European Journal of Haematology 93 (150–156)

ORIGINAL ARTICLE

Immature platelet fraction measured on the Sysmex XN

hemocytometer predicts thrombopoietic recovery after
autologous stem cell transplantation
Noreen van der Linden1, Lieke J.J. Klinkenberg1, Steven J.R. Meex1, Erik A.M. Beckers2,
Norbert C.J. de Wit1, Lenneke Prinzen1*
1
Central Diagnostic Laboratory, Maastricht University Medical Center, Maastricht; 2Department of Internal Medicine, Subdivision of Haematology,
Maastricht University Medical Center, Maastricht, the Netherlands

Abstract
Objectives: A period of thrombocytopenia is common after stem cell transplantation (SCT). To prevent serious
bleeding complications, prophylactic platelet transfusions are administered. Previous studies have shown that
a rise in immature platelets precedes recovery of platelet count. Our aim was to define a cutoff value for
immature platelets predicting thrombopoietic recovery within 2 d. Methods: Hematological parameters were
measured on the Sysmex XN hemocytometer. We calculated reference change values (RCV) for platelets in
eight healthy individuals as marker for platelet recovery. To define a cutoff value, we performed ROC analysis
using data from 16 autologous SCT patients. Results: RCV for platelet concentration was 14.1%. Platelet
recovery was observed 13 (median; range 9–31) days after SCT. Increase in immature platelet fraction (IPF)
before platelet recovery was seen in all autologous SCT patients. Optimal cutoff IPF was found to be 5.3% for
platelet recovery within 2 d (specificity 0.98, sensitivity 0.47, positive predictive value 0.93). Conclusions: We
identified an optimal cutoff value for IPF 5.3% to predict platelet recovery after autologous SCT within 2 d.
Implementing this cutoff value in transfusion strategy may reduce the number of prophylactic platelet
transfusions.

Key words immature platelet fraction; stem cell transplantation; thrombopoiesis; platelets; transfusion

Correspondence Noreen van der Linden, Central Diagnostic Laboratory, Maastricht University Medical Center, PO Box 5800, 6202
AZ Maastricht, the Netherlands. Tel: +31-(0)43-3872786; Fax: +31-(0)43-3874667; e-mail: [email protected]

*Present address: Sint Franciscus Gasthuis, Rotterdam, the Netherlands.

Accepted for publication 18 March 2014 doi:10.1111/ejh.12319

The average platelet count in humans ranges between 150 fraction (IPF) suggests increased thrombopoiesis, where-
and 350 9 109/L (1). Thrombocytopenia, that is, the state as low IPF suggests decreased thrombopoiesis (3, 4).
of decreased platelet concentration in peripheral blood, Immature platelets decrease in size and RNA content as
may be due to several causes, roughly divided into three they age, analogous to reticulocytes (5, 6). Their charac-
main mechanisms: (i) aberrant distribution of platelets due teristics allow us to differentiate immature from mature
to dilution, bleeding, or splenomegaly, (ii) increased platelets, not only by flow cytometry, but also on the
platelet destruction or consumption, and (iii) a decreased newest generation hemocytometers (3, 7–9). In 1969, In-
platelet production. To correctly diagnose the cause of gram and Coopersmith were the first to show an increase
thrombocytopenia, it is crucial to distinguish between in immature platelets in mature beagles subjected to severe
these three mechanisms (2). The assessment of thrombo- blood loss, reflecting increased thrombopoietic activity (5).
poietic activity may be useful in differentiating between Several more recent publications confirmed this observa-
these three. Immature platelets, newly released from tion (2, 10–12). This might also be of interest in patients
megakaryocytes in the bone marrow, are supposedly a undergoing stem cell transplantation (SCT) for hematologi-
marker of thrombopoietic activity: High immature platelet cal malignancies (7, 13–19). In the post-transplant period,

150 © 2014 The Authors. European Journal of Haematology Published by John Wiley & Sons Ltd.
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and
distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
van der Linden et al. IPF predicting thrombopoietic recovery

patients have a significantly lower concentration of plate- committee. Patients could object to the use of their medical
lets due to a decrease in thrombopoiesis. As platelets play information for research purposes. Two patients were
an essential role in primary hemostasis, severe thrombocy- excluded due to missing data (i.e., day of platelet recovery
topenia is associated with an increased risk of bleeding not recorded). Sixteen patients were included in the final
(20). Studies in thrombocytopenic patients have shown data analysis. During hospital stay, bleedings were reported
concordance between the onset of bleeding and platelet and classified according to the WHO bleeding scale (30).
count. A minimum platelet count of 5 9 109/L is thought to
be sufficient to maintain vascular integrity and prevent severe
Sample collection
bleeding (21, 22). Prophylactic platelet transfusions are
administered to prevent severe and life-threatening bleeding Venous whole-blood samples were collected in K2-EDTA
in thrombocytopenic patients (23). According to current con- Vacutainer tubes (BD Diagnostics, Plymouth, UK). Hemato-
sensus, prophylactic platelet transfusions are ordered when oncological patients admitted to the ward were routinely sam-
platelet count falls below a threshold of 10 9 109/L (23–25). pled daily around 8 AM. When samples were drawn more fre-
Disadvantages of prophylactic transfusions may be major and quently, only the 8 AM samples were used for our analysis.
minor adverse effects for patients, varying from mild transfu- Samples were stored at 4°C for a maximum of 51 h until
sion reaction to anaphylaxis or severe infection. Also, high analysis. A previous study showed that the IPF in samples
costs and the future shortage of donors should be kept in with K2-EDTA stored at 4°C stays within precision limits
mind (23). Furthermore, it is debatable whether this prophy- over 72 h (31). We confirmed this finding by an additional
lactic strategy is actually necessary. Some studies suggest that experiment. Patient data were collected between start of condi-
a therapeutic transfusion strategy is safe in a selected group tioning regimen and day of platelet recovery.
of stable patients without sepsis, infections or an increased
bleeding risk (23, 26). A previous study even suggested that
Measurement of immature platelets
platelet transfusions may inhibit thrombopoiesis by binding
thrombopoietin (27). A strategy to reduce platelet transfu- Whole-blood, EDTA anticoagulated, samples were measured
sions in thrombocytopenic patients after SCT may be based on both the Sysmex XE-5000 analyzer (Sysmex corporation,
on a better prediction of the thrombopoietic recovery. If a Kobe, Japan) and the Sysmex XN analyzer. Earlier Sysmex
reliable prediction of the natural recovery of the platelet num- hematology analyzers (such as the XE-5000) use the RET-
ber within a few days is feasible, a valid decision whether or channel for the measurement of immature platelets and the
not to give a prophylactic platelet transfusion would be facili- optic platelet count (PLT-O). Impedance (PLT-I) is an alter-
tated. The number of immature platelets could be used as native method for measuring platelets. The Sysmex XN has,
such a predictor, as various studies have described a rise in in addition, a novel PLT-F-channel for measurement of both
the number of immature platelets shortly preceding platelet mature and immature platelets. This PLT-F-channel was
recovery (13–17, 28). Some studies also proposed a cutoff introduced to specifically gate platelets for a more accurate
value for immature platelets predicting platelet recovery (18, platelet count (PLT) and IPF. On both analyzers, an algo-
29). Different definitions of platelet recovery were used in rithm decides which platelet count was reported. Measure-
combination with a less standardized, more time-consuming ment of immature platelets is similar on both analyzers and
method of measurement of immature platelets. A fundamental is based on the principle of hemocytometry. After perfora-
step toward the implementation of immature platelets in tion of platelet cellular membranes by reagents, nucleic acids
transfusion management after SCT is defining a reliable cut- are stained by fluorescent dyes: polymethine and oxazine
off value based on a standardized technique. The aim of this (XE-5000) or oxadine (XN; 31, 32). An algorithm defines
study was to define a cutoff value for immature platelets pre- the gating of mature and immature platelets. This algorithm
dicting thrombopoietic recovery using the novel Sysmex XN contains side fluorescence (reflection of RNA content), side
analyzer (Sysmex corporation, Kobe, Japan). scattered light (information on intracellular structure), and
forward scattered light (information on cell size).
Methods
Platelet transfusions
Patients
Platelet transfusions were given in a standardized fashion
For a period of 8 months (September 2012 through April according to the national guidelines. Prophylactic transfusion
2013), eighteen adult hemato-oncological patients, scheduled trigger was defined by a platelet concentration of
for autologous SCT in the Maastricht University Medical <10 9 109/L. In case of clinical complications, surgical
Center, were included. The number of venous blood draws intervention or the use of anticoagulants the transfusion trig-
and transfusion regime were not influenced by our observa- ger may be higher (33). Patients in the SCT protocol
tional study. Our study was approved by the local ethics received irradiated platelet products, prepared from the buffy

© 2014 The Authors. European Journal of Haematology Published by John Wiley & Sons Ltd. 151
IPF predicting thrombopoietic recovery van der Linden et al.

coats of five different ABO/D-matched whole-blood donors, A B C

resuspended in one unit of plasma to a volume of circa
350 mL and containing at least 250 9 109 platelets (33, 34).
None of the patients received HLA compatible apheresis
platelet concentrates. Some patients received pathogen-inacti-
vated platelets (treated with riboflavin/UVB-pathogen) (Mir-
asol; TerumoBCT, Lakewood, CO, USA; see Table 2).
Transfusion reactions were reported according to the stan-
dard protocol.
Figure 1 Biological variation. Mean concentrations (dots) and absolute
range (horizontal lines) of platelet count (PLT) (panel A), the immature
Biological variation, RCV, and platelet recovery
platelet fraction (IPF) (panel B) and the absolute number of immature
To assess biological within-person variation, PLT, IPF, platelets (IPA) (panel C) measured in healthy individuals (n = 8) on 5 d
and the absolute number of immature platelets (IPA) were in duplicate. Subjects are numbered in a random order.

measured in eight healthy subjects (four men and four

women, age 23–33) once a day at the same time on 5 d Table 1 Coefficients of variation and RCV
in duplicate on the Sysmex XN and XE analyzers. Two-
fold nested ANOVA was used to estimate, between-person PLT IPF IPA
(CVbp), biological (within-person) (CVwp), and analytical RCV95% 14.12 23.44 33.63
(CVa) coefficients of variation including 95% confidence CVa 0.58 (0.48–0.74) 4.04 (3.31–5.16) 4.20 (3.45–5.37)
intervals (35). RCV was calculated based on the biological CVwp 5.06 (4.06–6.70) 7.43 (5.69–10.14) 10.60 (8.33–14.27)
and analytical coefficients of variations found in this CVbp 17.21 (11.25–35.26) 45.86 (30.20–93.55) 32.91 (21.43–67.53)
experiment. RCV is calculated
 using the formula—
p Reference change values (RCV), analytical coefficient of variation
RCV ¼ Z  2  CVwp2 þ CVa2 —and reflects the mini- (CVa, 95% confidence interval), within-person coefficient of variation
mal difference required to define a statistically significant (CVwp, 95% confidence interval), and between-person coefficient of
increase or decrease compared with the previous result in variation (CVbp, 95% confidence interval) for platelet count (PLT),
that person. Z is the number of standard deviations appro- immature platelet fraction (IPF), and the absolute number of immature
platelets (IPA) based on our biological variation study.
priate to the desired probability (36, 37). In this study,
we used a Z-score of 1.96 for P < 0.05. Platelet recovery
was defined as the first day that platelet count increased coefficients of variation for PLT, IPA, and IPF. Results are
without transfusion and exceeding the RCV. shown in Table 1.

Statistical analyses Patient characteristics

Statistical analyses were performed with SPSS Statistics ver- Sixteen patients who underwent autologous SCT were
sion 20 (IBM corporation, New York, NY, USA). Sensitivity, included in the study. All of them received a peripheral
specificity, and positive predictive value (PPV) were calcu- blood SCT. One suffered WHO grade 2 bleeding, a combi-
lated as described by Emir et al. (38), where sensitivity, speci- nation of epistaxis and petechiae. One transfusion reaction
ficity, and PPV for our patient population are weighted was reported: a patient experienced chills after platelet trans-
averages. Weights we used are based on the number of patient fusion. No infectious cause was identified in this case.
samples as a control (no recovery within 2 d) or as a case Patient characteristics are shown in Table 2.
(recovery within 2 d). We analyzed sensitivity and specificity
of both IPA and IPF from day 8 after SCT until the day of
Platelet recovery
platelet recovery. Using these data, we constructed receiver
operating characteristics (ROC) curves. Platelet recovery (an increase in platelets greater than its
RCV and not due to platelet transfusion) was observed after
a median of 13 d in patients after autologous SCT. Before
Results
platelet recovery, we observed an increase in IPA exceeding
RCV, that is, more than 33.63%, in all patients. This
Biological variation
increase in IPA measured on the XN analyzer preceded
Figure 1 shows the mean concentrations and absolute ranges platelet recovery by a median of 3 d (range 1–6 d). Increase
for PLT, IPA, and the IPF of eight healthy individuals mea- in IPF exceeding RCV, that is, more than 23.44%, was also
sured as described in the methods section. Using these data, observed in all patients prior to platelet recovery. This
we calculated analytical, within-person, and between-person increase in IPF measured on the XN analyzer was seen by a

152 © 2014 The Authors. European Journal of Haematology Published by John Wiley & Sons Ltd.
van der Linden et al. IPF predicting thrombopoietic recovery

Table 2 Patient characteristics A

Autologous PBSCT (n = 16)

Male gender (%) 63

Age (yrs) 59 (44–66)
Diagnosis AL-amyloidosis (n = 2)
AITL (n = 1)
Follicular lymphoma (n = 2)
Hodgkin lymphoma (n = 1)
MM (n = 7)
DLBCL (n = 3)
Conditioning regimen BEAM (n = 6)
HDM (n = 9)
Cyclophosphamide, Busulfan (n = 1)
Days till platelet recovery 13 (10–16)
Number of platelet transfusions 1 (1–5)
per patient
Pathogen-inactivated 2
B
platelet transfusions
Bleeding complications WHO grade 2 (n = 1)

Median and range.

AITL, angioimmunoblastic T-cell lymphoma; BEAM, BCNU, cytarabine,
etoposide and melphalan; DLBCL, diffuse large B-cell lymphoma;
HDM, high-dose melphalan; MM, multiple myeloma; PBSCT, periph-
eral blood stem cell transplantation.

median of 4 d (range 2–7 d) before platelet recovery. Fig-

ure 2 shows the course of PLT, IPA, and IPF in a patient
after autologous SCT.

ROC analysis
Figure 2 Representative example of the course of platelet count
ROC curves were constructed for all patients. IPA and IPF, as (PLT), the absolute number of immature platelets (IPA), and immature
well as XN and XE data, were analyzed separately. ROC platelet fraction (IPF) in a patient from stem cell transplantation (SCT)
curve for IPF from day 8 till the moment of platelet recovery until recovery. Panel A shows the course of the PLT (black) and the
after autologous SCT measured with the XN analyzer showed IPA (gray). Panel B shows the course of PLT (black) and the IPF
(gray). Dots represent concentrations in morning samples. Dotted ver-
an area under curve (AUC) of 0.86. ROC curves for the IPA,
tical line indicates day of SCT. Continuous vertical lines indicate plate-
based on data from the Sysmex XE-5000, had lower AUCs.
let transfusions. Stars indicate an increase (in PLT, IPA, or IPF)
ROC curves are shown in Fig. 3. AUCs are shown in Table 3. exceeding its reference change value (RCV) not due to platelet trans-
Our aim was to define a cutoff value with high PPV. fusion.
ROC curve for IPF after autologous SCT measured on the
Sysmex XN showed the most optimal AUC. We determined Discussion
a cutoff value of 5.3% [sensitivity 0.47 (95% CI 0.29–0.65),
specificity 0.98 (95% CI 0.85–1.10), and PPV 0.93 (95% CI In patients awaiting thrombopoietic recovery after SCT, a
0.81–0.1.06)] to predict platelet recovery within 2 d strategy of prophylactic platelet transfusion is considered
measured on the Sysmex XN. best practice, despite questions and debates about dose and
lowest acceptable platelet counts (21, 22, 39). Platelet trans-
fusions, although generally accepted as safe, may cause vari-
Table 3 AUCs of ROC curves based on estimated sensitivity and
ous transfusion reactions and are considered a scarce and
specificity for the IPF and the absolute number of immature platelets
(IPA) measured on both the Symex XE-5000 and the XN analyzer
also costly resource (23). In search of sensitive predictors of
thrombopoietic recovery, the feasibility of immature platelets
AUC measured by the Sysmex XN hematocytometer was studied
IPA-XN 0.83 as a tool to detect early platelet recruitment.
IPA-XE 5000 0.71 An important step toward determination of a cutoff
IPF-XN 0.86 value for immature platelets is to objectively define platelet
IPF-XE-5000 0.66 recovery. We used a validated statistical approach based on

© 2014 The Authors. European Journal of Haematology Published by John Wiley & Sons Ltd. 153
IPF predicting thrombopoietic recovery van der Linden et al.

that IPF measured by the XN has a better precision than

when measured by the XE-5000.
We estimated a cutoff value of IPF 5.3% in patients after
autologous SCT to predict thrombopoietic recovery within
2 d, with a PPV of 0.93. Based on the large between-subject
variations of 32.9% and 45.9% for IPA and IPF, respec-
tively, found in our biological variation study, it would be
reasonable to define a cutoff value indicating the difference
between two measurements (or delta). However, ROC curves
based on both absolute and delta values for IPA and IPF did
not have acceptable AUCs, and this method was therefore
not further investigated (data not shown).
In only one other study, a cutoff IPF predicting platelet
recovery after autologous SCT was estimated (18). In this
Figure 3 ROC curves and AUCs of immature platelet fraction (IPF) study, containing 31 patients, an IPF greater than 7.0% on
measured on the Sysmex XE-5000 and XN analyzer after autologous day 8 after SCT predicted platelet recovery within 4 d (PPV
stem cell transplantation (SCT). 79%, sensitivity 76%). Because immature platelets were
assessed using flow cytometry, it is difficult to compare this
biological and analytical variation to objectify platelet recov- result with our findings.
ery based on RCV (36, 37). A possible limitation may be The cutoff value for IPF of 5.3%, with high PPV and
that we assumed biological within-subject variation to be specificity, is based on observational data. Theoretically, this
the same for healthy subjects as for patients after SCT. cutoff may be used to decide whether or not to give a plate-
However, this assumption appears valid because a previous let transfusion. Platelet transfusion may be omitted when
study showed that for the majority of parameters, biological platelet count is likely to recover within 2 d. This strategy
within-subject variation is of the same order in health and may reduce the number of platelet transfusions and its asso-
disease (40). This is likely not true for analytical variation. ciated risks and costs.
Nevertheless, Fraser (41) showed that a ratio of 0.50 of ana- The hemostatic capacity of immature platelets is a point
lytical variation to within-subject variation has an effect of of interest, because current prophylactic strategy is intended
only 12% on RCV. While within-subject variation is much to prevent serious bleeding incidents. Previous studies sug-
larger than analytical variation in our study, differences in gest that thrombocytopenic patients with high concentrations
analytical variation will have a negligible effect on RCV. of immature platelets of up to 40–50%, such as ITP patients,
Previous studies used different definitions for platelet have a smaller bleeding-tendency compared with thrombocy-
recovery, for example, platelet count ≥30 9 109/L for three topenic patients with low concentrations of immature
consecutive days, seven consecutive days with a platelet platelets, such as chemotherapy-treated patients (42). This is
count ≥20 9 109/L without platelet transfusions, or a spon- suggestive of a higher hemostatic capacity of immature
taneous increase in PLT count without platelet transfusions platelets. Clearly, clinical interventional studies are needed
(13–15, 18, 28). Despite the fact that we used RCV to define to address this issue proving more evidence for the value of
platelet recovery, the interval between increase in immature high IPF in platelet transfusions strategies.
platelets and platelet recovery we found appears similar, that Another argument to include a marker of thrombopoiesis
is, approximately 2 d (15, 16, 18, 28). in transfusion strategy is the observation that thrombopoiesis
To estimate sensitivity, specificity, and PPV, we used may be suppressed by transfused platelets (7, 15, 27, 43).
the approach proposed by Emir et al. (38) to use multiple However, while previous studies reported a decrease in IPF
time points for each patient. We included all morning after platelet transfusion (7, 15), we did not observe an
samples from day 8 after SCT until platelet recovery. This unequivocal effect of platelet transfusions in our population.
may be a limitation, as it led to a different number of A recent study by Bat et al. (44) showed that platelet trans-
controls per patient: more controls when recovery time is fusions lead to a decrease in IPF, but do not alter the IPA,
longer, which in turn led to higher specificity. Day 8 was suggesting dilution by an increase in circulating mature
chosen because we observed earliest platelet recovery 2 d platelets. Our finding was that immature platelets are
later (day 10). sometimes increased and sometimes decreased after platelet
ROC curves based on estimated sensitivity and specificity transfusion. The dilution effect of platelet transfusion on
showed differences in AUCs between IPF and IPA and immature platelets (e.g., increase or decrease) depends on
between measurements on the Sysmex XE-5000 and the both the immature platelet count before transfusion and the
Sysmex XN. ROC curve of IPF measured on the Sysmex immature platelet content of the platelet concentrate (17,
XN had a larger AUC compared to XE-5000. This reflects 45).

154 © 2014 The Authors. European Journal of Haematology Published by John Wiley & Sons Ltd.
van der Linden et al. IPF predicting thrombopoietic recovery

In summary, we demonstrated an increase in immature flow cytometry: an indirect thrombocytopoietic marker. Eur J
platelets measured on the Sysmex XN analyzer preceding Intern Med 2006;17:541–4.
platelet recovery. We showed that IPF cutoff of 5.3% is a 11. Ault KA. Flow cytometric measurement of platelet function
promising predictor of platelet recovery in patients after and reticulated platelets. Ann N Y Acad Sci 1993;677:293–
autologous SCT. However, a small number of patients were 308.
included in the final data analysis. Due to the observational 12. Sakakura M, Wada H, Abe Y, Nishioka J, Tomatsu H, Ham-
design of the study, the relationship between immature plate- aguchi Y, Oguni S, Shiku H, Nobori T. Usefulness of mea-
lets and bleeding risks could not be evaluated. Interventional surement of reticulated platelets for diagnosis of idiopathic
thrombocytopenic purpura. Clin Appl Thromb Hemost
clinical trials in a multicenter setting are required to validate
2005;11:253–61.
the cutoff value presented in this study and to establish its
13. Goncalo AP, Barbosa IL, Campilho F, Campos A, Mendes C.
role in IPF-based transfusion policies.
Predictive value of immature reticulocyte and platelet fractions
in hematopoietic recovery of allograft patients. Transplant
Acknowledgements Proc 2011;43:241–3.
14. Takami A, Shibayama M, Orito M, Omote M, Okumura H,
This study was funded by the Sysmex Corporation by sup- Yamashita T, Shimadoi S, Yoshida T, Nakao S, Asakura H.
plying the XN analyzer and reagents and paying the fee for Immature platelet fraction for prediction of platelet engraft-
open access publication. The sponsor had no role in data ment after allogeneic stem cell transplantation. Bone Marrow
collection, data analysis, data interpretation, or writing of Transplant 2007;39:501–7.
this manuscript. E.B., N.C.J.W. and L.P. did the experimen- 15. Zucker ML, Murphy CA, Rachel JM, Martinez GA, Abhyan-
tal design of the study; N.L. and L.P. performed experiments kar S, McGuirk JP, Reid KJ, Plapp FV. Immature platelet
and analyzed results; N.L., L.J.J.K. and S.J.R.M. performed fraction as a predictor of platelet recovery following hemato-
the statistical analysis; N.L. made the figures; and all authors poietic progenitor cell transplantation. Lab Hematol
wrote the article. The authors have no competing interests. 2006;12:125–30.
16. Martinelli G, Merlo P, Fantasia R, Gioia F, Crovetti G. Retic-
ulated platelet monitoring after autologous peripheral haemat-
References
opoietic progenitor cell transplantation. Transfus Apher Sci
1. Kaushansky K. Determinants of platelet number and regula- 2009;40:175–81.
tion of thrombopoiesis. Hematology Am Soc Hematol Educ 17. Michur H, Maslanka K, Szczepinski A, Marianska B. Retic-
Program 2009;2009:147–52. ulated platelets as a marker of platelet recovery after alloge-
2. Abe Y, Wada H, Tomatsu H, et al. A simple technique to neic stem cell transplantation. Int J Lab Hematol
determine thrombopoiesis level using immature platelet frac- 2008;30:519–25.
tion (IPF). Thromb Res 2006;118:463–9. 18. Chaoui D, Chakroun T, Robert F, et al. Reticulated plate-
3. Briggs C, Kunka S, Hart D, Oguni S, Machin SJ. Assessment lets: a reliable measure to reduce prophylactic platelet trans-
of an immature platelet fraction (IPF) in peripheral thrombo- fusions after intensive chemotherapy. Transfusion
cytopenia. Br J Haematol 2004;126:93–9. 2005;45:766–72.
4. Strauss G, Vollert C, von Stackelberg A, Weimann A, Gae- 19. Richards EM, Jestice HK, Mahendra P, Scott MA, Marcus
dicke G, Schulze H. Immature platelet count: a simple param- RE, Baglin TP. Measurement of reticulated platelets follow-
eter for distinguishing thrombocytopenia in pediatric acute ing peripheral blood progenitor cell and bone marrow trans-
lymphocytic leukemia from immune thrombocytopenia. Pedi- plantation: implications for marrow reconstitution and the
atr Blood Cancer 2011;57:641–7. use of thrombopoietin. Bone Marrow Transplant
5. Ingram M, Coopersmith A. Reticulated platelets following 1996;17:1029–33.
acute blood loss. Br J Haematol 1969;17:225–9. 20. Pihusch M. Bleeding complications after hematopoietic stem
6. Karpatkin S. Human platelet senescence. Annu Rev Med cell transplantation. Semin Hematol 2004;41(1 Suppl 1):93–
1972;23:101–28. 100.
7. Briggs C, Hart D, Kunka S, Oguni S, Machin SJ. Immature 21. Slichter SJ. Relationship between platelet count and bleeding
platelet fraction measurement: a future guide to platelet trans- risk in thrombocytopenic patients. Transfus Med Rev
fusion requirement after haematopoietic stem cell transplanta- 2004;18:153–67.
tion. Transfus Med 2006;16:101–9. 22. Hanson SR, Slichter SJ. Platelet kinetics in patients with bone
8. Kienast J, Schmitz G. Flow cytometric analysis of thiazole marrow hypoplasia: evidence for a fixed platelet requirement.
orange uptake by platelets: a diagnostic aid in the evaluation Blood 1985;66:1105–9.
of thrombocytopenic disorders. Blood 1990;75:116–21. 23. Estcourt LJ, Stanworth SJ, Murphy MF. Platelet transfusions
9. Lee LG, Chen CH, Chiu LA. Thiazole orange: a new dye for for patients with haematological malignancies: who needs
reticulocyte analysis. Cytometry 1986;7:508–17. them? Br J Haematol 2011;154:425–40.
10. Jimenez MM, Guedan MJ, Martin LM, Campos JA, Martinez 24. Schiffer CA, Anderson KC, Bennett CL, et al. Platelet trans-
IR, Vilella CT. Measurement of reticulated platelets by simple fusion for patients with cancer: clinical practice guidelines of

© 2014 The Authors. European Journal of Haematology Published by John Wiley & Sons Ltd. 155
IPF predicting thrombopoietic recovery van der Linden et al.

the American Society of Clinical Oncology. J Clin Oncol Intervals on Variance Components. New York, USA: Marcel
2001;19:1519–38. Dekker; 1992:79–91.
25. British Committee for Standards in Haematology BTTF. 36. Fraser CG. Reference change values. Clin Chem Lab Med
Guidelines for the use of platelet transfusions. Br J Haematol 2012;50:807–12.
2003;122:10–23. 37. Harris EK, Yasaka T. On the calculation of a “reference
26. Wandt H, Schaefer-Eckart K, Wendelin K, et al. Therapeutic change” for comparing two consecutive measurements. Clin
platelet transfusion versus routine prophylactic transfusion in Chem 1983;29:25–30.
patients with haematological malignancies: an open-label, 38. Emir B, Wieand S, Su JQ, Cha S. Analysis of repeated mark-
multicentre, randomised study. Lancet 2012;380:1309–16. ers used to predict progression of cancer. Stat Med
27. Shinjo K, Takeshita A, Nakamura S, Naitoh K, Yanagi M, Tobi- 1998;17:2563–78.
ta T, Ohnishi K, Ohno R. Serum thrombopoietin levels in 39. Cid J, Lozano M. Platelet dose for prophylactic platelet trans-
patients correlate inversely with platelet counts during chemo- fusions. Expert Rev Hematol 2010;3:397–400.
therapy-induced thrombocytopenia. Leukemia 1998;12:295–300. 40. Ricos C, Iglesias N, Garcia-Lario JV, et al. Within-subject
28. Yamaoka G, Kubota Y, Nomura T, Inage T, Arai T, Kitanaka biological variation in disease: collated data and clinical con-
A, Saigo K, Iseki K, Baba N, Taminato T. The immature sequences. Ann Clin Biochem 2007;44(Pt 4):343–52.
platelet fraction is a useful marker for predicting the timing of 41. Fraser CG, Hyltoft Petersen P, Libeer JC, Ricos C. Proposals
platelet recovery in patients with cancer after chemotherapy for setting generally applicable quality goals solely based on
and hematopoietic stem cell transplantation. Int J Lab Hema- biology. Ann Clin Biochem 1997;1(Pt 1):8–12.
tol 2010;32(6 Pt 1):e208–16. 42. Psaila B, Bussel JB, Frelinger AL, Babula B, Linden MD, Li
29. Wang C, Smith BR, Ault KA, Rinder HM. Reticulated plate- Y, Barnard MR, Tate C, Feldman EJ, Michelson AD. Differ-
lets predict platelet count recovery following chemotherapy. ences in platelet function in patients with acute myeloid leu-
Transfusion 2002;42:368–74. kemia and myelodysplasia compared to equally
30. Miller AB, Hoogstraten B, Staquet M, Winkler A. Reporting thrombocytopenic patients with immune thrombocytopenia. J
results of cancer treatment. Cancer 1981;47:207–14. Thromb Haemost 2011;9:2302–10.
31. Briggs C, Longair I, Kumar P, Singh D, Machin SJ. Perfor- 43. Kuter DJ, Rosenberg RD. The reciprocal relationship of
mance evaluation of the Sysmex haematology XN modular thrombopoietin (c-Mpl ligand) to changes in the platelet mass
system. J Clin Pathol 2012;65:1024–30. during busulfan-induced thrombocytopenia in the rabbit.
32. Meintker L, Haimerl M, Ringwald J, Krause SW. Measure- Blood 1995;85:2720–30.
ment of immature platelets with Abbott CD-Sapphire and Sys- 44. Bat T, Leitman SF, Calvo KR, Chauvet D, Dunbar CE. Mea-
mex XE-5000 in haematology and oncology patients. Clin surement of the absolute immature platelet number reflects
Chem Lab Med 2013;51:2125–31. marrow production and is not impacted by platelet transfu-
33. CBO. Richtlijn bloedtransfusie. Utrecht, the Netherlands: sion. Transfusion 2013;53:1201–4.
CBO, 2011. 45. Saigo K, Sakota Y, Masuda Y, et al. Automatic detection of
34. Sanquin. Bloedwijzer deel 1; Erytrocyten, Trombocyten, Vers immature platelets for decision making regarding platelet
bevroren plasma. Amsterdam, the Netherlands: Sanquin 2013. transfusion indications for pediatric patients. Transfus Apher
35. Burdick RK. Chapter 5.2. The balanced two-fold nested ran- Sci 2008;38:127–32.
dom model. In: Burdick RK, Graybill FA, eds. Confidence

156 © 2014 The Authors. European Journal of Haematology Published by John Wiley & Sons Ltd.