly benefited the enu-
meration of platelets in severe thrombocytopenia
1
2 E. MAURER-SPUREJ ET AL. ERRONEOUS AUTOMATED PLATELET COUNTS
(Briggs, Harrison & Machin, 2007). However, the accu-
racy of automated platelet counts has seen renewed
interest because of recent reports of erroneous low
platelet counts (Zandecki et al., 2007; Diquattro et al.,
2008) and the ongoing discussion to reduce the trans-
fusion trigger for prophylactic transfusions (Heddle
et al., 2006).
Prophylactic platelet transfusions are the standard
care for patients with acute leukemia when their plate-
let count reaches the transfusion trigger, generally 10
or 20 109/l (Schiffer et al., 1986; Heddle et al., 2006).
The determination of pretransfusion platelet counts in
patient samples is an important application for auto-
mated hematology analyzers although it has long been
recognized that the accuracy of automated hematology
analyzers decreases with decreasing platelet numbers
(Segal et al., 2005). It is well known that automated
hematology analyzers, irrespective of the measurement
technique, can fail to discriminate platelets from other
cell fragments and debris. The introduction of the
International Reference Method (IRM) (Harrison et al.,
2001) has been a major improvement in the calibra-
tion of automated platelet counts but it requires access
to expertise in immunofluorescence flow cytometry
(Segal & Harrison, 2006). Adding to the discussion on
the reliability of the optical platelet counts compared
with the impedance counts (Briggs & Machin, 2006)
we report here that optical platelet counts were falsely
increased when 1-lm sized foreign particles were
added to blood samples.
The maximal increase in platelet count in the reci-
pient of a platelet transfusion is expected to be equiv-
alent to the transfusion dose, which is the number of
transfused platelets corrected for the blood volume of
the recipient. Depending on the platelet product, the
platelet dose ranges from 9 109/l for three pooled
whole blood derived platelet units to 39 109/l for
apheresis platelets based on a 5 l blood volume. In a
recent pilot study we found that approximately 25%
of platelet transfusions (32/128) caused an increase of
the 1-h post-transfusion platelet count in recipients,
which significantly exceeded the expected limits
(19 5 109/l). To explain these discrepancies, we
tested (i) the possibility that the automated hematol-
ogy analyzer Sysmex XE-2100 includes particles other
than platelets in the platelet counts and (ii) whether
platelet concentrates could be the source for these
particles. We report the results of a proof-of-principle
study to determine whether additional particles can
be present in platelet transfusions and could cause the
observed platelet count abnormalities.
MATERIALS AND METHODS
Study population
This study was approved by the Institutional Ethics
Review Committee of the University of British
Columbia and Vancouver Hospitals and was con-
ducted in accordance with the Declaration of Helsinki.
Fourteen healthy volunteers gave written informed
consent prior to donating whole blood, which was
platelet depleted and analyzed in the absence or after
the addition of latex beads. Forty-nine patients with
acute leukemia were enrolled at the Vancouver Gen-
eral Hospital and gave written informed consent to
allow us access to their routine laboratory results.
Platelet counts 16 h before (precount) and 1 h after
the administration of a platelet transfusion (1 h post-
count) were recorded. We compared the measured
and the predicted platelet count increments for 128
transfusions.
Laboratory tests
The platelet count of normal whole blood samples
(EDTA vacutainer; Becton Dickinson, Oakville, ON,
Canada) was reduced by differential removal of plate-
lets and not by dilution. Whole blood was centrifuged
at 150 g for 12 min. Platelet poor plasma was obtained
by centrifugation of the supernatant platelet rich plasma
(PRP) at 2000 g for 15 min and was added back to the
red blood cell fraction. Differences in nal platelet
counts of these articially thrombocytopenic (range 20
60 109/l) or low-normal whole blood samples (range
133173 109/l) were probably due to donor variations
and the age of the blood samples. Subsequently, platelet
depleted samples were split and latex beads (Polybead,
1.0 lm diameter, cat. #07310; Polysciences, Warring-
ton, PA, USA) were added to one aliquot (+Beads)
while the other aliquot served as the control sample.
The automated hematology analyzer Sysmex XE-2100
(Sysmex, Mississauga, ON, Canada) was used in the
impedance as well as the optical mode. For the man-
ual platelet counts, EDTA-anticoagulated whole blood
was diluted with 1% ammonium oxalate (1 : 20),
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Journal compilation  2008 Blackwell Publishing Ltd, Int. Jnl. Lab. Hem.
 E. MAURER-SPUREJ ET AL.
mixed well and incubated for 5 min at room tempera-
ture, applied to the hemocytometer (VWR, Toronto,
ON, Canada) and allowed to settle for 1520 min
prior to cell counting on the light microscope (40
dry objective). Samples were also counted with the
Advia 120 hematology analyzer (Bayer, Tarrytown,
NY, USA), which uses an optical detection method
that is different from the Sysmex XE-2100 utilizing
two scattering detectors at 23 and 515.
One hundred and twenty-eight post-transfusion
remnants of platelet concentrates were sampled using
aseptic technique. Samples were fixed with buffered
paraformaldehyde (2% final concentration) and
imaged on a Leica DMRA2 microscope (Leica, Rich-
mond Hill, ON, Canada) with a differential interfer-
ence contrast (DIC) attachment, 100 oil-immersion
objective (NA = 1.4) and a digital camera (Retiga EXi
Fast Cooled Mono 12-bit; QImaging, Surrey, BC, Can-
ada). This setup was also used to image whole blood
samples in the absence and presence of latex beads
because this microscopy technique yields high resolu-
tion images suitable for publication.
Calculation of the transfusion dose
The following equations were used to calculate the
predicted 1 h post-transfusion platelet counts
(Table 2).
 
predicted 1 h postcount 109 =l precount platelet dose
1 spiked samples
  VT  platelet content  100
platelet dose 109 =l 2 Figure 1a shows a representative optical scatter plot of
BV VT
VT is the approximate volume of the platelet transfu-
sion. For a transfusion of pooled random donor platelet
rich plasma (PRP) units VT = n60 ml, with n being
the number of single units pooled. For apheresis
platelets or buffy coat pools VT = 360 ml. The mini-
mally required platelet content of platelet concentrates
derived from whole blood by the PRP method is
5.5 1010 platelets/unit (60 ml) and 2.8 1011 plate-
lets/pool (360 ml) for buffy coat platelets (Maurer-
Spurej & Chipperfield, 2007). The platelet content of
single donor platelets derived by apheresis was mea-
sured and ranged from 2.3 to 5.9 1011 platelets/unit
(360 ml). VT/(BV + VT) is the dilution factor for the
platelet content of the product after transfusion. BV is
the patient blood volume and is calculated as BV
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Journal compilation  2008 Blackwell Publishing Ltd, Int. Jnl. Lab. Hem.
ERRONEOUS AUTOMATED PLATELET COUNTS 3
(ml) = 70body weight (kg). For obese patients the
blood volume was calculated according to (Lemmens,
Bernstein & Brodsky, 2006):
70
BV q
  3
weight= 22  height2
The unit for weight is (kg) and for height is (cm).
Statistical analysis
Statistical analysis was performed using the statistics
software SPSS for Windows, version 16.0 (SPSS Inc.,
Chicago, IL, USA). The nonparametric Wilcoxon
signed ranks test for paired data was used to deter-
mine the statistical significance of the differences
between automated platelet counts of control and
spiked blood samples. This test gives exact probabili-
ties for small sample sizes without knowledge of their
distribution. For the calculation of the statistical differ-
ence between measured and predicted 1 h post-
transfusion platelet counts from thrombocytopenic
leukemia patients the Students t-test for paired sam-
ples was used. The normal distribution of the data sets
was verified with Q-Q plots.
RESULTS
Automated and manual platelet counts in control and
a thrombocytopenic whole blood sample (control)
obtained with the Sysmex XE-2100. The optical scat-
ter plots of thrombocytopenic whole blood samples
spiked with latex beads showed that nonfluorescent
beads were counted as platelets (Figure 1b). The
addition of approximately 20 109 latex beads /l
increased the platelet counts of 10 artificially throm-
bocytopenic and four samples with low-normal plate-
let counts (Figure 1c, black bars) compared with the
control samples (white bars). Latex beads were used
to mimic natural particles that could affect the optical
platelet count due to their size and shape. As
expected, the relative error caused by the added parti-
cles decreased with increasing platelet counts.
The Advia 120 also uses an optical method to enu-
merate platelets. However, the measured parameters
4 E. MAURER-SPUREJ ET AL. ERRONEOUS AUTOMATED PLATELET COUNTS
(a) (b)
(c)
Figure 1. Automated optical platelet counts obtained
with the Sysmex XE-2100 analyzer. Optical scatter
plots of representative thrombocytopenic whole
blood samples (RBC: red blood cells): control (plot a)
and spiked with approximately 20109/l latex beads
(plot b). Non-fluorescent beads were counted as
platelets (c). Artificially platelet-depleted whole blood
was analyzed before (white bars) and after spiking
with latex beads (black bars).
are 515 scatter vs. 23 scatter. The collected events
are gated by refractive index thresholds. Figure 2 shows
the representative scatter plots from a control (a) and a
spiked sample (b) obtained with the Advia 120. Differ-
ential interference contrast microscopy was used to
illustrate the difference between thrombocytopenic
whole blood samples without beads (c) and samples
with added beads (d). Beads did not affect the manual
platelet counts because of the visible difference. Experi-
enced laboratory technologists ignore particles that do
not resemble platelets and therefore, the manual plate-
let counts of the spiked and the control aliquots were
the same. Latex beads were also not counted by the
Sysmex XE-2100 impedance method, probably due to
the small volume and the solid composition of the
beads. The results from the different automated hema-
tology analyzers are summarized in Table 1. The Wilco-
xon signed ranks test was used to determine whether
the difference between the control samples and the
samples spiked with beads were statistically significant.
(a) (b)
(c) (d)
Figure 2. Advia 120 counts and DIC microscopy. Opti-
cal scatter plots of representative thrombocytopenic
whole blood samples in the absence (a) and presence
of latex beads (b). The Advia 120 counted 25% of
the added beads as platelets. Microscopic inspection
of control (c) and spiked samples (d) showed clear
differences between beads and platelets. The main
cell population seen in the micrographs are aged
RBC (mostly echinocytes). The scale bars represent
5 lm.
The platelet counts of artificially thrombocytopenic
(n = 10) and low-normal (n = 4) control samples
(without beads) were compared with the corresponding
samples spiked with latex beads (+Beads). Platelet
counts were obtained with the Sysmex XE-2100 (opti-
cal and impedance method) and the Advia 120 (optical
method). The delta (D) values are the differences
between the control and the spiked (+Beads) sample
counts. Although the Advia 120 included fewer beads
into the platelet count than the optical count from the
Sysmex XE-2100, both optical methods resulted in sta-
tistically significant erroneous platelet counts for
thrombocytopenic samples (Wilcoxon P < 0.05). In
contrast, no significant differences were found with the
impedance method or for higher platelet counts.
Unexpectedly high 1 h post-transfusion platelet counts
obtained with the Sysmex XE-2100
Using equation (1) the predicted 1 h post-transfusion
platelet counts were calculated and compared with the
measured counts. The pairs were grouped depending
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 E. MAURER-SPUREJ ET AL. ERRONEOUS AUTOMATED PLATELET COUNTS 5

Table 1. Automated platelet counts (109/l) and Wilcoxon signed ranks test

Sysmex XE-210 imped-

Sysmex XE-2100 optical ance Advia optical

Control +Beads D Control +Beads D Control +Beads D

Thrombocytopenic samples 20 47 27 24 24 0 24 28 4
22 42 20 22 24 2 22 24 2
25 52 27 28 29 1 23 29 6
25 53 28 25 26 1 24 31 7
26 52 26 31 30 )1 28 34 6
31 60 29 38 37 )1 33 39 6
39 64 25 44 45 1 43 47 4
40 58 18 45 45 0 46 55 9
48 74 26 50 54 4 49 61 12
60 82 22 65 64 )1 58 63 5
N 10 10 10 10 10 10
Mean 33.6 58.4 37.2 37.8 35 41.1
SD 12.9 12.2 13.8 13.8 13 14.4
Significance (2-tailed) 0.005
0.272 0.005
Low-normal platelet count 133 146 13 161 155 )6 152 141 )11
138 157 19 164 156 )8 157 148 )9
164 176 12 186 180 )6 175 160 )15
173 186 13 200 197 )3 191 185 )6
N 4 4 4 4 4 4
Mean 152 166.25 177.75 172 168.75 158.5
SD 19.5 18.1 18.6 20.3 17.8 19.3
Significance (2-tailed)
0.066 0.066 0.068

the measured and predicted 1 h post-transfusion plate-

Table 2. Paired t-test analysis of 1 h post-transfusion let counts showed no statistically significant difference
platelet counts (109/l) measured with the Sysmex
(P = 0.504 at the 95% confidence level).
XE-2100 compared with predicted values

1-h post- Significance

transfusion t-test Detection of additional particles in platelet transfusions
Platelet Count N Mean SD t (2-tailed) by microscopy

Differential interference contrast (DIC) microscopy

Measured 96 35.1 11 0.671 0.504
Predicted 96 34.7 10 images were taken from residual post-transfusion
Measured 32 57.4 18 14.415 0.0001 samples of the platelet concentrates to demonstrate
Predicted 32 37.2 16 that additional events could originate from transfu-
sions. Figure 3 shows DIC micrographs representa-
on whether their difference was  or >11 (two stan- tive of 32 similar samples that caused unexpectedly
dard deviations of the mean estimated platelet dose to high 1 h post-transfusion platelet counts. Arrow-
allow a margin that accounts for the estimation errors heads mark small, smooth particles in contrast to
in equation 2). In 25% of cases (32/128) an unexpect- platelets. Panel (a) shows that platelet concentrates
edly high 1 h post-transfusion platelet count was mea- might contain a high number of particles other than
sured with the Sysmex XE-2100 (paired t-test platelets and panel (b) illustrates the size and mor-
P = 0.0001 at the 95% confidence level). The results phological differences between platelets and these
are summarized in Table 2. In 75% of cases (96/128) particles.
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Journal compilation  2008 Blackwell Publishing Ltd, Int. Jnl. Lab. Hem.
6 E. MAURER-SPUREJ ET AL. ERRONEOUS AUTOMATED PLATELET COUNTS
(a)
Figure 3. Differential interference contrast (DIC) micrographs of a post-transfusion platelet concentrate sample rep-
resentative for 32 similar samples that caused unexpectedly high post-transfusion platelet counts. Arrowheads mark
particles that differ from platelets to illustrate their relative abundance (a) and the difference in size and morphol-
ogy (b). The scale bars represent 5 lm (a) and 1 lm (b).
DISCUSSION
The maximal post-transfusion platelet recovery in the
recipient is not expected to exceed the platelet con-
tent of the transfusion. The platelet content of platelet
concentrates produced by different methods has to
meet regulatory requirements (Moroff, 2008).
Although, at the time of transfusion, the actual num-
ber of single platelets in a platelet concentrate is
unknown, a close estimate of the platelet dose con-
tained in a transfusion can be calculated. The presence
of other particles in a platelet concentrate is equally
unknown but, if transfused to a thrombocytopenic
patient, might falsely elevate post-transfusion platelet
counts.
Latex beads are not physiological particles but
they offer the possibility for controlled spiking exper-
iments by adding known concentrations of single
particles to blood samples. Latex beads have to be
selected carefully for surface characteristics that pre-
vent spontaneous aggregation in physiological salt
solutions. The beads used in this study slowly formed
dimers but did not interact with platelets. The vol-
ume of 1-lm diameter beads is approximately
0.510)15 l, according to 4r3p/3 (r being the radius
of the sphere). This is well below the volume cut-off
for the impedance detection. In addition, the solid
plastic material of the latex beads causes their imped-
ance signals to be very different from the signals
originating from platelets because the measure of
impedance is a combination of particle resistance,
(b)
inductance, and capacitance to the alternating cur-
rent passing through the particle.
On the contrary, the optical size measurement is
based on the scattering intensity, with large particles
scattering much more light in forward direction than
smaller particles. However, another important factor is
the optical contrast, or difference in refractive indices,
between the particles and the surrounding medium.
The refractive index difference of cells and the
surrounding plasma primarily depends on the cellular
composition. Solid latex beads or nucleated cells have
a higher contrast leading to a higher scattering
intensity than platelets. This can also be observed
microscopically by the high brightness of the
beads compared with the anucleated, low-contrast
platelets (Figure 2d). Thus, despite the smaller size of
the beads (approximately 0.510)15 l vs. approxi-
mately 910)15 l) the scattering intensity from the
beads is well in the range of the platelet scattering
(height along the y-axis in Figure 1b). Even the latex
bead dimers can be seen as an individual population
in the scatter plot above the main population of beads
(Figure 1b), which was verified by microscopy (data
not shown).
The inaccuracy of automated platelet counts from
thrombocytopenic samples is well documented
(Harrison et al., 2001; Briggs, Kunka & Machin, 2004).
However, unexpectedly high post-transfusion platelet
counts have so far not been addressed. Spontaneous,
transient, and unexpectedly high post-transfusion
count increments seen in approximately 25% of our
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 E. MAURER-SPUREJ ET AL. ERRONEOUS AUTOMATED PLATELET COUNTS 7

patients with acute leukemia cannot be explained
with platelet redistribution triggered by platelet trans-
fusion, or by marrow recovery. While intravenous
immunoglobulin treatment of thrombocytopenic
patients is known to release platelets from the liver
and other stores (Heyns Adu et al., 1986; Tanaka et al.,
2005), unexpectedly high post-transfusion platelet
counts are likely caused by erroneous counts of addi-
tional particles contained in the transfusion. These
particles could be blood cell fragments, bacterial con-
taminants or protein aggregates (von Ahsen et al.,
2001; Latif et al., 2003; Sysmex Europe Science,
2005; Branda & Kratz, 2006). Their characterization is
a focus of our ongoing research.
Our data support the previously reported finding
by Briggs, Kunka and Machin (2004) that optical
platelet counts obtained with the Sysmex XE-2100 are
less accurate than the impedance counts. We recom-
mend the use of the Sysmex XE-2100 impedance
counting method for samples from thrombocytopenic
leukemia patients because the risk for erroneously
high platelet counts by the optical method increases
with decreasing transfusion trigger. The introduction
of a gate in the Sysmex scatter plot could reduce the
spurious counts. Our data also highlight the necessity
for pretransfusion quality determinations of platelet
concentrates. New technology for noninvasive, pre-
transfusion testing exists but is not yet commercially
available (Maurer-Spurej et al., 2006; Maurer-Spurej
& Chipperfield, 2007; Maurer-Spurej, Labrie & Brown,
2008).
ACKNOWLEDGEMENTS
We thank Prof. Paul Harrison for helpful comments
on the size exclusion of particles in automated hema-
tology analyzers. This work was supported by a
research grant from Canadian Blood Services to EMS.

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