British Journal of Haematology, 2000, 108, 228235

Immunoplatelet counting: a proposed new reference procedure

PAUL H A R R I S O N ,* A L L A N H O RTO N , D ONNA G R AN T,* C A RO L B R I G G S * A ND S AM M AC H I N * *Haemostasis Research,
Department of Haematology, 98 Chenies Mews, University College London WC1E 6HX, UK, and Gulf Coast Pathology,
Cellular Analysis Division, Fort Myers, FL, USA
Received 7 October 1999; accepted for publication 11 October 1999

Summary. Given the high degree of interoperator error and

poor precision of manual platelet counting, it has recently
been proposed that an immunoplatelet counting method
could become the new reference procedure. Platelets are
identied immunologically with a suitable monoclonal
antibody, and the platelet count is derived from the ratio of
uorescent platelet events to collected red blood cell (RBC)
events that are also counted by a reliable and calibrated
standard impedance counter (RBC ratio). In this study, we
have set up a rapid and simple method for immunoplatelet
counting and simultaneously compared the RBC ratio with
the bead ratio derived from two different preparations of
commercial calibration beads (Trucount and FlowCount
beads). Comparison of the level of imprecision of the RBC
ratio with either the manual count or bead ratios revealed a
superior coefcient of variation of < 5% even in samples with

a platelet count < 20 109/l. The RBC ratio correlated

extremely well with the existing manual phase reference
method (r2 093) and especially well with three different
commercial impedance counters and a dual-angle optical
counter (r2 098099). However, at < 100 109/l, the
correlation of the RBC ratio with the dual-angle optical
count (ADVIA 120) (r2 096) was superior to all
impedance counters. This suggests that automated optical
counting methods may be more accurate at determining
platelet counts in thrombocytopenic samples. As the RBC
ratio is rapid, cheap and relatively easy to perform, we
propose that this method could replace the manual count as
a new international reference method.

There is an increasing clinical need for reducing the use of

prophylactic platelet transfusions. This has driven the
platelet transfusion threshold down from 20 109/l to
10 109/l but without a signicant increase in spontaneous
bleeding risk (Rebulla et al, 1997; Ancliff & Machin, 1998;
Norfolk et al, 1998). Given the expense of platelet transfusions, this has resulted in a signicant decrease in blood
bank expenditure. It has also been proposed that the decision
threshold could be reduced further to as low as 5 109/l
provided the clinicians are condent in the reliability of the
count for assessing bleeding risk (Gmur et al, 1991; Murphy,
1992; Ancliff & Machin, 1998). Therefore, in order to
guarantee that the correct and safe clinical decision is made,
it is becoming more critical that platelet counts in severe
thrombocytopenia are not only precise but accurate.
Although modern impedance automated cell counters are
very precise, their inability to resolve platelets from cell
debris and other particulate matter often results in an
overestimate of the platelet count, particularly in severe

thrombocytopenia (Rowan, 1991; Hammerstrom, 1992;

Dickerhoff & von Ruecker, 1995; Springer et al, 1998). In
order to calibrate and assess which type of instrument can
perform the most reliable platelet counts, particularly at
< 20 109/l, one requires an accurate and precise reference
counting procedure (Ault, 1996).
The current international reference method for platelet
counting is performed by a manual procedure using phasecontrast microscopy (England et al, 1988). This method has
a number of signicant limitations and suffers from imprecision, with a typical interoperator coefcient of variation (CV)
in the order of 1025%. Recently, a new immunoplatelet
counting procedure has been advocated as a possible
alternative reference method and is under review by the
International Council for Standardization in Haematology
(ICSH) (Dickerhoff & Von Ruecker, 1995; Tanaka et al, 1996;
Ault et al, 1997; Davis & Bigelow, 1999). The principle of the
methodology involves labelling a whole-blood sample with
an antiplatelet monoclonal antibody (conjugated to a
uorophore) and detecting the platelets by ow cytometry.
The platelet count is simply calculated from the ratio of
uorescent platelet events to the number of red cells detected

Correspondence: Dr Paul Harrison, Haemostasis Research, Department of Haematology, 98 Chenies Mews, London WC1E 6HX, UK.

228

Keywords: immunoplatelet count, CD61, optical platelet

count, impedance platelet count, quality control.

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Immunoplatelet Counting
(RBC ratio). To derive the count, one simply multiplies this
number by the known RBC count in the sample, as
determined by a standard impedance analyser. The main
advantage of the RBC ratio is that, providing the blood
sample is mixed and that coincident events (RBC/RBC and
RBC/platelet) are eliminated by optimal dilution, then the
count obtained is totally independent of potential pipetting
and dilution artifacts. An alternative immunoplatelet
counting procedure uses a known amount of added
uorescent calibration beads to derive the platelet count
(bead ratio) (Dickerhoff & Von Ruecker, 1995; Matzdorff et al,
1998). However, unlike the RBC ratio, this method is
dependent upon very accurate and precise pipetting.
In this study, the aim was to establish an optimized, rapid
and simple immunoplatelet counting procedure that could
simultaneously determine the RBC ratio, with the bead ratio
derived from two different preparations (lyophilized and
suspension) of commercially available calibration beads
(Trucount and FlowCount beads). To compare the efcacy
of these three proposed immunoplatelet counting procedures, data were analysed using three different automated
impedance cell counters and a recently available automated
blood cell counter using two angles of laser light scatter
(ADVIA 120), and also the current manual reference
method.
METHODS
Calibration beads. Trucount absolute count tubes (Becton
Dickinson, Oxford, UK) contain a lyophilized pellet of 42 mm
uorescent beads and are thus very stable. FlowCount beads
(Beckman Coulter, High Wycombe, Bucks, UK) are a suspension of 10 mm uorescent beads and were prepared as
follows. Briey, four bottles of FlowCount were diluted with
an equal volume of Isoton, allowed to stand overnight and
centrifuged at 1000 g for 10 min. The supernatant was
removed, and the pellets were resuspended in one-third of
the original volume and stored at 48C.
Immunoplatelet counting method. All samples were obtained
from standard vacutainer K3EDTA anticoagulated peripheral
whole-blood samples and kept at room temperature for 24 h
or less. All quantities were aliquoted using calibrated positive
displacement pipettes, and the outside of the plastic tip was
carefully wiped with tissue paper to remove excess blood or
solution. Mixed blood (2 ml) was pipetted into the bottom of a
Falcon polystyrene tube (Becton Dickinson). Anti-CD61
uorescein isothiocyanate (FITC; 2 ml; 6 mg/ml; Becton
Dickinson), an antiglycoprotein IIIa monoclonal antibody
(clone RUU-PL 7F12) (Wong & Springer, 1995), was then
immediately pipetted into the same tube but next to and not
mixed with the blood sample. Isoton (6 ml; Beckman Coulter,
High Wycombe, Bucks, UK) was then added, and the blood
was mixed with both antibody and Isoton by pipetting gently
up and down. The tube was then incubated for exactly 1 min
and diluted to exactly 2 ml with Isoton. The relatively short
incubation time of 1 min was demonstrated not to affect the
derived platelet count when compared with longer incubation periods of up to 30 min (data not shown). After gentle
mixing, 05 ml was removed and added to a Trucount tube

229

(Becton Dickinson). Isoton (04 ml) was then added, followed

by 100 ml of the preprepared suspension of FlowCount
(Beckman Coulter).
The nal dilution of whole blood was therefore 1:2000.
Flow cytometric analysis was performed within 2 h using a
Coulter XL ow cytometer (Beckman Coulter). Samples were
analysed at medium ow rate with a forward scatter
discrimination of 10 for either 30 s or, alternatively, until
1000 platelet events (in thrombocytopenic samples) or
50 000 RBC events had been collected.
Flow cytometric data analysis. Plateletred blood cell (RBC)
ratios or plateletbead ratios were calculated from plotted
histograms of cell size (log forward scatter) vs. granularity
(log side scatter) (Fig 1) and uorescence (log FL1 or FL3) vs.
cell size (log forward scatter) (Fig 2). Figure 1 clearly shows
the platelet (A), RBC (B) and two different bead populations
(C and D). FlowCount and Trucount beads are located above
(C) and below the RBC cloud (D) respectively. Figure 2
demonstrates that uorescent platelets (gate C) are resolved
from either noise/debris (gate G), RBC (gate E) and platelet/
RBC coincident events (gate F).The nal platelet counts
determined via the RBC ratio were calculated by dividing
the number of uorescent platelet events (in gate C, Fig 2) by
the number of red blood cell events (gate E, Fig 2) and
multiplying by the known RBC count determined on a
calibrated Coulter STKS automated impedance cell counter
(Beckman Coulter). The nal platelet counts determined
from the bead ratios were also determined in parallel by
multiplying the number of uorescent platelet events (gate C,
Fig 2) by the ratio of total bead number to collected bead
events (Trucount beads are in gate D, Fig 2; FlowCount beads

Fig 1. Flow cytometry scattergram (log FS vs. log SS) of all detected
events (Coulter XL with a FS discriminator of 10) within a blood
sample diluted 1:2000. The platelet cloud is shown on the left-hand
side (A) and is clearly resolved from either the red blood cell cloud (B)
or the two discrete bead populations. FlowCount beads (10 mm) and
Trucount beads (4 mm) are located above (C) and below (D) the red
blood cell cloud (B) respectively.

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P. Harrison et al

Fig 2. Flow cytometry histogram (log FL1 vs. log FS) of all events
collected in Fig 1. The uorescent platelets (gate C) are clearly
resolved from either noise/debris (gate G), RBC (gate E), platelet/RBC
coincident events (gate F) and Trucount beads (gate D). The nal
platelet counts determined by the RBC ratio were calculated by
dividing the number of uorescent platelet events (gate C) by the
number of RBC events (gate E) and multiplying by the known RBC
count. The bead ratio counts were calculated by multiplying the
number of platelet events (gate C) by the ratio of total bead number
to collected Trucount (gate D) or FlowCount bead events (gate H; not
shown) followed by multiplication by the dilution factor of 2000.

are in gate H of a plot of log FL3 vs. log FS; not shown)
followed by multiplication by the dilution factor of 2000.
Manual platelet counts. Two experienced operators performed manual platelet counts, using a standard phase
microscopy method (Brecher et al, 1953; England et al,
1988). A 1:20 dilution of EDTA anticoagulated whole blood
was prepared in a diluent of 1% ammonium oxalate. The
suspension was then mixed on a mechanical mixer for 10
15 min to allow for the complete lysis of all red cells. A clean
dust-free Neubauer chamber was lled with the suspension
and left in a moist chamber for 20 min to allow the platelets
to settle. The preparation was then examined using the 40
objective, the platelets appearing as small but refractive
particles. The total number of platelets appearing in 80 small
squares on the chamber were counted and were equivalent
to the platelet count 109/l. Both sides of the chamber were
counted to check for reproducibility. The nal count was
then taken as the mean value of all four counts.
Automated platelet counts. Platelet counts using an impedance technique were determined on the Coulter STKS, Coulter
Gen-S (Beckman Coulter) and SE-9500 (Sysmex UK) automated cell counters. In the impedance method, a specic
amount of diluted blood ows through a small aperture
located between two sensing electrodes. The red cell and
platelet counts are performed on the same cell suspension
with the platelets being analysed by the number of pulses of
cell size between 2 and 20 (red cells being over 36 ).
Analysis of pulses for the exact platelet count varies in the

different analysers. Indeed, the same manufacturer may

analyse impedance counts slightly differently within distinct
machine models. The Coulter STKS and GEN-S (Beckman
Coulter) apply log normal conversion of the data and
extrapolate the curve to cover the normal size range up to
70 . In contrast, the SE-9500 (Sysmex UK) produces a platelet
size single histogram using three thresholds or discriminators.
One is xed at 12 and the other two hunt the lower (between
2 and 6 ) and upper (between 12 and 30 ) ends of the platelet
population within these limits. These thresholds allow the
analyser efciently to distinguish platelets from debris at the
lower end and red cell fragments at the upper end.
An optical platelet count was determined on the ADVIA
120 (Bayer Diagnostics, Newbury, Berkshire, UK). This
analyser simultaneously measures laser light scatter (using
a solid-state laser diode) at low angle (238) and higher
angle (5158) in the forward direction, in a similar manner
to their red cell analysis. Platelets are analysed between
refractive index values of 135 and 14 up to a size of 60 .
This allows larger platelets to be included in the platelet
count (Stanworth et al, 1999).
RESULTS
Elimination of coincidence events
One of the potential problems with immunoplatelet counting
using ow cytometry is the inability of the ow cell to
discriminate platelet/RBC and/or RBC/RBC coincidence
events (Ault, 1996; Davis & Bigelow, 1999). In order to
optimize the procedure, a series of studies was performed to
determine the inuence of sample dilution and acquisition
rate on both the RBC and bead ratio counts. For example,
low sample dilutions were associated with higher numbers of
platelet/RBC coincidence events and RBC events (Table I).
Dilution curves from 1:125 to 1:8000 at three different ow
rates demonstrated that a 1:2000 dilution with a medium
acquisition rate (data from low and high ow rates are not
shown) are the optimum conditions required to eliminate
coincidence events and thus give an accurate platelet count
(Table I). This is in close agreement with the gure of 1:1800
suggested by Ault (1996).
Precision study
The precision of the RBC and bead ratios was determined by
analysis ( 10) of three samples with low (18 109/l),
normal (181 109/l) and high platelet counts (751 109/l).
A comparison of the 30 s analysis time with data collection
of 1000 platelet events or 50 000 RBC events was also
undertaken. The results are summarized in Table II. The data
clearly show that the CV of the RBC ratio is superior (< 5% in
the normal sample) to either the Trucount ratio or the
FlowCount ratio. With the thrombocytopenic sample, the
RBC ratio CV was excellent (28%) and again superior to
both bead-derived counts (44% and 56%), but only when
1000 platelet events were collected. As a result, any samples
with a platelet count < 50 109/l were subsequently reanalysed to collect at least 1000 platelet events. As the RBC ratio
exhibited superior precision to the bead ratios, all subsequent
analyses were undertaken using this parameter.

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Immunoplatelet Counting

231

Table I. Inuence of blood dilution on the number of total events, RBC events, platelet events, RBC/platelet coincident events (expressed as
absolute number or %), calculated RBC ratio and platelet counts.
Dilution
factor

Events/
second

RBC
events

Platelet
events

RBC
ratio

Calculated
platelet count

RBC/platelet
coincident events

RBC/platelet
(% platelets)

125
250
500
1000
2000
4000
8000

ND
4501
3576
1978
1342
866
636

ND
120887
93136
46173
29230
14407
8272

ND
7329
5785
2719
1642
799
291

ND
0061
0062
0059
0056
0055
0035

ND
296
302
288
273
268
171

ND
802
362
136
54
28
14

ND
109
63
50
33
35
48

A normal sample was analysed at medium ow rate for 30 s. ND, not determined.

counts. However, below 100 109/l, the correlation of the

ADVIA 120 optical count with the RBC ratio was clearly
superior (r2 096). One grossly lipaemic sample, which
was excluded from the correlation, gave very high impedance counts (135, 103 and 183 109/l) relative to both the
immunocount (43 109/l) and ADVIA 120 (59 109/l).

Comparison with manual counts

Any proposed reference method should ideally be compared
with the existing procedure. Forty-two samples with a wide
range of platelet counts (7506 10 9/l) were analysed by
manual, RBC and bead-derived counts. Figure 3 compares
the correlation of the manual method with the RBC ratio.
Figure 3A shows all data (n 42), and Fig 3B focuses at
platelet counts below 100 109/l (n 20). The interobserver
CVs between the four manual determinations varied from 2%
to 48%, with an overall mean of 16%, clearly demonstrating
the problem with the existing reference method.

Examples of aberrant impedance counting

Figure 5 illustrates a total of 41 examples with platelet
counts below < 50 10 9/l, which were found to exhibit
differences between the RBC ratio and impedance counts.
The graph illustrates the effect of two different transfusion
thresholds set at either 10 or 20 109/l on clinical decisionmaking. Making a reasonable assumption that the immunocount was the truest value, then with the transfusion
threshold set at 20 109/l, seven patients would have been
given unnecessary transfusions, as they were underestimated by the impedance counter. Conversely, eight patients
would not have been transfused, as they were overestimated
by the impedance counter and therefore may have exhibited
clinical bleeding problems. In contrast, if the transfusion

Comparison with impedance counters and the ADVIA 120

Eighty-seven samples with a range of 4794 109/l (Coulter
STKS) were analysed by the RBC ratio, bead ratios and the
three different available impedance cell counters. Figure 4
compares the correlations of RBC ratio with the impedance
counters and the ADVIA 120 over the whole range (Fig 4A)
and < 100 109/l (Fig 4B). All three impedance counters
and the ADVIA 120 all correlated extremely well (r2 098
099) with the RBC ratio over the entire range of platelet

Table II. Comparison of coefcients of variation (CVs) obtained with three different
samples with platelet counts of 18, 181 and 751 109/l.
Platelet count

RBC ratio (%)

Trucount ratio (%)

FlowCount ratio (%)

30 s analysis
18
181
751

83
21
50

81
53
74

81
36
73

1000 platelet events

18
28
181
40
751
56

44
71
64

56
71
76

50 000 RBC events

18
75
181
389
751
653

89
714
56

104
699
44

The RBC ratio is compared with the bead ratios obtained with Trucount or
FlowCount. CVs are compared when acquisition was stopped after 30 s, or 1000
platelet events or 50 000 RBC events had been collected.
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P. Harrison et al

Fig 3. Comparison of the immunoplatelet count (derived from the

RBC ratio) with the existing manual phase reference method. The
correlations are shown for all samples (A) and for below 100 109/l
(B).

threshold was set at 10 109/l, then only three patients

would have been overtransfused and four patients undertransfused. Table III illustrates specic examples in which the
impedance platelet count was either overestimated or
underestimated because of the presence of either noise or
large platelets, resulting in counting errors compared with
the RBC ratio count.
DISCUSSION
The goal of this study was to set up an optimized platelet
immunocounting procedure and demonstrate its potential as
an international reference method. Indirect platelet immunocounts can be derived from either the RBC or bead ratios,
which require either an accurate RBC count (performed by a
calibrated impedance counter; Dickerhoff & Von Ruecker,
1995; Tanaka et al, 1996; Ault et al, 1997; Davis & Bigelow,
1999) or the addition of a known quantity of added calibration
beads (Matzdorff et al, 1998). Platelets are identied immunologically with a suitable monoclonal antibody (e.g. antiCD61-FITC) and are thus resolved from both noise/debris
and red cell events. Providing the sample is optimally diluted
(1:2000) before analysis at the medium acquisition rate on
the Coulter XL, the number of coincidence events is reduced
to a level that does not inuence the nal derived platelet
count signicantly. Ault (1996) has previously recommended a
dilution factor of at least 1:1800, whereas Davis & Bigelow
(1999) have recently demonstrated that blood should ideally

be diluted by at least 1:400 with an acquisition rate of

< 4000 cells/s. The precision of the RBC ratio was superior to
that of the bead ratio derived from either a lyophilized
preparation (Trucount) or solution of beads (FlowCount). As
the RBC ratio is independent of potential pipetting and
dilution artifacts that may inuence the bead ratio, this was
not totally unexpected. Even within thrombocytopenic
samples, providing at least 1000 platelet events are collected,
the level of precision was excellent (< 5%). Upon comparison
of the RBC ratio with the current reference procedure
(manual count), both methods correlated well (Fig 3),
although the manual procedure exhibited a mean CV of
16% even when two experienced laboratory technologists
counted the samples in duplicate. Comparison of the
proposed RBC ratio method with three different existing
commercial impedance cell counters and one optical counter
demonstrated that this method has potential as a new
calibration reference method (Fig 4A). However, compared
with impedance counts, this method will not be interfered
with by the presence of cellular debris, which can often result
in the impedance counter overestimating the count,
especially in severely thrombocytopenic samples (< 20
109/l; see Fig 5 and Table III). Conversely, in patients with
macrothrombocytopenia and ITP, most impedance counters
cannot discriminate large platelets, often resulting in an
underestimate of the platelet count (see Fig 5 and Table III).
Interestingly, the ADVIA 120 optical count, which not only
eliminates noise but also counts large platelets, appears to
compare favourably with the immunocount, especially at
low counts of < 100 109/l (Fig 4B). This is in agreement
with previous studies using the CELL-DYN 4000 (Abbott),
which demonstrated a superior correlation between
immunocounts and optical counts (Ault, 1996; Ault et al,
1997).
Although the immunoplatelet count derived from the RBC
ratio has clear advantages over the existing reference
method, there are a number of potential, albeit rare,
problems that must be recognized. Samples containing
signicant numbers of platelet aggregates, platelet white
cell complexes, microparticles and very large platelets may
result in gating problems. Upper and lower platelet gates
within the log FS vs. FL1 plot can be placed to discriminate
single platelets from aggregates, cell complexes or microparticles (Matzdorff et al, 1998). Also, depending upon the
target antigen of the antibody used to measure the
immunoplatelet count (e.g. CD41, CD42b or CD61), where
there is absence of certain glycoproteins (e.g. Bernard Soulier
syndrome and Glanzmann's thrombasthenia), then no
uorescent platelets will be detectable. Furthermore, it is
also possible that, when patients have signicant levels of
platelet autoantibodies or are being treated with antiplatelet
glycoprotein therapy (e.g. anti-GpIIb/IIIa antibodies such as
Reopro), these could interfere with the assay. Although these
problems are rare, possible strategies include using a novel
antibody that targets a unique epitope with no autoantibody
specicity or using a panel of antibodies to a number of
different antigens on the platelet surface.
In summary, the immunoplatelet counting procedure
described is a rapid, simple, reproducible and cheap method,

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P. Harrison et al
which could be adopted by any laboratory with suitable ow
cytometric experience. It is suggested that this methodology
could replace the existing manual reference method and
provide important information concerning the accuracy of
platelet counting by conventional impedance and optical
counters in severe thrombocytopenia. Providing accuracy
can be guaranteed at around the current platelet transfusion
threshold of 10 109/l, then the most appropriate clinical
decision is more likely with regard to prophylactic platelet
support (Kickler et al, 1998). Furthermore, improved
accuracy of platelet counts at this level may demonstrate
that the transfusion threshold could be decreased to as low
as 5 109/l without any increase in the risk of spontaneous
bleeding. A further reduction in this threshold would result
in a dramatic reduction in costs by decreasing the frequency
of unnecessary platelet transfusions.

Fig 5. Illustration of the effect of inaccurate impedance counts on

platelet transfusion decision-making. Aberrant samples were compared by immunoplatelet counting (derived from the RBC ratio) and
a conventional impedance counter. The graph illustrates two
different transfusion thresholds set at either 20 10 9/l (dotted
lines) or 10 10 9/l (solid lines). Impedance counts that were either
underestimated or overestimated are highlighted by arrows.

Table III. Examples of impedance counting errors when compared

with the immunocount derived from the RBC ratio.

ACKNOWLEDGMENTS
The authors are grateful to Beckman Coulter, Miami, FL,
USA, for providing FlowCount beads. We are also indebted to
Nigel Llewellyn-Smith (Becton Dickinson, Oxford, UK) and
David Warunek (Becton Dickinson, Franklin Lakes, NJ, USA)
for providing Trucount tubes and the anti-CD-61 (FITC)
antibody. We would also like to thank Sue Mead (Bayer
Diagnostics, Newbury, UK), Andy Hay (Sysmex, Milton Keynes,
UK) and Sandy Piepho (Beckman Coulter, Miami, FL, USA) for
the provision of their respective automated cell counters.
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33

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q 2000 Blackwell Science Ltd, British Journal of Haematology 108: 228235