International Journal of Laboratory Hematology

The Official journal of the International Society for Laboratory Hematology

ORIGINAL ARTICLE INTERNAT IONAL JOURNAL OF LABORATO RY HEMATO LOGY

Evaluation of the immature platelet fraction as an indicator of

platelet recovery in dengue patients
T. DADU*, K. SEHGAL*, M. JOSHI*, S. KHODAIJI*

*Hematology Laboratory,
Department of Laboratory
Medicine, PD Hinduja National
Hospital and MRC, Mumbai,
India
Correspondence:
Tina Dadu, Hematology Labora-
tory, Department of Laboratory
Medicine, PD Hinduja National
Hospital and MRC, Mumbai,
India. Tel.: +91 22 24447945;
Fax: +91 22 24449151;
E-mail: dadu.tina@gmail.com
doi:10.1111/ijlh.12177
Received 27 August 2013;
accepted for publication 31
October 2013
Keywords
Platelets, immature platelet
fraction, dengue, thrombocyto-
penia, blood transfusion
S U M M A RY
Introduction: Thrombocytopenia is a common complication in many
disorders (such as aplastic anemia, ITP, dengue fever,), the etiology
being multifactorial. Immature platelet fraction (IPF) is a new
parameter which is a measure of reticulated platelets that reflects
the rate of thrombopoiesis. We tried to evaluate IPF as an indicator
to predict the recovery of platelets in patients with dengue.
Methods: A total of 32 patients suffering from dengue fever (as con-
firmed by NS1 antigen or IgM antibody positivity for dengue) were
taken for the study. The platelet count and IPF value of all these
patients were evaluated on a daily basis.
Results: It was found out that IPF has a strong correlation with the
recovery of platelet counts in patients with dengue. 84.3% patients
showed recovery within 24 h after attaining the peak IPF, 93.75% of
the patients showed recovery within 24–48 h of the rise of the IPF
compared with the previous day’s value, and 100% patients showed
a recovery within 24 h of the fall of the IPF compared with the previ-
ous days. It was also observed that 93.75% of the patients show
platelet recovery within 24–48 h if the IPF was more than 10%.
Conclusion: Based on our study, we concluded that IPF can be used
to evaluate the recovery of platelets in patients with dengue. It
holds a great promise of becoming a reliable future guide for deci-
sions concerning platelet transfusions.

The immature platelet fraction (IPF) is a new

INTRODUCTION
Pathogenesis of thrombocytopenia is multifactorial,
thus making it difficult to diagnose the cause and
institute treatment. It is important that enhanced
destruction of platelets is differentiated from decreased
production of platelets as in postchemotherapy, aplas-
tic anemia (AA) [1], or amegakaryocytic thrombocyto-
penia patients [2].
parameter which is an automated measure of reticu-
lated platelets in peripheral blood. Reticulated plate-
lets contain RNA and are newly released platelets that
are larger, more physiologically active and are the
analog of the red cell reticulocyte [3]. The number of
reticulated platelets reflects the rate of thrombopoiesis
[4]. IPF levels rise as bone marrow production of
platelets increases, and therefore, its measurement

© 2013 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2014, 36, 499–504 499
500 T. DADU ET AL. | IPF-INDICATOR OF PLATELET RECOVERY IN DENGUE

provides an assessment of bone marrow platelet pro-

duction from a peripheral blood sample, in a similar
way as a reticulocyte count provides a measure of red
cell production [3, 5].
Thrombocytopenia in patients with dengue may
cause a steep fall in platelet count, warranting platelet
transfusion.
However, unnecessary transfusions are best
avoided due to heightened risk from alloimmuniza-
tion, immunosuppression, transmission of infectious
diseases, and graft-vs.-host disease. Clearly, a transfu-
sion may be avoided when the platelet count is set to
rise. This brings us to the issue of how we can reliably
predict the rise in the platelet count. IPF count holds
great promise of being this predictor.
The aim of this study was to establish the relationship
between IPF and increase in platelet count in patients Figure 1. Optical (fluorescence) platelet scattergrams
with dengue who suffer from thrombocytopenia. with forward scattered light on the y-axis and
fluorescence on the x-axis.

M AT E R I A L S A N D M E T H O D S
Sample collection
Peripheral blood samples were collected in K2EDTA
(Beckton Dickinson, Franklin Lakes, NJ, USA). All
samples were analyzed within 4 h after collection.
reticulocyte mode in which the IPF value was
obtained. These values (all routine full blood count
parameters including platelets within the healthy ref-
erence range) were used to establish a normal refer-
ence range for the IPF%.

Samples for reproducibility

Method (Instrument)
The Sysmex XE-2100 (Sysmex, Kobe, Japan) was
used to measure the IPF. This fully automated hema-
tology analyzer uses a carefully designed gating sys-
tem in the optical (fluorescence) reticulocyte/platelet
channel to reliably quantitate the IPF. The cells are
stained with a proprietary fluorescent dye containing
polymethine and oxazine, which then pass through a
semiconductor diode laser beam, and the resulting
forward scatter light (cell volume) and fluorescence
intensity (RNA content) are measured. A computer
algorithm discriminates the mature and IPF by the
intensity of forward scattered light and fluorescence
[6] (Figure 1).
A reproducibility assay was run on three samples, one
with normal IPF and normal platelet count, and two
with low Platelet count and high IPF. IPF value of
each sample was measured 11 times on the analyzer,
and reproducibility was calculated.
Samples for stability
For determining stability of the parameter, six samples
were taken; three with normal IPF and normal plate-
let count and three with low platelet count and high
IPF values. All the samples were stored at 4–8 °C and
were warmed for 20 min before running them in the
analyzer. The IPF value of each of these samples was
measured at 0, 6, 12, 24, 48, and 72 h.

Normal samples
To establish the reference range, 100 samples from
apparently healthy adults who reported for routine
health check to the hospital OPD were run for CBC in
Patients samples
The patient group consisted of 32 patients who satis-
fied the following criteria: (i) patients with clinical

© 2013 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2014, 36, 499–504
 T. DADU ET AL. | IPF-INDICATOR OF PLATELET RECOVERY IN DENGUE 501

suspicion of dengue and a positive dengue test (NS1

Table 1. Reproducibility results (three samples
or IgM antibody), (ii) patients with platelet count of analysed 11 times each)
<1 50 000/lL and a falling platelet trend, and (iii)
patients who had not received blood transfusion or Mean Plt Mean CV (%)
any other treatment. The platelet counts of these (9 103/lL) IPF (%) SD for IPF
patients were followed up until the platelet counts Normal Plt, IPF 193.45 3.55 0.358 10.09
started recovering. Patients who had normal platelet Low Plt, 29.18 11.33 1.14 10.02
counts in spite of a positive test for dengue (NS1 or High IPF 1
IgM antibody) or in whom only IgG antibody against Low Plt, 25.81 16.41 1.29 7.89
High IPF 2
dengue virus was found positive were excluded from
the study. The exclusion criteria based on only IgG CF, coefficients of variation; IPF, immature platelet frac-
positivity was to avoid including false-positive cases in tion.
the study, as the serodiagnosis of past, recent, and
present dengue virus infections is difficult based on
only IgG, due to the long persistence of IgG antibodies
Reproducibility and precision
for months [6]. However, as IgG is also a predictor of
secondary dengue infection, in practice, patients hav- Reproducibility of the method was calculated with the
ing only IgG positivity may be considered as having three chosen samples by 11 repeat analyses on Sys-
dengue infection and IPF can be used to monitor the mex XE 2100. The CVs for IPF varied from 7.89% to
platelets in these. 10.09% in samples with high IPF to samples with nor-
mal IPF, respectively (Table 1).
R E S U LT S
Stability results
Normal reference range
The IPF values were found to be stable for 6 h at 4–
The normal reference range of IPF, which was calcu- 8 °C. At 12 h, most of the samples were stable except
lated using 100 normal samples, was 0.7–4.3% (2.5– for two which showed >10% change in the IPF. After
97.5 percentiles). There was no statistical difference in 12 h, there was >10% change in the value of the IPF
the normal range for males and females. in all the samples except one (Figure 2).

Figure 2. Graph showing stability

of immature platelet fraction
(IPF) measurement from 0 h to
72 h.

© 2013 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2014, 36, 499–504
502 T. DADU ET AL. | IPF-INDICATOR OF PLATELET RECOVERY IN DENGUE
Patients samples
Thirty-two patients were enrolled in the study and
followed up until the platelet count started rising. Of
the 32 patients, the lowest platelet count ranged from
8 9 103 to 57 9 103/lL and the time period until
there was a stable rise in the platelet count varied
from 2 to 3 days. The IPF count was measured simul-
taneously with the platelet count in each sample.
We analyzed the relationship of the IPF count to
platelet recovery by looking at different points in
time/time points as follows:
• When the IPF reached its peak value.
• When IPF showed a rising trend.
• When it showed a falling trend.
• When IPF crossed 10%.
Figure 3 uses an example to demonstrate the time
taken for the recovery of the platelets with respect to
the IPF value at different time points.
Peak refers to the maximum IPF value reached
while monitoring IPF on a daily basis. The time
required for the platelets to start recovering after IPF
reached its peak value was then calculated. It was
observed that 84.3% patients showed recovery within
24 h and the rest between 24 and 48 h (Figure 4).
Rising/Falling trend was defined as an increase or
decrease, respectively, in IPF by more than 10% (CV
of IPF) from its previous value. If only two values
Figure 3. Relationship of platelet recovery to peak, rising trends and falling trends in immature platelet fraction
(IPF).
were recorded on a patient, then the second sample
displayed either a rising or falling trend. Our study
demonstrated that 93.75% of the patients showed
recovery within 24–48 h of the rise, and 100%
patients showed a recovery within 24 h of the fall
(Figure 4).
We also attempted to calculate the recovery time
on a single data point with a cut-off value of >10%
and found that 93.75% of the patients show platelet
recovery within 24–48 h if the IPF was more than
10% (Figure 4).
DISCUSSION
The immature platelet fraction (% IPF) is a new
parameter which is an automated measure of reticu-
lated platelets in peripheral blood. In our study, the
reference range was found to be 0.7–4.3%, which is
similar to a reference range derived by Briggs [7] of
1.1–6.1% of the total platelet count.
In our study, we found that the CV of a normal
sample with IPF in the normal range was 10.09%. For
samples with low platelet count and high IPF, the CV
ranged from 7.89% to 10.02%. Lower CV was
obtained when samples had a high IPF%, as expected.
The results of our study were very similar to that of
Briggs [7] where the coefficients of variation (CV) for
IPF in the three samples from individuals with platelet
counts within the healthy reference range varied from
© 2013 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2014, 36, 499–504
 T. DADU ET AL. | IPF-INDICATOR OF PLATELET RECOVERY IN DENGUE
6.82% to 11.39%, and for the three low platelet
count samples with a high IPF, the CV ranged from
6.92% to 14.27%.
Our study clearly demonstrates that the % IPF was
significantly affected by storage even at 4–8 °C, which
is the recommended temperature for storage of CBC
samples [8]. We found that there was an insignificant
difference in the mean values of IPF within 6 h. After
6 h, two samples showed a change of >10% from the
previous IPF value. However, after 12 h, there was a
significant increase in the IPF values. There was a
steady upward trend in the % IPF value after that.
Our results were very similar to that of Andrew Osei-
Bimpong [9], who also found a significant rise in the
mean IPF after 12 h. Thus, we consider it an unde-
pendable tool after 12 h of blood collection. The
results of our study, however, contrasted with the
study by Briggs [7] where the IPF remained stable
over 2 days when blood samples were stored at room
temperature, and there was no clinically significant
increase or decrease in the IPF values from 0.5 to
48 h after sampling. This contrast may be due to the
difference in the storage temperature at which the
samples were stored, that is, storage of sample at
room temperature in Briggs study viz-a-vie at 4–8 °C
in our study. However, as the sample size in both
studies was small, a large sample size is required to
find the appropriate storage temperature for IPF.
Figure 4. Percentage of patients
showing platelet recovery at
different points in time.
© 2013 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2014, 36, 499–504
503
Immature platelet fraction has many applications
in clinical practice. Many studies have been carried
out to find the utility of IPF in predicting platelet
recovery. Most of the studies are on patients receiving
chemotherapy. However, to the best of our knowl-
edge, there is no such study carried out in patients
with dengue. Thrombocytopenia of moderate degree
is a usual finding associated with dengue, the reasons
for which are multifactorial, which include early tran-
sient marrow suppression with damage to megakaryo-
cytes [10], platelet aggregation to endothelial cells
targeted by dengue fever viruses [11], hemophagocy-
tosis [12, 13], and finally, immune destruction of
platelets, with dengue antibody complexes being
found on their membrane [14]. Prophylactic platelet
transfusion may be given at level of <20 9 103/lL
absence of bleeding manifestations [15]. However,
according to WHO, it should be noted that prophylac-
tic platelet transfusions for severe thrombocytopenia
in otherwise hemodynamically stable patients have
not been shown to be effective and are not necessary
[16]. Because no clear criteria exist for management
of thrombocytopenia in patients with dengue, IPF can
be used by the treating physician to predict the recov-
ery of platelets in patients with dengue, so as to avoid
unnecessary blood transfusion.
Our study shows that after the reaching the peak
IPF, 84.3% patients recovered within 24 h and rest
504 T. DADU ET AL. | IPF-INDICATOR OF PLATELET RECOVERY IN DENGUE

within 24–48 h. Usually in patients with dengue, at a
certain point, the IPF starts rising even though the
platelets might be falling. This is due to a combination
of pathogenetic mechanisms; the peripheral destruc-
tion, which lowers the platelet count, stimulates the
marrow to produce more platelets which causes an
increase in the IPF. We found that when the IPF starts
going up (rising trend), 93.75% of the patients
showed recovery within 24–48 h of the rise. After a
certain point when the IPF has peaked, then the
platelets start coming up and IPF starts falling. If the
patient is caught at this stage, where the first value
was the peak value, it is obvious that the value on
second day will be less than the peak (falling trend).
This fall in the IPF is a strong predictor of an impend-
ing rise in platelet count. So if the platelets have not
started recovering, then the time lag for recovery of
platelets in such cases is <24 h. It was observed that
100% patients show a recovery within 24 h of the
fall. Assuming that we may not have follow-up IPF
data for all patients in a clinical scenario, we also
evaluated the platelet recovery time based on a single
time point IPF cut-off value of 10% and found that
93.75% of the patients show platelet recovery within
24–48 h if the IPF is more than 10%.
Studies carried out in patients with cancer undergo-
ing chemotherapy and hematopoietic stem cell trans-
plant show a transient increase in IPF% 1–11 days
prior to platelet recovery (>30 9 109/L). In patients
undergoing chemotherapy with a peak IPF% >10%,
platelet recovery occurred significantly earlier than in
those with IPF% peak values <10% (median periods
were 2 and 5 days; P < 0.05) [17]. IPF can also be a
sensitive measure for evaluating thrombopoietic recov-
ery during aplastic chemotherapy [5].
Therefore, IPF shows great promise of becoming a
reliable future guide for decisions concerning platelet
transfusions. There appears to be a direct corelation
between increases in base IPF levels and correspond-
ing increases in platelet count. The time lag between
increased IPF levels and corresponding increases in
platelet count appears to be around 24–48 h in
patients with dengue. Therefore, measurement of IPF
should be considered as routine practice to evaluate
and monitor thrombocytopenia in patients with
dengue.

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© 2013 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2014, 36, 499–504