DOI 10.1515/cclm-2014-0165 Clin Chem Lab Med 2014; 52(8): 1107–1117

Review

Johannes J.M.L. Hoffmann* Reticulated : analytical aspects and clinical utility

Abstract: Reticulated platelets are immature platelets Formation and maturation circulating in ; they reflect the activity of megakary- opoiesis in the bone marrow. Therefore, they can be used of as a non-invasive test in patients with thrombocytopenia in various clinical conditions. The preferred method of The study of blood platelets or thrombocytes started in analysis is by flow cytometry. However, there is an evident the late 19th century, when Wright had developed his lack of analytical standardization, making it difficult to variant of the Romanovsky stain that allowed detailed compare results obtained in different laboratories. Cur- observations of the smallest blood elements [1]. Plate- rently, two types of hematology analyzers are on the mar- lets represent the terminal stage of megakaryopoiesis, ket offering fully automated measurement of reticulated a highly complex sequence of events that can be traced or immature platelets: the high end analyzers manufac- back to the generation of the pluripotent hematopoietic tured by Sysmex (XE- and XN-series) and Abbott (CELL- stem cell in bone marrow. This pluripotent stem cell pro- DYN Sapphire). Although the methods are essentially liferates and differentiates via several intermediates into different and cannot be used interchangeably, both have a megakaryoblast and eventually into a been proven to have clinical utility. Reticulated or imma- (Figure 1). Megakaryopoiesis is regulated by various ture assays are useful for the differential diagno- growth factors and cytokines, of which thrombopoietin sis of thrombocytopenia and for monitoring bone marrow (TPO) is the most important. TPO stimulates the number, recovery after chemotherapy or stem cell transplanta- the size and the ploidy of megakaryocytic cells and is the tion. These assays may aid clinicians in platelet transfu- key regulator of platelet concentration in the circulation sion decisions when recovery from thrombocytopenia is [2]. Once the megakaryoblast stage is reached, the cell imminent. In addition, preliminary findings indicate that loses its proliferative capacity and starts the maturation there is a rationale for reticulated or immature platelets process. Megakaryopoiesis has a unique way of matu- for risk stratification in acute coronary syndromes and for ration that does not occur in other cell lines: endomi- monitoring the effect of treatment with antiplatelet drugs tosis. This is nuclear division without cell division; the in patients with coronary artery diseases. The aim of this cell is multiplying its nuclear material and increases paper is to present the present technology available for its cytoplasm, all within the same cell. Finally, matura- measuring reticulated platelets as well as an overview tion results in a megakaryocyte that possesses multiple of the current status of clinical application. This over- nuclear copies and abundant cytoplasm. The nuclear view also indicates that more research is needed before ploidy of a megakaryocyte is normally between 8N and reticulated or immature platelet assays can be applied in 64N (median 16N), whereas higher and lower ploidy other clinical conditions than thrombocytopenia and after may occur in various pathological conditions. Depend- transplantation. ing on the organism’s need of new platelets, endomitosis stops and the formation of platelets commences (Figure Keywords: diagnostic use; immature platelets; megakaryo­ 1). First, there is intracytoplasmic formation of channel- poiesis; reticulated platelets; thrombocytopenia. like structures composed of lipids, called the membrane demarcation system [3]. These lipids later assemble into membrane bilayers and eventually form the cell mem- *Corresponding author: Dr. Johannes J.M.L. Hoffmann, Abbott branes of platelets when the megakaryocyte cytoplasm Diagnostics Division, Abbott GmbH and Co. KG, Max-Planck-Ring 2, 65205 Wiesbaden-Delkenheim, Germany, starts to disintegrate. In this phase there is also active E-mail: [email protected] protein synthesis and ­cytoskeletal assembly. 1108 Hoffmann: Reticulated platelets

Differentiation Maturation

Endomitosis Hematopoietic Myeloid Megakaryoblast 2N 4N 8N 16N 32N stem cell stem cell

Megakaryocytes

Bone marrow

Blood

Platelets

Figure 1 Schematic representation of megakaryopoiesis. After Kuter [2].

Platelet production by Physiology of reticulated platelets megakaryocytes When platelets are released from the megakaryocyte cyto- plasm, they still contain a small amount of RNA. For long Eventually, megakaryocytes form pseudopodia-like exten- it has been thought that this RNA was a vestigial remnant sions protruding into sinuses and release platelets into of megakaryocytic RNA, but there are recent indications the extracellular space. How this exactly takes place is not that platelets are able using this RNA for protein synthesis fully elucidated [4–6]. A healthy human produces approx- [9]. Anyway, they represent the youngest platelets in the imately 1–2 million platelets per second. In response to circulation and are named reticulated platelets (retPLT), in thrombocytopenia, platelet production can be accelerated analogy with in [10]. The con- by up to a 10-fold increase [7]. centration of retPLT in bone marrow is on the average 2–3 Megakaryocyte cytoplasmic volume expands syn- times higher than in peripheral blood, where they corre- chronized with nuclear ploidy and this eventually deter- late with megakaryocyte numbers [11]. And whereas plate- mines the number of platelets that a megakaryocyte will lets persist in the circulation for 7–10 days, retPLT have a produce. A single megakaryocyte can generate up to 5000 much shorter lifespan ( < 1 day). Therefore they can act as platelets [1, 2]. In steady-state conditions, the platelet a marker of megakaryopoietic activity in the bone marrow production rate is aimed at keeping the total circulating [12, 13], which gives retPLT clinical and diagnostic utility. platelet mass (platelet number × mean platelet volume, also called plateletcrit) constant [1]. In conditions of stress, platelets are released from megakaryocytes at an earlier stage, which results in platelets that are larger than normal. Reticulated platelet methods Each individual has its own personal setpoint for – flow cytometry platelet count and platelet volume; these are largely under genetic control [8]. As a consequence, intra-individual The initial description of retPLT dates back to 1969, when variations in platelet count are quite small in comparison Ingram and Coopersmith studied a canine model of acute with the population reference ranges. In the normal pop- blood loss and observed coarsely punctuated reticulum ulation, platelet count is inversely correlated with mean in platelets after supravital staining of blood with new platelet volume (MPV), and consequently the total circu- methylene blue [14]. This technique was initially also used lating platelet mass is less variable between individuals in human blood, but it is obviously not well suited for than platelet count. routine applications. Hoffmann: Reticulated platelets 1109

Kienast and Schmitz initiated a breakthrough in the since two manufacturers offer hematology analyzers that field when they described a flow cytometric technique for are capable of measuring reticulated or immature plate- analyzing retPLT, based on RNA staining by thiazole orange lets (Table 1). [15]. In subsequent years, several research groups published their findings in a wide variety of conditions like thrombo- cytopenia [16–21], thrombocytosis [22, 23], after stem cell transplantation [24–27], hereditary platelet diseases [28, 29], Immature platelet methods – thrombo-embolic disorders [30, 31], kidney disease [32–34], Sysmex hematology analyzers preeclampsia [35], hyperthyroidism [36] and in healthy and thrombocytopenic neonates [37, 38]. The overall conclu- The first available fully automated method for measur- sion from these studies is that retPLT in blood represent a ing reticulated platelets was in the R-3000, a dedicated useful non-invasive marker of megakaryopoietic activity in analyzer developed by Toa Medical (later the bone marrow. However, it also became evident that the Sysmex) [46, 47]. The method used auramine O as a fluo- flow cytometric assay was prone to methodological vari- rescent RNA dye and a 488 nm Argon laser. By plotting ation, which made it difficult to compare results obtained forward light scatter (representing cell size) against fluo- with different assays. For example, the normal reference rescence (RNA content), reticulated platelets could be range was reported to range between 1% and 15% [23, 39]. distinguished from mature platelets. In normal healthy This wide range can be explained by lack of standardization individuals the mean retPLT count was 0.98%–1.27% and of the methods. Many factors have been identified that con- thrombocytopenic patients with a variety of diseases had tribute to this analytical issue: the type and concentration increased concentrations, in accordance with the flow of fluorescent dye, incubation time and temperature, fixa- cytometric results mentioned above [18, 47, 48]. Notably, tion, RNAse treatment and the flow cytometric data analy- a strong positive correlation existed between percent- sis, including gating and threshold settings [17, 18, 39–41]. age retPLT and the fraction of large platelets, except in One of the major problems is that platelets show non-RNA- patients with aplastic anemia [47]. Furthermore, the large specific binding of fluorescent dye, resulting in background platelet fraction highly correlated with MPV; data on the staining, which is size-dependent [39, 42, 43]. This issue correlation between retPLT and MPV were not provided, can be solved by optimizing the assay conditions and, in but one can reasonably assume that such correlation was particular, by applying a two-dimensional gating process present. [39]. Nevertheless, standardization attempts for improving Automated measurement was later integrated into concordance between laboratories were unsuccessful, even the Sysmex XE-2100 and XE-5000 hematology analyzers, when using the same protocol [9]. Recently, new initiatives as a part of the reticulocyte determination. This enabled were undertaken that are aimed at developing a method automated quantification of what from then on was called with the potential to become a future international reference immature platelet fraction (IPF) [49]. As these instruments method [44, 45]. However, even if a standardized method employ a 633 nm diode laser as the light source, other dyes would be available, it would carry the disadvantage of not were needed and a mixture of polymethine and oxazine was being well suited for routine clinical applications, since flow selected. As before, a forward scatter versus fluorescence cytometry requires a great deal of expertise and is hardly scatterplot defined platelets with the highest fluorescence available for patient care on a 24/7 basis. For the time intensity as immature platelets (Figure 2A). The reference being, a surrogate level of standardization is possible, values in a healthy population were clearly higher than with

Table 1 Overview of some key characteristics of methods for measuring reticulated or immature platelets.

Flow cytometry Sysmex IPF Abbott retPLT

Sample preparation Variable: none, fixation or None None isolation of platelets Fluorescent dye Thiazole orange, acridin orange Polymethine (XE-series); oxazine (XN-series) CD4K530 Incubation time Variable 15 min–2.5 h Not specified 47 s Precision at normal platelet count, CV Not specified 7%–11% < 12% Precision in thrombocytopenia, CV Not specified 9%–36% 11%–32% Reference range,% Highly variable 1%–15% 1.1–6.6 0.5–6.0 Reference range, 109/L Mean 3.2 2–17 1–18 1110 Hoffmann: Reticulated platelets

RETC B C A WBC

retPLT RETC FL1 RNA Forward scatte r Forward scatte r RBC PLT

Fluorescence intensity Fluorescence intensity 7°

Figure 2 Scatterplots of the automated assays of immature and reticulated platelets currently available. In the Sysmex XE-series, immature platelet fraction (IPF) is determined in the reticulocyte assay, using forward scatter against fluorescence (A). The Sysmex XN-series measure IPF as part of the fluorescent platelets assay, again using forward scatter against fluorescence (B). In the Abbott CELL-DYN Sapphire reticulocyte assay, platelets and red blood cells are first separated using a side scatter against intermediate angle scatterplot (not shown) and then reticulated platelets are determined using fluorescence (FL1) against intermediate angle scatter (7°), correcting for size-dependent background staining (C). IPF, immature platelet fraction; PLT, platelets; RBC, red blood cells; RETC, reticulo- cytes; retPLT, reticulated platelets; WBC, white blood cells. the previous R-3000 method: mean 3.4% (range 1.1%–6.1%) separation of platelets and red blood cells. The embedded [49]. These reference values were later confirmed by others algorithm defines retPLT in an FL1 versus 7° scatterplot [50–54]. In all studies, patients with idiopathic thrombocy- (Figure 2C). This approach enables correcting for the size- topenia (ITP) had increased IPF; in some patients IPF was dependent background fluorescence of platelets [39]. In even strongly increased, up to 50%–60% [49, 54, 55]. Such healthy individuals, mean retPLT are between 1.4% and extreme values were later recognized to be artifacts due to 2.2%, and reported reference ranges are 0.4%–2.8% [59], interference by fragments [56]. 0.4% to 4.45%–6.0% [60–62] and 1.0%–3.8% [63]. With the introduction of the XN-series hematology In the CELL-DYN Sapphire, the correlation between analyzers, Sysmex changed the IPF method. It is now part retPLT and MPV was investigated in relatively small of the fluorescent platelet assay, which uses a proprietary groups and was described as not significant [64] or only oxazine-based dye for staining RNA. Still, IPF is derived weak [63]. However, in a large group of subjects with from the forward scatter (cell size) versus sideward fluo- normal platelet counts, we found a significant negative rescence (RNA content) scatterplot [56, 57], as shown correlation between retPLT and MPV [62]. in Figure 2B. The moderate correlation in IPF between XE-2100 and XN is explained, at least partially, by reduced interference in the XN system [56]. Preliminary data suggest that the XN reference values of IPF are compara- Comparison of reticulated and ble with the XE-2100 method [56]. immature platelet methods

Due to the lack of a standardized reference method for retPLT, assessing the performance of the automated Reticulated platelet methods – methods is difficult; only a few side-by-side comparisons Abbott CELL-DYN Sapphire are available. Studies where Sysmex XE-2100 IPF was compared Apart from the Sysmex method discussed above, the with reference flow cytometric retPLT indicated low or Abbott CELL-DYN Sapphire is currently the only hematol- moderate correlations [65, 66]. Remarkably, the coeffi- ogy analyzer capable of measuring retPLT. The assay is an cients of correlation differed between patients groups, integral part of the reticulocyte assay, which is based on the from no correlation in healthy individuals to relatively fluorescent dye CD4K530 [58] that is excited by a 488 nm high correlations in patients with thrombocytopenia due solid state laser. Three angles of scattered light plus fluo- to peripheral destruction [66, 67]. One study using the rescence are recorded, which allows multi-dimensional Sysmex XT-2000iV, which uses an identical method as Hoffmann: Reticulated platelets 1111

XE-2100, demonstrated reasonable correlation between that IPF#, but not relative IPF predicts imminent platelet IPF and reference flow cytometry, albeit with a significant recovery in chemotherapy-induced thrombocytopenia in systematic bias [68]. children [76]. Importantly, IPF# seems not to be influenced Direct comparison between Sysmex XE-2100 or by platelet transfusions, whereas IPF% decreases, most XE-5000 IPF and CELL-DYN Sapphire retPLT resulted likely due to dilution [77]. Other authors found IPF# not in weak or modest correlations [63, 64]. Sapphire was helpful for assessing platelet turnover [63]. More studies found to have a distinctively narrower reference range are needed in order to fully appreciate whether the abso- than XE-5000, enabling higher sensitivity for separating lute IPF or absolute retPLT count have additional value to normal and abnormal patients [63]. relative counts. These weak correlations and the different relation- It is well accepted that accelerated megakaryopoie- ships between IPF/retPLT and MPV mentioned previously, sis is associated with increased MPV. As also IPF is often lead to the conclusion that although both parameters increased in this condition, this has led to the widespread harbor information of platelet turnover, they do reflect belief that immature platelets are synonymous with large different aspects of thrombopoiesis. As a consequence, platelets [47, 78]. This notion seems to be reinforced by the two parameters cannot be used interchangeably [63]. high correlations between IPF and MPV [64, 66, 79–81]. This correlation is only present in Sysmex analyzers, most likely as a result of how IPF is derived: the scatterplots indeed suggest that immature platelets are the largest Pre-analytical aspects platelets [49, 54, 82]. In contrast, no significant correlation was found between retPLT and MPV in flow cytometry [17] Obviously, immature or reticulated platelets are normally and neither in CELL-DYN Sapphire [63, 83]. It is true that measured in blood samples collected into K2-EDTA, as for platelets produced in response to stress megakaryopoie- other hematologic parameters. One single study advised sis are on the average larger, but there is no evidence that that citrate-theophylline-adenosine-dipyridamole solu- this can be extrapolated to normal megakaryopoiesis. In tion (CTAD) was to be preferred over EDTA, as IPF meas- healthy individuals with steady-state platelet production, ured using a Sysmex XE-5000 remained more stable in we found a significant negative association between retPLT blood from patients with chronic ITP [69]. and MPV [62]. This is more in keeping with the concept of Regarding storage temperature before analysis, most a constant circulating platelet mass, which would require authors agree that XE-2100 IPF is stable for 24–48 h after more small platelets or fewer larger platelets [1]. So, reticu- blood collection, provided the samples are kept at ambient lated platelets are not necessarily large platelets. temperature [49, 70, 71]. Others, however, reported stabil- ity for 3–8 h only [50, 51]. When blood samples are kept at 4 °C, IPF is rather unstable [69, 72], but the increase in IPF is apparently so predictable that it can be corrected for by Clinical utility of reticulated/ a simple algorithm [73]. immature platelets Stability data on retPLT as measured with CELL-DYN Sapphire range between < 6 h [63] and at least 26 h [62]. In the earlier years, retPLT research focused on a pos- sible differential diagnostic aid in patients with throm- bocytopenia. Since megakaryopoietic activity is low in patients with bone marrow failure, the assumption was Post-analytical aspects that consequently retPLT would be low, too. In contrast, conditions with peripheral platelet destruction like ITP Traditionally, IPF is expressed in relation to the PLT count, are characterized by accelerated megakaryopoiesis and as a percentage. The absolute IPF (IPF#), the concentra- hence the retPLT count would be increased. Many studies tion of immature platelets (in 109/L), might better reflect have now confirmed that retPLT or IPF are valuable in real-time platelet production in analogy with what abso- establishing the cause of thrombocytopenia: decreased lute reticulocyte count does for erythropoietic activity [67]. production can reliably be distinguished from peripheral There are indeed some reports indicating the usefulness destruction (see Table 2). of IPF# in neonatal infections [74], in chronic liver disease Another well-documented application of retPLT is [51] and in differentiating acute ITP and from thrombocy- monitoring the thrombocytopenic phase after chemother- topenia due to acute [75]. It has been reported apy and transplantation for hematological malignancies 1112 Hoffmann: Reticulated platelets

Table 2 Clinical conditions where retPLT or IPF can be regarded as established diagnostic or prognostic tools.

Condition Intended goal References retPLT References IPF

Low platelet count Thrombocytopenia of unknown etiology Differentiating hypoproduction [15, 63, 66, 84, 85] [49, 55, 63, 66, 75] from accelerated destruction Chemotherapy Predicting platelet recovery [86, 87] [76] Bone marrow or peripheral stem cell Predicting platelet recovery [17, 88–90] [91–95] transplantation Normal or high platelet count Thrombocytosis Estimating platelet turnover [22, 23, 96]

(Table 2). Generally, an increase in retPLT precedes the well as for monitoring drug treatment of coronary artery recovery of platelet count by 2–3 days. This creates the disease [78, 79, 105–107]. However, larger and randomized opportunity to defer platelet transfusions that would be studies are necessary for proving that these concepts are given when transfusion decisions are based on platelet effective and safe in clinical practice. There are also indi- counts only. Until present, limited clinical evidence sup- cations that it is preferentially immature platelets that are porting this concept has been reported and there is an recruited into arterial thrombi [106] and if confirmed, this evident need for randomized, controlled clinical studies finding may have consequences for treatment with anti- in this field [82, 97, 98]. A highly interesting observation platelet drugs [107]. that also needs to be confirmed is that prophylactic trans- Apart from the above disease states, there are several fusions with high IPF platelet concentrates seem to be other conditions where retPLT are supposed to play a role more effective than low IPF platelets [99]. (Table 3). Most of these associations are only described in Researchers in the field of cardiovascular diseases a single paper or are based on small patient numbers, thus have recently gained interest in retPLT (Table 3). The the scientific evidence is still weak. Therefore, additional current literature seems to provide preliminary support research studies are absolutely required for obtaining for the clinical utility of retPLT or IPF determinations, for independent confirmation on the utility retPLT or IPF in risk assessment in acute coronary syndrome [100–104] as these settings.

Table 3 Clinical conditions where retPLT or IPF may have potential clinical utility, but further studies are required for confirmation.

Condition Possible application References retPLT References IPF

Low platelet count without marrow dysfunction ITP Predicting treatment response [84] Disseminated intravascular coagulation Assessing prognosis [108] Thrombotic thrombocytopenic purpura Assessing disease activity and adjusting therapy [49, 109] Cyclic thrombocytopenia Predicting next thrombocytopenia phase [110] [111] Assessing prognosis in cases with aberrant karyotype [98, 112] Normal or high platelet count Essential thrombocytemia and Investigate possible linkage with Jak2 mutation [113] polycythemia vera Therapy with thrombopoietic drugs Assessing treatment effect [67] Altered platelet function Acute coronary syndrome Assessing role of platelet activation in prognosis [100, 104] [101–104] Drug treatment of coronary artery Predicting treatment response [78, 106, 107] [79, 105] disease Chronic uremia Assessing effect of hemodialysis on platelet kinetics [32] [114] Miscellaneous Chronic liver disease Differentiating between hepatitis and cirrhosis [51] ICU patients Predicting development of sepsis [115] Neonates Predicting degree of thrombocytopenia [116, 117] Diabetes mellitus Predicting cardiovascular complications [81] Platelet transfusions Supporting transfusion decisions [97] [82, 98, 99] Hoffmann: Reticulated platelets 1113

or IPF may be useful for risk assessment as well as Highlights and conclusions therapy monitoring –– Some other indications look promising, but need Summarizing the literature reviewed above, it can be con- further investigation before routine use of reticulated cluded that: platelet assays is warranted –– Reticulated platelets are the most immature platelets in the circulation and they reflect the megakaryopoietic Acknowledgments: I thank my colleagues Ting Yu and Nigel activity in bone marrow Llewellyn-Smith for their valuable comments and advice. –– Reticulated platelets are not necessarily large platelets –– The two commercially available methods (Sysmex IPF and Abbott reticulated platelets) are not Conflict of interest statement interchangeable and each require their own reference Author’s conflict of interest disclosure: The author stated values and clinical decision limits that there are no conflicts of interest regarding the publi- –– There is an urgent need for a standardized method cation of this article. Employment and leadership played that can serve as an international reference for no role in the study design; in the collection, analysis, and assessing the performance of reticulated/immature interpretation of data; in the writing of the report; or in the platelet methods decision to submit the report for publication. –– Measuring reticulated/immature platelets is of Research funding: None declared. proven clinically usefulness in patients with Employment or leadership: The author is a scientific thrombocytopenia of unknown etiology and for employee of Abbott Diagnostics. monitoring bone marrow recovery after chemotherapy Honorarium: None declared. and stem cell transplantation –– Reticulated platelets seem to play a role in the etiology of coronary arterial diseases. If confirmed in large- Received February 16, 2014; accepted April 8, 2014; previously scale trials, measurement of reticulated platelets ­published online May 7, 2014

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Johannes (Hans) Hoffmann started his career in clinical chemistry in 1976 as a trainee. Once certified as a specialist he became the head of the hematology laboratory in a large tertiary-care teaching hospital in the Netherlands, where he later was also appointed director of the Department of Clinical Laboratories. In 1992 he obtained his PhD in medical sciences at Leiden University, the Netherlands, on a thesis in the field of fibrinolysis. Since 2008 he is responsible for scientific affairs in hematology with Abbott Diagnostics in Europe. His scientific work comprises over 100 papers in peer-reviewed journals, mainly focused on general hematology, flow cytometry, coagulation and fibrinolysis. He is also author and co-author of several books on laboratory medicine and hematology. He gave numerous oral and poster presentations in congresses and other scientific events. He acts as a reviewer for various journals, including Clinical Chemistry and Laboratory Medicine, where he currently serves his last term as an Editorial Board member. He is a member of several international committees and working groups on standardization in laboratory hematology.