Reticulated Platelets: Analytical Aspects and Clinical Utility

DOI 10.1515/cclm-2014-0165 Clin Chem Lab Med 2014; 52(8): 1107–1117 Review Johannes J.M.L. Hoffmann* Reticulated platelets: analytical aspects and clinical utility Abstract: Reticulated platelets are immature platelets Formation and maturation circulating in blood; they reflect the activity of megakaryopoiesis in the bone marrow. Therefore, they can be used of megakaryocytes 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 megakaryocyte been proven to have clinical utility. Reticulated or imma- (Figure 1). Megakaryopoiesis is regulated by various ture platelet 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 cytoplasm, 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 reticulocytes in erythropoiesis [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 thrombocytopenia [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 reticulocyte 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

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