Reticulated Platelets

Version	Summary	Created by	Modification	Content Size	Created at	Operation
1		Muataz Ali Hamad	+ 1349 word(s)	1349	2021-05-18 11:29:09	\|
2	Format correct	Vicky Zhou	Meta information modification	1349	2021-05-19 04:15:06	\|

This entry is adapted from the peer-reviewed paper 10.3390/cells10051172

Reticulated platelets (RP) are the youngest platelet fraction released into the circulation. These immature platelets have increased RNA content, a larger cell volume, more dense granules, higher levels of surface activation markers and are thought to be more reactive compared to their mature counterparts. RP have been associated with cardiovascular disease, diabetes and increased mortality. Yet only a few animal studies investigating RP have been conducted so far and further investigations are warranted. Established methods to count RP are flow cytometry (staining with thiazole orange or SYTO13) or fully automated hematology analyzers (immature platelet fraction, IPF). IPF has been established as a diagnostic parameter in thrombocytopenia, cardiovascular disease and, in particular, the response to antiplatelet therapy.

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1. Introduction

Platelets are anucleate cell fragments derived from megakaryocytes (MK) in the bone marrow (BM) at a range of 150,000 to 400,000 cells/μL and play significant roles in hemostasis, thrombosis, and inflammation ^[1]. Reticulated platelets (RP) are the youngest platelets released into the circulation and contain a residual amount of megakaryocyte-derived RNA ^[2]. RP thus have increased RNA content compared to mature platelets and are characterized by a larger cell volume, more dense granules and higher levels of surface activation markers. They are considered to show increased reactivity and are associated with impaired response to antiplatelet therapy ^[2]^[3]^[4]. Higher levels of RP have been linked to a higher risk of major adverse cardiovascular events ^[5]^[6] and are associated with a higher risk of death in patients with acute coronary syndrome ^[7]. Platelets are refractory to many techniques that are commonly used in cell culture, which increases the need for animal models to manipulate and study platelets in different disease settings. The mouse model has been one of the most commonly used animal models to investigate platelets and serves to imitate a variety of pathological conditions. Murine and human platelets are functionally very similar with some differences in structure. While the usefulness of mouse models is clear, some inherent differences must be acknowledged and appreciated in the interpretation of the data.

2. Methods for Reticulated Platelets Determination

2.1. Flow Cytometry

The most commonly used method to evaluate RP is flow cytometry (FCM) with nucleic acid binding fluorochromes such as thiazole orange (TO) or SYTO13. The higher RNA content in RP makes it possible to discriminate these from mature platelets when staining against RNA with a nucleic acid-specific dye. TO is such a nucleic acid-specific dye that exhibits several thousand-fold increases in fluorescence emission upon binding to RNA or DNA ^[8]. TO is excitable at 488 nm which makes it suitable for most flow cytometers, easily permeates the cell membrane, and has a high quantum yield when bound to nucleic acids. When used in RP studies, there is a direct dose–response relationship between the amount of TO added and the number of RP observed, reaching a plateau at 5 μg TO per 5 μL of whole blood, while 12 μg was found to be the optimum ^[9]. Increasing the amount of TO would produce a population shift or mean channel shift that needs to be accounted for when setting gates for TO positive events ^[9]. Using a platelet-specific antibody (such as CD41, CD61) combined with TO is therefore an accurate method of counting RP.

The nucleic acid dye SYTO 13 is an alternative for staining RP which proves to have several advantages over TO ^[10]. SYTO13 shows high stability over time, facilitating extended experimental analysis (for instance making it more suitable for sorting) and higher quantum yield. For both dyes, it is important to note that nonspecific labeling may occur by staining of mitochondria or dense granules with both dyes.

2.2. Fully Automated Analyzers

Besides FCM, automated cell counters have been developed to measure human RP as a fraction of the total platelet count such as the Sysmex hematology analyzer, Abbott CELL-DYN Sapphire, and Mindray analyzer.

2.2.1. Sysmex Analyzers

The Sysmex XE-2100 and 5000 hematology analyzers allow the counting of RP together with reticulocytes using dedicated software and fluorescent dye (polymethine). The immature platelet fraction (IPF) is expressed as both a percentage of total platelets (%IPF) and in the absolute count (#IPF). The new generation of the Sysmex device (XN) uses a specific platelet channel (PLT-F), and a different fluorescent dye (phenoxazine) ^[11]. Some studies have compared the two generations XE and XN and concluded that the XN generation has higher and broader reference intervals of %IPF ^[12]^[13]^[14]. The %IPF in adult, healthy humans range from 0.7–10.1 with some sex-specific reference intervals ^[15]^[16]^[17]. The concept that both generations of analyzers use is the measuring of forward scatter (cell volume) and fluorescence intensity (RNA content), and a computer algorithm discriminates between the mature and immature platelets on these bases.

2.2.2. Abbott Analyzer

The Abbott CELL-DYN Sapphire is a hematology analyzer capable of measuring RPs. The measurement of RP is based on the fluorescent dye CD4K530 as part of the reticulocyte assay. The platelets are separated from the red blood cells by recording three angles of scattered light plus fluorescence. The algorithm used defines RPs in a scatterplot of FL1 versus light at a 7° angle ^[18].

2.2.3. Mindray Analyzer

The Mindray BC-6800 measures IPF by asymmetric cyanine-based dye for staining RNA. IPF is expressed as a percentage and is derived from forward scatter vs. sideward fluorescence scatterplot. The IP/RP can be reported as an absolute number (×10⁹/L) calculated by multiplying the platelet count by the value percentage ^[19].

RP are often investigated in more detail using flow cytometry. IPF is obtained with automated hematology analyzers. The term “young platelets” is more descriptive. These values when compared show a modest correlation with a similar but numerically different trend. The different results obtained with different methods may be attributed to several analytical and pre-analytical reasons. The used stains (solution ready to use or home-preparation), contamination from other blood cells such as leukocyte or the unspecific binding of the dyes and, the different gating strategies are all possible explanations for the differences between the results obtained with different methods. For instance, the flow cytometer gate is usually set at 1%, while for Sysmex it is proprietary and not modifiable ^[19]. Therefore, although the terms RP and IPF are frequently considered synonymous, in practice the two parameters cannot be used interchangeably and it is important to distinguish between them as they only partially overlap ^[20].

In basic research, if mouse platelets are analyzed using automated cell counters that are designed for human platelets, it is necessary to adjust the discriminators due to the smaller size. Very low platelet counts might be attributed to improper blood collection or improper adjustments on the automated cell counters. Another variable that might affect platelet counting is the presence of cellular fragments with a size similar to platelets.

3. Conclusions

In conclusion, the youngest RPs in the circulation are more reactive and show greater tendency to recruit other platelets and immune cells to the site of injury. Containing residual RNA makes it possible to stain RPs and differentiate them from reticulocytes and mature platelets. RPs have been associated with higher CVR in ACSs, major adverse outcomes and, most importantly, increased mortality. Not only do they spike in STEMI and all forms of MI, but diabetes also promotes reticulated thrombopoiesis. Additionally, IPF is utilized as a disease and prognosis modifying parameter in a variety of different conditions. Whether RP/IPFs only serve as prognostic markers or whether RPs themselves are drivers of disease and pose a targetable threat still needs to be investigated in depth. Only a few animal studies on RP have been carried out to this day and most of our current knowledge is derived from observational studies in humans. Although mouse platelets are in many ways similar to those of humans, there are still some questions to be raised when using murine platelets, for example, whether platelets isolated from different sites are similar or whether the method of collection can influence platelet activation. Further considerations limiting research reproducibility are potential differences across strains or the presence of undesirable pathogens. Nevertheless, mouse models have successfully mimicked human diseases and have provided insights into their underlying pathology. Interpreting the results as relevant to a specific experimental setting is crucial and there is no ‘best’ model for every study.

References

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