Keywords
reticulated platelets - immature platelet fraction - staining
Introduction
Reticulated platelets (RPs), also known as immature platelets, are a fraction of the
platelet pool, mostly representing the youngest platelets released from the bone marrow.[1]
[2] They are characterised by some—known so far—distinct features such as: increased
ribonucleic acid (RNA) content, higher volume, more dense granules, higher levels
of surface activation markers and probably higher platelet reactivity.[1]
[3]
[4]
[5]
[6] Until now, several studies have shown an association of RP levels and cardiovascular
events or mortality.[7]
[8]
[9]
[10] Several laboratory methods with distinct features are used to determine RP in clinical
settings. Initially, analysis of RP was performed by means of light microscopy using
supravital staining of blood with new methylene blue, but this method is obviously
not well suited for extended applications.[11] Currently, flow cytometry is the method preferred by the majority of clinical laboratories.
Kienast and Schmitz reported a flow cytometric assay based on thiazole orange (TO)
staining for analysing RP.[12] TO binds to platelet RNA and various intracellular components.[13] The amount of unspecific non-RNA binding and hence varying fluorescence intensity
is affected by staining conditions, such as incubation time, temperature or fixation.[1] Consequently, this method is hampered by an evident lack of analytical standardization,
which disables a direct comparison of data obtained by different laboratories.[4]
[12]
[14]
[15]
[16] Moreover, when prolonged sample work-up is inevitable, for example, in the setting
of cell sorting with a low number of events, TO staining is difficult as it leads
to unspecific TO-positive platelets and increasing fluorescence intensity over time.
An alternative option to measure RP is a fully automated assay using a fluorescent
polymethine dye which has been established on the SYSMEX XE-2100 (SYSMEX, Kobe, Japan)
analyser. The immature platelet fraction (IPF, %) provides the fraction of immature
platelets within the whole platelet pool, while immature platelet count provides the
number of immature platelets (103/µL)—both determined using a cut-off pre-defined by the manufacturer. By means of
this method, an association between RP and clinical events or drug response, for example,
in patients treated with thienopyridines, has previously been shown.[5]
[8]
[9]
[10]
[17] However, as the SYSMEX assay is a fully automated closed system, assay modifications
for experimental purposes can hardly be implemented and more in-depth investigations
on RP surface markers are not possible.
Therefore, the aim of our present work is to establish a new platelet staining protocol
which overcomes some of the limitations in RP analysis mentioned before. A potential
staining dye is SYTO 13 which is a cell-permeant green fluorescent dye with high potency
and affinity to RNA showing a large fluorescence enhancement after binding. SYTO 13
has an absorption maximum at 491 nm and an emission maximum at 514 nm in the presence
of RNA.
Materials and Methods
Staining of Platelets
We developed a laboratory protocol for SYTO 13 staining of platelets. First, washed
platelets (WPs) were obtained from citrate-anticoagulated blood (Sarstedt, Nümbrecht,
Germany). Blood was centrifuged at 150 × g for 10 minutes and platelet-rich plasma (PRP) was manually separated. To avoid activation
and aggregation of platelets, 10 µL prostaglandin I2 (10 µg/mL; Sigma-Aldrich, St. Louis, Missouri, United States) were added to 490 µL
PRP and thoroughly mixed. After pelleting (380 × g, 20 minutes), platelets were re-suspended
in 1,000 µL Tyrode's-HEPES buffer (pH 7.4; 145 mMol NaCl [Sigma-Aldrich], 2.9 mMol
KCl [Sigma-Aldrich], 10 mMol HEPES [Sigma-Aldrich], 1 mMol MgCl2 [Sigma-Aldrich], 5 mMol glucose [Sigma-Aldrich]), pelleted again (380 g, 20 minutes)
and re-suspended in 500 µL Tyrode's-HEPES buffer. Platelets were diluted to a final
concentration of 5 × 104 platelet/µL with Tyrode's-HEPES buffer. To stain RNA, 10 µL of SYTO 13 (1 µM, final
concentration 12.5 nM; Thermo Fisher, Waltham, Massachusetts, United States) were
added to a suspension of 100 µL WP diluted in 690 µL phosphate-buffered saline (PBS;
pH 7.4; 137 mMol NaCl, 2.7 mMol KCl, 10 mMol Na2HPO4 [Sigma-Aldrich], 1.8 mMol KH2PO4 [EMD Millipore, Burlington, Massachusetts, United States]). For TO staining, 70 µL
of Retic-Count (Becton Dickinson, Heidelberg, Germany) was added to a suspension of
100 µL WP diluted in 630 µL PBS. As negative controls, 100 µL WP were diluted in 700
µL PBS. After respective periods of incubation at room temperature in the dark, median
fluorescence intensity (MFI, arbitrary units) of all platelets was measured using
a SONY SH800Z cell sorter (SONY, Tokyo, Japan).
Furthermore, modifiability of assays is a desirable criterion for laboratory experiments.
Hence, we compared staining of WP (a), fixed WP (b), PRP (c) and whole blood (d) to
confirm adaptability of SYTO 13 staining of platelets:
-
WP were prepared and stained for 90 minutes as aforementioned.
-
After fixation of WP with 1% formaldehyde (Polysciences, Hirschberg, Germany) for
10 minutes, 100 µL fixed WPs were stained with 10 µL SYTO 13 (1 µM) in 690 µL PBS
for 90 minutes.
-
50 µL PRP were stained with 10 µL SYTO 13 (1 µM) in 690 µL PBS for 90 minutes.
-
5 µL whole blood were double-stained with 5 µL CD41 allophycocyanin (APC) (Becton
Dickinson) as a platelet-specific marker and 10 µL SYTO 13 (1 µM) in 690 µL PBS for
90 minutes.
All steps were performed at room temperature unless indicated otherwise. Fluorescence
was quantified in the FL-1 fluorescein isothiocyanate channel (500–550 nm) after platelet
gating by forward scatter/backward scatter characteristics and doublet exclusion.
For whole blood staining, platelets were additionally identified by gating for CD41-positivity
in the FL-4 APC (650–680 nm) channel. Fluorescence intensities of unstained platelets
remained stable over at least 24 hours. The sorter was calibrated with automatic setup
beads (SONY) every day before use.
Platelet RNA Stability at Different Incubation Temperatures
RNA of unstained WP from three healthy donors (19.5 × 106, 21 × 106, 23 × 106 platelets) was isolated immediately after WP preparation as well as after 5 hours
of storage at 22°C or 37°C, respectively. 500 µL WP were diluted 1:4 with TRIzol LS
reagent (Thermo Fisher). 200 µL chloroform (Carl Roth, Karlsruhe, Germany) per 750
µL TRIzol LS were added. After 3 minutes of incubation, samples were centrifuged for
15 minutes at 12,000 × g at 4°C. The aqueous phase was manually separated. Thereafter, RNA isolation from
the aqueous phase was accomplished by use of a miRNeasy Micro Kit (Qiagen, Venlo,
Netherlands) per protocol of the manufacturer. The extracted RNA amount was determined
by means of a RNA 6000 Pico Assay on a 2100 Bioanalyzer (Agilent, Santa Clara, California,
United States).
Correlation of TO/SYTO 13 Staining to IPF
Blood samples obtained from 21 patients after transcatheter aortic valve implantation
(TAVI) were analysed. Their median age was 83 years (interquartile range [IQR]: 80–86).
Eighteen patients received Edwards-Sapien 3 aortic valves and 3 patients received
Evolute R aortic valves, respectively. Demographic and clinical data of these patients
are provided in [Supplementary Table S1] (available in the online version). The study was approved by the ethics committee
of the University of Freiburg (Germany) and all patients gave written informed consent
before any study procedure. Blood was collected 1 day following the TAVI procedure
and WP were stained with TO and SYTO 13 as described afore. Polymethine-based RP analysis
as well as IPF were determined using the automated blood cell counter SYSMEX XE-2100.
Spearman's ρ was used to assess correlations between IPF and MFI of TO- and SYTO 13-stained platelets.
RNA Quantification according to SYTO 13 Staining Intensities
For internal validation, the association of RNA content and SYTO 13 staining intensities
was investigated. SYTO 13-stained platelets obtained from a drug-free healthy subject
were divided into quintiles in the FL-1 channel according to increasing fluorescence
intensities. A total of 8 × 106 platelets were sorted from each gate and RNA was extracted as described before. Due
to the high amount of fluid, the first centrifugation step in the Qiagen protocol
was modified and a vacuum chamber was used instead for separation of RNA. The next
steps were performed per protocol of the manufacturer. The extracted RNA amount was
determined in duplicate as described before.
RNA Quantification of RNAlow and RNArich Platelets
As a second internal validation, the RNA amount of 8 × 106 platelets sorted from the outer quintiles of SYTO 13 fluorescence intensities (termed
RNAlow and RNArich platelets) was quantified in WP from eight healthy donors. RNA
was extracted and quantified as described before. Paired t-test (GraphPad Prism 7, San Diego, California, United States) was used to compare
RNA amounts of RNAlow and RNArich platelets. All values are expressed as median with
IQR unless otherwise indicated.
Results
Staining of Washed Platelets
SYTO 13 staining of platelets showed a continuous shift in the FL-1 channel with—compared
with TO staining—a remarkable 10-fold increase in mean fluorescence intensity ([Fig. 1]). Doublet gating was used to avoid staining events with false positive high fluorescence
intensity ([Supplementary Fig. S1], available in the online version).
Fig. 1 Flow cytometry plots after an incubation time of 90 minutes of (A) unstained, (B) TO-stained and (C) SYTO 13-stained unfixed washed platelets from a patient after
TAVI. Abbreviations: BSC, backward scatter; SSC, side scatter; FL-1, 500–550 nm; TAVI,
transcatheter aortic valve implantation; TO, thiazole orange.
Staining under Various Conditions
Furthermore, staining of fixed WP, PRP and whole blood showed that SYTO 13 staining
is feasible under various conditions. Each tested condition yields an increase of
fluorescence in the FL-1 channel ([Fig. 2], left two columns). In sorting experiments, SYTO 13 staining of platelets enables
adequate sorting as confirmed by re-analysis of flow cytometry plots for RNArich and
RNAlow platelets in different FL-1 gates ([Fig. 2], right two columns). Platelet fixation resulted in loss of MFI as compared with
the other conditions. Depending on the planned specific laboratory experiments, one
might choose the most appropriate condition.
Fig. 2 Staining of washed platelets, fixed washed platelets, platelet-rich plasma and whole
blood with SYTO 13 for 90 minutes. Platelets were sorted according to the RNAlow and
RNArich gates and re-analysed. MFI, median fluorescence intensity (arbitrary units).
Stability of Staining Intensity
Having demonstrated that SYTO 13 staining, as compared with TO staining, is feasible
after 90 minutes of staining, the stability of staining intensity over prolonged incubation
times was investigated. After staining for 90 minutes, fluorescence intensity remained
stable for at least 5 hours ([Fig. 3A]). RNA amount remained stable up to at least 5 hours of incubation at room temperature
(22°C), while a substantial amount of RNA was degraded after 5 hours of incubation
at 37°C ([Fig. 3B]).
Fig. 3 (A) Stability of SYTO 13 and thiazole orange (TO) staining over time from 3 healthy
subjects (median and range; h = hours). (B) Representative example of a Bioanalyzer electropherogram of washed platelet ribonucleic
acid (RNA) from a healthy donor immediately after preparation of washed platelets
and at 5 hours of storage at 22°C and 37°C, respectively. Abbreviation: MFI, median
fluorescence intensity (arbitrary units).
Correlation of TO and SYTO 13 Staining with IPF
MFI of SYTO 13-stained platelets correlated well with the established IPF measurement
by SYSMEX analysed 30 minutes (r = 0.665, p = 0.001) as well as 90 minutes (r = 0.668, p < 0.001) after staining, whereas a moderate correlation with MFI of TO-stained platelets
was obtained solely after 30 minutes (r = 0.478, p = 0.029; [Fig. 4]).
Fig. 4 Correlation of immature platelet fraction (IPF) and MFI of unfixed washed platelets
after 30 minutes (upper panels) and 90 minutes (lower panels) of staining with (A) thiazole orange and (B) SYTO 13 ; n = 21 patients on day 1 after transcatheter aortic valve implantation. Abbreviation:
MFI, median fluorescence intensity (arbitrary units).
RNA Quantification according to SYTO 13 Staining Intensities
RNA quantification of sorted platelets according to the quintiles of MFI showed a
strong association of SYTO 13 staining intensities and extracted RNA amount ([Fig. 5]). [Fig. 5B] shows Bioanalyzer electropherograms of sorted platelets from each quintile indicating
an increase in the amount of total RNA with higher fluorescence intensity. 18S and
28S ribosomal peaks can only be observed in the electropherogram of the quintile with
highest staining intensity which is indicative for the high RNA amount in this quintile.
Fig. 5 (A) Gating strategy for sorting unfixed washed platelets in the FL-1 channel according
to quintiles ranging from low to high fluorescence intensities after SYTO 13 staining
(incubation time: 90 minutes). (B) Bioanalyzer electropherograms of extracted platelet ribonucleic acid (RNA) from
each quintile. (C) RNA amount (fg per platelet) of sorted platelets from each quintile; platelets from
a drug-free healthy subject measured in duplicate.
RNA Quantification of RNAlow and RNArich Platelets
RNA quantification resulted in a RNA amount of 0.35 (0.23–0.52) fg per platelet in
the 20% of platelets with lowest staining intensity (RNAlow platelets) and 0.88 (0.78–1.14)
fg per platelet in the 20% of platelets with highest staining intensity (RNArich platelets;
p = 0.016).
Discussion
Our approach was based on certain features: (1) the staining dye should be specific
for a single component of the platelet, (2) the staining procedure should yield a
clear increase in fluorescence intensity, (3) the staining conditions should enable
additional staining of extra- and intracellular components with other dyes or antibodies,
(4) advanced processing of platelets by cell sorting for further analysis should be
feasible and (5) the staining should be comparable to the well standardized and clinically
established method, the SYSMEX polymethine dye-based assay. We selected a nucleotide
staining dye since this seems to fulfil all requirements outlined above.
RNA extraction and quantification of platelets divided into quintiles by fluorescence
intensity after SYTO 13 staining showed a clear association of RNA amount and fluorescence
intensity. Distinct ribosomal peaks could only be determined in the electropherogram
of the quintile with highest fluorescence intensity indicating that RPs are present
in this quintile. Hence, the quintile with highest fluorescence intensity may be adopted
as a gate for dedicated sorting of RP. RNA extraction and quantification of sorted
RNAlow and RNArich platelets confirm highly different amounts of RNA in the respective
populations. Therefore, our staining and gating method indeed identifies different
platelet populations having low and high RNA levels correctly. In contrast to TO staining,
the stability of SYTO 13 MFI facilitates analysis of stained platelets in extended
experimental settings, for example, during cell sorting with low event rates.
Despite the strong association of SYTO 13 fluorescence intensities and extracted RNA
amount, we cannot exclude potential unspecific labelling of other cell components,
for example, dense granules or mitochondria. From the current data, similar to other
methods with continuous staining distribution, the true rate of newly released platelets
per day remains undetermined. Detailed in vivo experiments, for example, radiolabelling,
would be necessary to define specific cut-off values for platelet turnover. In summary,
our results indicate that SYTO 13 seems to be an attractive alternative staining dye
for experiments on RNA-rich platelets and thereby also for RP.
What is known about this topic?
-
A higher immature platelet fraction (IPF) is linked to higher platelet reactivity.
-
Reticulated platelets are associated with increased cardiovascular events and mortality.
-
There is no generally accepted standardised protocol for the determination of reticulated
platelets using thiazole orange (TO) despite this is the so far most frequently used
non-automated laboratory method.
What does this paper add?
-
An alternative and highly stable staining method for reticulated platelets using SYTO
13 is described.
-
Fluorescence intensities of SYTO 13-stained platelets are associated with the clinically
so far best standardised laboratory parameter IPF.
-
In contrast to TO staining, SYTO 13 staining facilitates analysis of platelets in
extended experimental settings.
