Clin. Lab. Haem.
2001, 23, 181±186
A. GIACOMINI*,
P. LEGOVINI ,
G. GESSONI*,
F. ANTICO*,
S. VALVERDE*,
M.M. SALVADEGO*,
F. MANONI*
Platelet count and parameters determined
by the Bayer ADVIATM 120 in reference
subjects and patients
*Department of Clinical Pathology, Chioggia Hospital, Venice, Italy and
Department of Pure and Applied Mathematics, University of Padua, Padua, Italy
Summary
In order to study the behaviour of traditional and new platelet parameters determined
by the ADVIATM 120 Hematology System, ®ve hundred samples from reference subjects, divided for sex and age, were processed. Signi®cant variations on the basis of sex
and age were found. Reference ranges as 95% con®dence limits were therefore calculated for each age class, and platelet parameters proved to have speci®c variations
during lifetime. Moreover, one hundred samples from thrombocytopenic patients were
processed by the ADVIATM 120 System. When compared with those of reference
subjects matched for sex and age, all platelet parameters, except mean platelet component (MPC), showed signi®cant differences.
Keywords
Platelets, mean platelet component, reference range, ADVIATM 120 System,
thrombocytopenia
Introduction
Thanks to recent advances in automatic blood cell
counting, a fast, accurate count and volume determination of platelets can be made. Moreover, platelet parameters (Chapman et al., 1996) such as platelet-crit (PCT),
mean platelet component (MPC) and mean platelet mass
(MPM), can be determined together with their distribution
widths. In particular, a modi®ed version of the two-angle
laser light scattering ¯ow-cytometric method used by
Bayer H* System Hematology analysers (Bayer Corporation, Tarrytown, NY) for RBC analysis has been developed
in the ADVIA System Hematology (Bayer Corporation,
Tarrytown, NY) to measure platelet parameters (Stanworth et al., 1999; Kunicka et al., 2000).
With this method, platelets can be distinguished from
RBC on the basis of size and refractive index. They are
identi®ed and then classi®ed according to their individual
Accepted for publication 11 June 2001
Correspondence: Alda Giacomini, MD, Laboratorio Analisi, Ospedale
Civile, Via Madonna Marina 500, 30019, Chioggia (VE), Italy.
Tel.: + + 39 0415534408/15; Fax: + + 39 0415534401; E-mail:
legovini@math.unipd.it
Ó 2001 Blackwell Science Limited
scattering patterns. The pair of scattering signals collected
for each platelet is converted into a corresponding pair of
cell volume and refractive index values by applying Mie's
scattering theory (Kerker, 1969). The platelet refractive
index is related linearly to platelet density, which is a
measure of the overall concentration of components within
a platelet, and, indirectly, is also an index of the platelet
activation state (Zelmanovic & Hetherington, 1998).
The new method determines cell-by-cell MPC and MPM
and displays their frequency histogram. The platelet count
and MPV can also be recorded and a platelet size
frequency histogram displayed. By their nature, platelets
are a sensitive indicator of health and disease in vivo but
can be technically dif®cult to analyse in vitro owing to
rapid changes in dimension, shape and content. It has
been claimed that this two-dimensional platelet analysis is
more reliable in obtaining accurate platelet counts
(Dorfman et al., 1997) and permits better discrimination
between platelets of all sizes and other particles (Fisher
et al., 1997). It has also been suggested that MPC is a
potentially useful screening test for platelet activation and
hence an indicator of patients at risk of thrombosis or
bleeding complications (Macey et al., 1998).
181
182
Platelet parameters in reference subjects and patients
ADVIATM 120 Hematology System is a relatively new
analyser; little is known about the reference ranges of its
new platelet parameters in reference subjects as well as
patients (Brummitt & Barker, 2000). The aim of the
present study was therefore to investigate the behaviour of
traditional and new platelet parameters, determined by
the ADVIATM 120 Hematology System, in reference
subjects divided on the basis of age and sex, to de®ne
reference ranges for clinical practice and to compare the
performance of platelet parameters between reference
subjects and thrombocytopenic patients.
liver cirrhosis and the other 6 were patients in chemotherapy. Samples had a platelet count of less than 100
(109/l).
Venous blood samples were drawn into 4.5 ml Terumo
Venoject Tubes containing K3EDTA anticoagulant and
tested within one hour of collection to minimize variations due to sample age. Samples were kept at room
temperature and platelet parameters were determined as
presented in Table 1. For a technical description of the
two-dimensional platelet analysis see Kunicka et al.
(1998).
Materials and methods
Statistics
Five hundred samples from reference subjects, who had
given their fully informed consent before participating in
the study, were processed for full blood count analysis
using the ADVIATM 120. The group consisted of 250
males and 250 females aged 1±85 years (mean age
36 years). Subjects aged 18±65 years were blood donors;
the other subjects were selected in accordance with the
National Committee for Clinical Laboratory Standards
H20-T speci®cations (NCCLS, 1992) from the following
groups: 1±10 years: screening in the open population
for thalassemia syndrome, 10±18 years: certi®cation for
competitive sport activities, over 65 years: subjects
attending our Laboratory for periodic check-up.
Reference subjects were divided into 10 groups on the
basis of sex and age. Age classes were: 1±10 years,
10±18 years, 18±45 years, 45±65 years, and over
65 years. Each class was divided in two groups: 50 males
and 50 females.
Patient samples were taken from Hospital inpatients ±
50 males and 50 females ± aged 25±85 years, on the basis
of platelet count. Of these patients, 94 were affected by
In order to select the appropriate test for comparing
platelet parameters between sex and/or age class, and
between reference subjects and patients, the ShapiroWilks W statistic was used to test samples for normality.
The normality hypothesis could not be rejected for only
two indices, namely PDW and PCDW. Non-parametric
tests were therefore chosen for inference and percentiles
were adopted to construct reference limits.
Differences due to age and sex, and between reference
subjects and patients were detected using the Mann±
Whitney U-test.
Statistical calculations were performed using the SAS
SystemÓ v6.12 (SAS Institute Inc., Cary, NC, USA), the
analysis tools of Excel97Ó (Microsoft, Redmond, CA, USA)
and the Excel add-in Morefun.xll v2.61 (L. Longre, OSR
RhoÃne-Alpes, France).
Results
Platelet parameter distributions were found not to be
Gaussian. Non parametric tests were therefore used to
Table 1. Platelet parameters determined by the ADVIATM 120 Hematology System. Modi®ed from Kunicka et al. (1998)
Parameter
Abbreviation
Units
Description
Platelet count
PLT
109/l
Mean platelet volume
Platelet volume
distribution width
Platelet-crit
Mean platelet component
MPV
PDW
¯
%
Platelets are identi®ed based on cell volume (0±60¯) and refractive index
values (r.i. = 1.35±1.40)
MPV is calculated from the platelet volume histogram; range 0±60 ¯
Distribution width of platelet volume histogram
PCT
MPC
%
g/dl
PCDW
g/dl
Percent of blood volume occupied by platelets
MPC is calculated from the platelet component histogram re¯ective of
platelet density: range 0±40 g/dl
Distribution width of PC histogram
MPM
PMDW
pg
pg
MPM is calculated from the platelet dry mass (PM) histogram; range 0±5 pg
Distribution width of PM histogram
Platelet component
distribution width
Mean platelet dry mass
Platelet dry mass
distribution width
Ó 2001 Blackwell Science Ltd., Clin. Lab. Haem., 23, 181±186
A. Giacomini et al.
compare for sex in each of the age classes. Signi®cant
differences were found for PLT, PCT and MPC in the
45±65 years group only, where females presented higher
PLT and PCT and lower MPC values than males.
A comparison between successive age classes using the
Mann±Whitney U-test showed that all platelet parameters, except PCDW, presented signi®cant differences
(P < 0.001) in the ®rst three age groups. Comparisons
involving the 45±65 years group were performed separately for sex because of the signi®cant sex-related
differences found previously.
MPC and MPM were signi®cantly (P < 0.001) lower in
males over 65 than in subjects 45±65 years old. For
females only MPC was signi®cantly lower in both
comparisons and MPM was signi®cantly lower when
comparing the 18±45 and 45±65 age groups. No
signi®cant differences in platelet parameter distribution
Figure 1. Boxplots of PLT, MPV and
MPC, separate for sex for each age class.
Hinges show the ®rst and third quartiles,
the inside cross-line indicates the median,
the whiskers extend from the 2.5 to the
97.5 percentile; P-values stem from the
Mann±Whitney test; y, years.
Ó 2001 Blackwell Science Ltd., Clin. Lab. Haem., 23, 181±186
widths, on the basis of sex and age, were found with
PCDW; whereas PDW and PMDW produced signi®cant
variations when comparing the ®rst three groups on the
basis of age.
Our results con®rm that platelet parameters change
with age. PLT (Figure 1) and PCT decrease between the
1±10 years and 18±45 years groups and afterwards
remain stable, whereas MPV (Figure 1) increases between
the 1±10 years and 18±45 years groups, remaining
steady afterwards. MPC and MPM increase between the
1±10 years and 18±45 years groups and decrease afterwards.
Reference ranges for clinical laboratory practice were
then de®ned as 95% con®dence limits for values between
the 2.5 and 97.5 percentiles. They were de®ned for each
age class considering males and females separately only in
the 45±65 age group (Table 2).
183
184
Platelet parameters in reference subjects and patients
Table 2. Reference limits for platelet indices. These are presented separately for males (M) and females (F) in the 45±65 years class, in
view of signi®cant sex-dependent differences
Age class years
PLT (109/l)
MPV (¯)
PDW (%)
PCT (%)
MPC (g/dl)
PCDW (g/dl)
MPM (pg)
PMDW (pg)
1±10
10±18
18±45
45±65 ± M
45±65 ± F
> 65
220±422
165±396
159±376
156±300
156±351
139±363
8.3±10.9
8.6±12.1
8.5±12.8
8.6±12.0
9.0±12.9
8.8±12.4
30.5±48.8
30.9±54.7
35.6±56.1
37.6±55.1
36.6±56.4
36.4±55.5
0.19±0.41
0.16±0.39
0.17±0.38
0.15±0.30
0.17±0.34
0.15±0.35
17.1±24.1
18.9±24.6
20.2±25.0
20.8±25.1
18.6±24.5
17.8±23.5
4.2±5.7
4.2±5.5
4.4±5.5
4.3±5.6
4.3±5.5
4.1±5.6
1.7±2.2
1.8±2.4
1.9±2.6
1.8±2.7
1.8±2.6
1.8±2.4
0.6±0.9
0.6±1.0
0.7±1.1
0.7±1.0
0.7±1.1
0.7±1.0
Table 3. Median semi-interquartile range of two samples, matched for age and sex, of reference (R) and thrombocytopenic (T)
subjects. For each index, P-values are reported from the Mann±Whitney U- test comparing reference with pathological samples
Sample (n 100) PLT (109/l)
MPV (¯)
PDW (%)
PCT (%)
MPC (g/dl)
PCDW (g/dl)
MPM (pg)
PMDW (pg)
R
T
U-test
9.9 0.5
11.2 0.8
0.0001
43.7 3.3
53.1 4.7
0.0001
0.2 0.04
0.1 0.02
0.0001
22.0 0.8
22.2 0.8
0.3098
5.1 0.2
5.1 0.3
0.0217
2.1 0.1
2.3 0.1
0.0001
0.8 0.1
1.0 0.1
0.0001
235 38
64 22
0.0001
In thrombocytopenic patients, no sex-based difference in
platelet parameters was found using the Mann±Whitney
U-test. Comparing thrombocytopenic patients with a
sex- and age-matched sample of reference subjects resulted
in signi®cant differences for all platelet parameters ± with
the exception of MPC (Table 3).
Discussion
Platelets have been studied for a considerable time, but
only in relation to thrombosis and haemostasis. However,
in recent years it has become clear that they play an
important role in several disorders characterized by
vascular pathology, including coronary artery disease
(Cahill et al., 1996a; Knight et al., 1997), Alzheimer's
disease (Davies et al., 1997), myeloproliferative disorders
(Thibert et al., 1995; Cahill et al. 1996b), diabetes
(Tschoepe et al., 1997), pre-eclampsia (Bleker et al.,
1999), in¯ammatory bowel disease (Collins et al., 1994)
and glomerular disease (Barnes, 1997). Activated platelets
have been identi®ed in most of these disorders and
antiplatelet therapy has been valuable in the management
of some conditions, including diabetic vascular disease
(ETDRS Investigators, 1992), ischaemic heart disease and
cerebrovascular disease (Anti-Platelet Trialists' Collaborative Study, 1994). Moreover these parameters, in our
experience (Giacomini et al., 2001), are also useful in the
quality control of platelet concentrates.
There is now much interest in developing methods and
instruments for an accurate platelet count and the
determination of platelet parameters in these pathologies.
In this study we analysed the platelet count and platelet
parameters of the ADVIATM 120 Hematology System
using samples from reference subjects and patients. The
two-dimensional platelet analysis of this haematology
analyser is a method by which both the volume and
refractive index of individual platelets are determined on
a cell-by-cell basis. Two simultaneously collected lightscatter measurements are converted into volume (platelet
size) and refractive index (platelet density) values using
Mie's scattering theory. This permits a much higher level
of platelet discrimination. In addition to standard platelet
parameters, which are available in most current haematology analysers such as PLT, PCT, MPV and PDW, the
ADVIATM 120 Hematology System offers novel parameters. Among these is MPC, a measure of the cell refractive
index related to platelet activation state (Macey et al.,
1999), and MPM, a parameter calculated as the product of
platelet volume by refractive index on a cell-by-cell
analysis. These new parameters may be useful in improving analyses of platelet state.
In utilizing the ADVIATM 120 Hematology System to
measure platelet parameters in our clinical laboratory we
®rst analysed reference subjects.
The reference ranges we found are in accordance with
the literature (Brummitt & Barker, 2000) for PLT and
MPM. However, they are lower for MPC and higher for
MPV and PCT; but this difference could be due to different
series.
A sex-dependent difference in PLT, PCT and MPC was
found in the 45±65 years group only, where separate
reference ranges are provided. This ®nding is in agreement
Ó 2001 Blackwell Science Ltd., Clin. Lab. Haem., 23, 181±186
A. Giacomini et al.
with Brummitt & Barker (2000), who found signi®cantly
higher PLT in females, although, in our experience,
differences were only found in 45±65 years-old subjects.
As most platelet parameters presented signi®cant variations in relation to age class, we chose to de®ne ageassociated reference ranges, unlike the above-mentioned
authors. Platelet parameters proved to vary speci®cally
during lifetime: PLT and PCT decrease from childhood to
adult age and are steady afterwards, whereas MPV
displays the opposite behaviour. MPC and MPM increase
from childhood to adult age and decrease afterwards. In
particular, as MPC appears to be a useful parameter for
studying platelet activation (Macey et al., 1999), we
suggest taking the modal-shaped age evolution of MPC
into account in clinical practice, in order to relate this
®nding to the increased thrombotic events in the elderly.
When comparing reference and thrombocytopenic subjects, MPV and MPM are signi®cantly higher in patients,
whereas MPC does not change signi®cantly. In addition to
the inverse relation between platelet number and volume ±
widely documented in the literature (Bessman et al.,
1981; Levin & Bessman, 1983) ± this platelet parameter
behaviour is referable to the pathogenesis of thrombocytopenia in cirrhosis. This pathology includes multifactorial
thrombocytopenia, partly due to a consumptive disorder
with increased and ineffective thrombocytopoiesis
(Parker Levine, 1999; Peck-Radosavljevic, 2000), concurrent with a peripheral mild platelet activation (Ingeberg
et al., 1985). Therefore, the lack of increase in mean
platelet component in thrombocytopenic patients could be
due to low granular production and to partial peripheral
degranulation. Clinical studies are necessary to verify
platelet functioning in thrombocytopenic subjects and, in
general, to determine the importance and practical
usefulness of the reference values presented in this study.
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