Clinica Chimica Acta 439 (2015) 143–147
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Invited critical review
Clinical applicability of reticulated platelets Luci Maria SantAna Dusse ⁎, Letícia Gonçalves Freitas Department of Clinical and Toxicological Analysis, Faculty of Pharmacy-Universidade Federal de Minas Gerais, Brazil
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Article history: Received 1 April 2014 Received in revised form 7 October 2014 Accepted 15 October 2014 Available online 23 October 2014 Keywords: Immature platelet Reticulated platelets Thrombopoiesis Thrombocytopenia
a b s t r a c t Background: Reticulated platelets (RPs), immature platelets newly released from the bone marrow into the circulation, have a high content of ribonucleic acid and are larger and more active in thrombus formation. Objective: This review compiles articles that evaluated RP in order to establish their clinical significance. Discussion: RPs increase when platelet production rises and decrease when production falls. As such, the measurement of circulating RPs allows the assessment of thrombocytopenia, i.e., bone marrow production or peripheral destruction. Conclusion: RPs are a promising laboratory tool for evaluation of idiopathic thrombocytopenia (differentiating hypoproduction from accelerated platelet destruction), chemotherapy and after stem cell transplantation (predicting platelet recovery) and thrombocytosis (estimating platelet turnover). Additional randomized and well controlled clinical studies are required to clearly establish the significance of circulating RPs in other clinical conditions. © 2014 Elsevier B.V. All rights reserved.
Contents 1. 2.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Properties of reticulated platelets . . . . . . . . . . . . . . . . . . . 2.1. Clinical applicability of RPs in thrombopoietic disorders . . . . . . 2.1.1. Diagnosis of thrombocytopenias . . . . . . . . . . . . 2.1.2. Thrombocytopenia management in pregnancy . . . . . . 2.1.3. Thrombocytopenia in preeclampsia . . . . . . . . . . . 2.1.4. Thrombocytosis and essential thrombocythemia . . . . . 2.2. Clinical applicability of reticulated platelets in other conditions . . 2.2.1. Coronary artery disease . . . . . . . . . . . . . . . . 2.2.2. Myocardial revascularization with cardiopulmonary bypass 2.2.3. Recovery hematopoietic after marrow transplantation . . 2.2.4. Vaso-occlusive crisis in sickle cell anemia . . . . . . . . 2.2.5. Kidney dysfunction . . . . . . . . . . . . . . . . . . 2.2.6. Predicting sepsis in critically ill patients . . . . . . . . . 2.2.7. Veterinary medicine . . . . . . . . . . . . . . . . . . 2.2.8. Monitoring exposure to ionizing radiation . . . . . . . . 3. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction ⁎ Correspondent author at: Departamento de Análises Clínicas e Toxicológicas, Faculdade de Farmácia, Universidade Federal de Federal Minas Gerais, Av. Antônio Carlos, 6627, Pampulha CEP: 31270-901, Belo Horizonte, Minas Gerais, Brazil. Tel.: +55 31 3409 6880/6900; fax: +55 31 3409 6985. E-mail address: [email protected] (L.M.S. Dusse).
http://dx.doi.org/10.1016/j.cca.2014.10.024 0009-8981/© 2014 Elsevier B.V. All rights reserved.
Reticulated platelets (RPs) or immature platelets are similar to reticulocytes newly released by the bone marrow into the circulation [1,2]. RPs were identified in 1969 by Ingram and Coopersmith who observed increased circulating immature platelets in dogs after acute
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blood loss [3,4]. These platelets exhibited residual RNA when stained with methylene blue. During maturation, cellular RNA is unstable and degrades within 24 h [5,6]. Our understanding of the mechanisms associated with platelet production, ie, thrombopoiesis, has improved recently. Identification of the growth factor thrombopoietin (a critical hematopoietic cytokine) and its receptor c-Mpl (mainly expressed on megakaryocytes) represented an important advance in the field [7]. Further, two erythroid transcription factors, nuclear factor erythroid-2 (NF-E2) and GATA1, were found to be essential for megakaryocytes to complete maturation and platelet release [8,9]. These findings and the availability of recombinant thrombopoietin have encouraged other studies aiming to clarify fundamental aspects of thrombopoiesis [10]. Thrombopoietin is produced by the liver and it is the primary regulator of megakaryocyte progenitor expansion and differentiation. Thrombopoietin, through cMpl, activates several signaling pathways, promoting cellular survival and proliferation. Due to its central role in hematopoiesis, alterations of thrombopoietin or its receptor contribute to the occurrence of diseases; as congenital and acquired thrombocytosis, thrombocytopenia and aplastic anemia [11]. Circulating platelets depends on thrombopoiesis stimulation and platelet removal from the bloodstream. It is known that activationdependent mechanisms enhance platelet turnover and contribute to the presence of RPs in circulation. Therefore, assessment of RP could differentiate peripheral destruction vs suppression of bone marrow thrombopoiesis [12]. Although described in 1969, RPs were not clinically assessed for many years. Lack of appropriate methods and inability to compare results complicated these studies. Many factors contributed to this problem analytically, ie, types and concentration of fluorescent dyes, incubation time and temperature, fixation, RNAse treatment and the flow cytometric data analysis, including gating and threshold settings [13]. A major technical problem was that platelets show non-RNA specific binding to fluorescent dye resulting in background staining that is size-dependent [14]. 2. Properties of reticulated platelets The number of circulating RPs reflect the rate of thrombopoiesis, increasing with increased synthesis and decreasing with decreased production. RP exhibits a greater mass and higher prothrombotic potential compared to smaller platelets. Their count might reflect increased platelet consumption during the evolution of thrombosis or a prelude to the thrombus development [15]. Recently, McBane et al. [16], reported that newly formed platelets synthesized various granule and membrane glycoproteins, including glycoprotein (GP) Ib-IX-V and GP IIb/IIIa (integrin αIIbβ3) following stimulation. These observations suggest that young platelets are preferentially recruited for thrombus participation vs older ones. These researchers have postulated that younger RPs have an increased propensity for thrombus participation under high shear conditions compared to mature platelets. An increased receptor density of integrin β3 in younger platelets may probably justify this finding. RP aggregates faster with collagen and have increased P-selectin and thromboxane A2 [17]. P-selectin has an essential role in the recruitment of leukocytes to the inflammatory focus [18]. It has been suggested that RPs are present in inflammation and interact with leukocytes and endothelial cells [19]. Moreover, RPs have reduced response to antiplatelet drugs, ie, aspirin, possibly due to their greater reactivity and impaired cyclooxygenase-1 and -2 inhibition [20]. Although analysis of peripheral RPs have emerged as a powerful tool to estimate bone marrow production, this approach has some inherent limitations. Poor analytic standardization as well as the lack of appropriate internal and external quality control makes it difficult to compare inter-laboratory results. Currently, two Hematology analyzers, Sysmex (XE- and XN-series) and Abbott (CELL-DYN Sapphire),
are commercially available to measure RPs. Despite different methodologies, both have showed clinical utility [15]. Two prospective observational studies assessed RPs count for diagnosis of thrombocytopenia and concluded that this method showed high sensitivity (93%) and specificity (85%) [21,22]. In-house methods have established the normal range of RPs in healthy subjects to be 8.6 ± 2.8% [23] and 1.1–6.1% [4]. In a recent review, Hoffmann [15], reported that the normal reference range of 1– 15%. Unfortunately, poor standardization makes it difficult to establish a well-defined normal range [23]. Recently, several studies have assessed RPs normal ranges using Hematology analyzers. Comparable results were obtained. For example, the Abbott (CELL-DYN Sapphire) range was 0.4–2.8% [24], or 0.4–6.0% [25,26] whereas the Sysmex (XE- and XN-series) range was 0.8–6.3% [27] or 0.70–5.50% for males and 0.90–5.30% for females [28]. Mangalpally et al. [29], investigated platelet activation patterns relative to size and compared the inhibitory effects of aspirin. These researchers found that a higher proportion of large platelets (density, N1.055; mean volume, 12 μ3) bound fibrinogen and von Willebrand factor following stimulation. These large platelets also expressed Pselectin and integrin αIIbβ3 in absence or presence of aspirin. Similarly, Guthikonda et al. [30,31], demonstrated that in healthy subjects and patients with coronary artery disease, RPs were associated with impaired aspirin effectiveness ex vivo. These researchers concluded that this finding could be due to RPs increased activity vs senescent platelets. Hoffmann [15], using a Sysmex Hematology analyzer, demonstrated a strong positive correlation between RP count and fraction of large platelets, except in aplastic anemia. Although the large platelet fraction appeared highly correlated to mean platelet volume (MPV), data was not provided. On the other hand, Meintker et al. [32], using Abbott CELL-DYN found that RPs and MPV were poorly correlated in relatively small study groups. They concluded, however, that these parameters reflect different aspects of thrombopoiesis and are not interchangeable. In fact, in a large group of subjects with normal platelet counts, they found a significant negative correlation between RPs and MPV effectively demonstrating that RPs are not necessarily large. 2.1. Clinical applicability of RPs in thrombopoietic disorders 2.1.1. Diagnosis of thrombocytopenias Because thrombocytopenia can be caused by decreased bone marrow production, excessive peripheral destruction or abnormal storage, it is not always simple to define etiology. Methods currently used to evaluate thrombopoiesis are complex, expensive and invasive. These include bone marrow aspiration, quantification of IgG-bound platelets and platelet survival studies [23]. RPs count seems to be a promising alternative to assess bone marrow production [21]. The percentage of RPs in patients with idiopathic thrombocytopenic purpura (ITP) was significantly higher (8.41 ± 5.35%) than healthy subjects (1.92 ± 1.27%) [23]. Thomas-Kaskel et al. [33], evaluated RPs in ITP with respect to treatment and its usefulness in identifying complex causes of thrombocytopenia in allogeneic stem cell transplantation. They concluded that the RPs were a viable tool to improve the diagnosis and prognosis. Thrombocytopenia frequently occurs in liver disease and even more frequently in hepatitis. Possible mechanisms of thrombocytopenia in hepatitis are immune-mediated platelet destruction (due to antiplatelet antibodies or immune complexes), hypersplenism and decreased production by the bone marrow (due to reduced hepatic thrombopoietin synthesis or viral effect on megakaryocytes). Interferon therapy for hepatitis may also induce thrombocytopenia and finding which can justify suspension of antiviral therapy. Mechanisms of thrombocytopenia vary and some have multiple causes [34]. A new thrombopoietin receptor agonist has recently been reported to increase platelet counts in hepatitis C virus related cirrhosis, thereby allowing a subset of
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untreatable patients to receive antiviral therapy. Peripheral destruction and sequestration are major mechanisms of thrombocytopenia in chronic hepatitis. Hypersplenism is an obvious possible cause for peripheral sequestration in these patients. Splenomegaly with cirrhosis and portal hypertension was significantly more common in thrombocytopenic subjects than in those with normal platelet counts [35]. Using an automated method, Koike et al. [36], evaluated RP count and reported that, despite some limitations, RPs were a promising and reliable clinical laboratory parameter. 2.1.2. Thrombocytopenia management in pregnancy Suitable thrombocytopenia management in pregnancy is essential for the well-being of both mother and fetus. The healthy-appearing mother with mild thrombocytopenia may have either gestational benign thrombocytopenia, which does not produce fetal thrombocytopenia, or immune-mediated thrombocytopenia, which can produce fetal thrombocytopenia. The etiology of pregnancy-associated thrombocytopenia must be differentiated to facilitate clinical decision making [37]. Uhrynowska et al. [38], evaluated RP count in the second and third trimester and at delivery to discriminate pregnancy-related thrombocytopenia, immunologic thrombocytopenia and hereditary thrombocytopenia. They concluded that this method was useful for preliminary differentiation of pregnancy-related and immunologic thrombocytopenia in the second trimester. Higher RP count was informative as to the probability of immunologic thrombocytopenia in pregnancy and in newborns at delivery [38]. 2.1.3. Thrombocytopenia in preeclampsia Preeclampsia (PE) is a complex disorder characterized by hypertension and proteinuria occurring after the 20th week of pregnancy with no previous symptoms. PE is associated with increased platelet activation, which may be involved in its pathogenesis not only by promoting coagulation, but also as an important inflammatory mediator [39,40]. PE, frequently associated with thrombocytopenia, is probably due to consumption because activated platelets would be trapped in the platelet plug [41]. In response, young platelets would be released into the peripheral circulation. An early study suggested that RPs were useful for monitoring PE and may reflect increased platelet consumption during the evolution of placenta thrombosis or contribute to PE [42]. Everett et al. [43], recently demonstrated direct evidence that increased circulating RPs in PE was associated with increased consumption. 2.1.4. Thrombocytosis and essential thrombocythemia Thrombocytosis is defined as a platelet count greater than 400 × 109/L in peripheral blood. It can be secondary, ie, due to inflammation, infection, neoplastic or stressful processes (reactive thrombocytosis) or primary, ie, myeloproliferative disorder, caused by a clonal cellular defect (essential thrombocythemia). Although it is crucial to distinguish etiology to initiate clinical intervention, this is generally very difficult to achieve. For example, Moles-Moreau et al. [44], evaluated RP count, CD36 expression and platelet microparticles in differentiation of reactive thrombocytosis and essential thrombocythemia. They concluded that these markers, even when used in combination, were unable to discriminate these conditions. 2.2. Clinical applicability of reticulated platelets in other conditions 2.2.1. Coronary artery disease Guthikonda et al. [31], evaluated the relationship among RPs, platelet volume and function in coronary artery disease treated with antiplatelet agents, aspirin and clopidogrel. They concluded that RP was strongly correlated to antiplatelet therapy response. Larger platelets showed increased reactivity even in patients under antiplatelet therapy. Similarly, high RP number was associated with increased platelet reactivity despite prasugrel treatment [45]. A rise of RPs in patients
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with thrombotic disease without thrombocytosis and acute coronary syndromes, especially in the acute phase of myocardial infarction, has been reported [46]. 2.2.2. Myocardial revascularization with cardiopulmonary bypass McCabe et al. [47], assessed the biochemical, hormonal and cellular changes resulting from myocardial revascularization in cardiopulmonary bypass (CPB) surgery. The evaluation was performed pre-operatively, immediately at the end of CPB, 24 and 48 h postoperatively. Decreased platelet count was observed immediately after the end of CPB. Platelet trans-endothelial migration probably resulted from endothelial dysfunction during CPB, mainly from the interaction between endothelial cells and neutrophils activated by inflammatory molecules. This can result in adhesion molecule activation, which promotes migration and displacement of platelets to various tissues, favoring thromboembolic events. Inflammatory effects associated with CPB should be evaluated to achieve the best post-operative outcome. Thus, RP count could be an important and promising noninvasive laboratory parameter to assess platelet turnover in these patients [48, 49]. Recently, Marguerite et al. [50], evaluated platelets and bleeding risk in patients undergoing coronary artery bypass graft surgery or mitral valve repair using CPB. They concluded that RP count was a powerful tool to assess bone marrow recovery post-operatively. 2.2.3. Recovery hematopoietic after marrow transplantation Currently, the evaluation criteria for hematopoietic recovery after allogeneic marrow transplantation include a neutrophil count greater than 0.5 × 109/L (three consecutive days) and a platelet count above 20 × 109/L (seven consecutive days) [51]. Michur et al. [52], observed that increased RP paralleled these parameters suggesting its usefulness post-transplantation. Studies aiming to evaluate the RP kinetics in patients who received allogeneic progenitor cells have shown an association between increased circulating RPs and transplantation success [53]. As such, RP count may be useful to assess early marrow function following transplantation. Early estimation of bone marrow platelet regeneration is important to avoid prophylactic platelet transfusion thus minimizing the possibility of infection and other associated risks [1,54]. The immature reticulocyte fraction (IRF) and the immature platelet fraction (IPF) predict early hematopoietic recovery. Neutrophil count and platelet recovery were significantly correlated, suggesting that serial IRF and IPF measurement be used to trace hematopoietic restoration after bone marrow transplantation [46]. Mitani et al. [54], investigated whether RP count before apheresis could predict the number of peripheral blood stem cells that could potentially be collected from patients with hematologic malignancies or as an alternative to CD34+ cell counts. Unfortunately, they concluded that RP count was not reliable in predicting CD34+ cell harvest-time. 2.2.4. Vaso-occlusive crisis in sickle cell anemia Noronha et al. [55], demonstrated that patients with sickle cell vasoocclusive crisis had higher circulating RP vs patients in stable phase. 2.2.5. Kidney dysfunction Himmelfarb et al. [56], revealed that patients undergoing peritoneal or hemodialysis have markedly increased circulating RPs, indicating accelerated platelet turnover. Increased platelet activation and turnover may, however, contribute to platelet dysfunction in these patients. Recently, Würtz et al. [57], evaluated the antiplatelet effect of aspirin in patients with moderately reduced renal function and concluded that the reduced effect of aspirin could be explained by the increased RPs. Several mechanisms can be responsible for cardiovascular complications, i.e., hyperlipidemia and hypertension, in renal transplantation. Increased circulating RPs has been proposed as an important mechanism
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associated with cardiovascular risk in these patients [20]. A significant association between RP count and platelet function supports the role for platelets in cardiovascular complications in kidney transplantation [58]. 2.2.6. Predicting sepsis in critically ill patients It has recently been demonstrated that critically ill ICU patients have increased circulating RPs prior to the onset of sepsis [59]. 2.2.7. Veterinary medicine RP count has been widely studied in veterinary medicine. It was shown useful in assessing toxic myeloproliferation or drug induced myelosuppression in rats [60]. The percentage of RPs has also been used for differential diagnosis of thrombocytopenia in dogs thus avoiding bone marrow aspiration [61,62]. RPs count can help optimize platelet transfusion in murine models, particularly in immunemediated platelet destruction [63]. 2.2.8. Monitoring exposure to ionizing radiation The biologic effect of high level radiation exposure is fairly well known, but the effect of low level exposure is difficult to define given the lack of clinical findings. RPs count was measured in subjects exposed to a low level radiation to assess bone marrow thrombopoiesis [64]. Results demonstrated that RPs may be used as an early indicator of early bone marrow exposure. 3. Conclusions Current evidence indicates that RPs evaluation is a promising laboratory parameter to estimate bone marrow production and assess etiology of thrombocytopenia. Despite some limitations in standardization [65], RPs count has emerged as a rapid, non-invasive approach to investigate the megakaryopoietic activity. RPs can be of diagnostic or prognostic value in idiopathic thrombocytopenia (differentiating hypoproduction from accelerated destruction), chemotherapy and after stem cell transplantation (predicting platelet recovery) and thrombocytosis (estimating platelet turnover). Certainly, the routine delivery of this parameter for hematology analyzers will bring greater knowledge about their utility in other clinical conditions. Despite these promising findings, it is clear that additional, randomized and well-controlled clinical studies involving larger groups are required to effectively establish the clinical significance of circulating RPs. Acknowledgment The authors thank FAPEMIG and CNPq/Brazil. LMSD is grateful for CNPq Research Fellowship (PQ). References [1] Briggs C, Hart D, Kunka S, Oguni S, Machin SJ. Immature platelet fraction measurement: a future guide to platelet transfusion requirement after haematopoietic stem cell transplantation. Transfus Med 2006;16:101–9. [2] Briggs C, Harrison P, Machin SJ. Continuing developments with the automated platelet count. Int J Lab Hematol 2007;29:77–91. [3] Ingram M, Coopersmith A. Reticulated platelets following acute blood loss. Br J Haematol 1969;17:225–9. [4] Briggs C, Kunka S, Hart D, Oguni S, Machin SJ. Assessment of an immature platelet fraction (IPF) in peripheral thrombocytopenia. Br J Haematol 2004;126:93–9. [5] Ault KA, Knowles C. In vivo biotinylation demonstrates that reticulated platelets are the youngest platelets in circulation. Exp Hematol 1995;23:996–1001. [6] Harrison P, Robinson MS, Mackie IJ, Machin SJ. Reticulated platelets. Platelets 1997; 8:379–83. [7] Kaushansky K. Thrombopoietin: the primary regulator of platelet production. Blood 1995;86:419–31. [8] Shivdasani RA, Rosenblatt MF, Zucker-Franklin D, et al. Transcription factor NF-E2 is required for platelet formation independent of the actions of thrombopoietin/MGDF in megakaryocyte development. Cell 1995;81:695–704.
[9] Shivdasani RA, Fujiwara Y, McDevitt MA, Orkin SH. A lineage-selective knockout establishes the critical role of transcription factor GATA-1 in megakaryocyte growth and platelet development. EMBO J 1997;16:3965–73. [10] Schulze H, Shivdasani RA. Mechanisms of thrombopoiesis. J Thromb Haemost 2005; 3:1717–24. [11] Hitchcock IS, Kaushansky K. Thrombopoietin from beginning to end. Br J Haematol 2014;165:259–68. [12] Martinelli G, Merlo P, Fantasia R, Gioia F, Crovetti G. Reticulated platelet monitoring after autologous peripheral haematopoietic progenitor cell transplantation. Transfus Apher Sci 2009;40:175–81. [13] Matic GB, Chapman ES, Zaiss M, Rothe G, Schmitz G. Whole blood analysis of reticulated platelets: improvements of detection and assay stability. Cytometry 1998;34:229–34. [14] Robinson MS, Mackie IJ, Khair K, et al. Flow cytometric analysis of reticulated platelets: evidence for a large proportion of non-specific labelling of dense granules by fluorescent dyes. Br J Haematol 1998;100:351–7. [15] Hoffmann J. Reticulated platelets: analytical aspects and clinical utility. Clin Chem Lab Med 2014;52:1107–17. [16] McBane RD, Gonzalez C, Hodge DO, Wysokinski WE. Propensity for young reticulated platelet recruitment into arterial thrombi. J Thromb Thrombolysis 2014;37:148–54. [17] Tschoepe D, Roesen P, Kaufmann L, et al. Evidence for abnormal platelet glycoprotein expression in diabetes mellitus. Eur J Clin Invest 1990;20:166–70. [18] Lowenberg EC, Meijers JC, Levi M. Platelet-vessel wall interaction in health and disease. Neth J Med 2010;68:242–51. [19] Hartwig JH, Italiano-Jr JE. Life of the blood platelet. In: HR I, LS E, ST P, editors. Blood: principles, practice of hematology. Philadelphia: J. B. Lippincott; 1995. p. 959–1080. [20] Zanazzi M, Cesari F, Rosso G, et al. Reticulated platelets and platelet reactivity in renal transplant recipients receiving antiplatelet therapy. Transplant Proc 2010;42: 1156–7. [21] Abe Y, Wada H, Tomatsu H, et al. A simple technique to determine thrombopoiesis level using immature platelet fraction (IPF). Thromb Res 2006;118:463–9. [22] Pons I, Monteagudo M, Lucchetti G, et al. Correlation between immature platelet fraction and reticulated platelets. Usefulness in the etiology diagnosis of thrombocytopenia. Eur J Haematol 2010;85:158–63. [23] Ogata H. Measurement of reticulated platelets in thrombocytopenia—methodology and clinical utility. Kurume Med J 1998;45:165–70. [24] Costa O, Van Moer G, Jochmans K, Jonckheer J, Damiaens S, De Waele M. Reference values for new red blood cell and platelet parameters on the Abbott Diagnostics CellDyn Sapphire. Clin Chem Lab Med 2012;50:967–9. [25] dlIS Gordillo M, Lemes A, Lopez Brito J, Garcia Bello M, Molero T. Reticulated platelets (RP) counts by Cell-Dyn Sapphire (Abbott) method. Preliminary data. Int J Lab Hematol 2011;33(Suppl. 1):118. [26] Lang SDR, Cohen J. Reticulated platelet normal ranges on CELL-DYN Sapphire. J Lab Hematol 2013;35(Suppl. 1):112. [27] Pekelharing JM HO, De Jonge R, Lokhoff J, Sodikromo J, Spaans M, Brouwer R, De Lathouder S, Hinzmann R. Haematology reference intervals for established and novel parameters in healthy adults. Sysmex diagnostic perspectives, 1; 2010 1–11. [28] Hoffmann JJ, van den Broek NM, Curvers J. Reference intervals of reticulated platelets and other platelet parameters and their associations. Arch Pathol Lab Med 2013; 137:1635–40. [29] Mangalpally KK, Siqueiros-Garcia A, Vaduganathan M, Dong JF, Kleiman NS, Guthikonda S. Platelet activation patterns in platelet size sub-populations: differential responses to aspirin in vitro. J Thromb Thrombolysis 2010;30:251–62. [30] Guthikonda S, Lev EI, Patel R, et al. Reticulated platelets and uninhibited COX-1 and COX-2 decrease the antiplatelet effects of aspirin. J Thromb Haemost 2007; 5:490–6. [31] Guthikonda S, Alviar CL, Vaduganathan M, et al. Role of reticulated platelets and platelet size heterogeneity on platelet activity after dual antiplatelet therapy with aspirin and clopidogrel in patients with stable coronary artery disease. J Am Coll Cardiol 2008;52:743–9. [32] Meintker L, Haimerl M, Ringwald J, Krause SW. Measurement of immature platelets with Abbott CD-Sapphire and Sysmex XE-5000 in haematology and oncology patients. Clin Chem Lab Med 2013;51:2125–31. [33] Thomas-Kaskel AK, Mattern D, Kohler G, Finke J, Behringer D. Reticulated platelet counts correlate with treatment response in patients with idiopathic thrombocytopenic purpura and help identify the complex causes of thrombocytopenia in patients after allogeneic hematopoietic stem cell transplantation. Cytometry B Clin Cytom 2007;72:241–8. [34] Dou J, Lou Y, Wu J, Lu Y, Jin Y. Thrombocytopenia in patients with hepatitis B virusrelated chronic hepatitis: evaluation of the immature platelet fraction. Platelets 2014;25(6):399–404. [35] Zucker ML, Hagedorn CH, Murphy CA, Stanley S, Reid KJ, Skikne BS. Mechanism of thrombocytopenia in chronic hepatitis C as evaluated by the immature platelet fraction. Int J Lab Hematol 2012;34:525–32. [36] Koike Y, Miyazaki K, Higashihara M, et al. Clinical significance of detection of immature platelets: comparison between percentage of reticulated platelets as detected by flow cytometry and immature platelet fraction as detected by automated measurement. Eur J Haematol England 2010:183–4. [37] Schwartz KA. Gestational thrombocytopenia and immune thrombocytopenias in pregnancy. Hematol Oncol Clin North Am 2000;14:1101–16. [38] Uhrynowska M, Maslanka K, Kopec I, et al. Reticulated platelet count used for differentiation of the type of thrombocytopenia in pregnant women. Ginekol Pol 2012;83:265–9. [39] Szarka A, Rigo Jr J, Lazar L, Beko G, Molvarec A. Circulating cytokines, chemokines and adhesion molecules in normal pregnancy and preeclampsia determined by multiplex suspension array. BMC Immunol 2010;11:59.
L.M.S. Dusse, L.G. Freitas / Clinica Chimica Acta 439 (2015) 143–147 [40] Pennington KA, Schlitt JM, Jackson DL, Schulz LC, Schust DJ. Preeclampsia: multiple approaches for a multifactorial disease. Dis Model Mech 2012;5:9–18. [41] Holthe MR, Staff AC, Berge LN, Lyberg T. Different levels of platelet activation in preeclamptic, normotensive pregnant, and nonpregnant women. Am J Obstet Gynecol 2004;190:1128–34. [42] Rinder HM, Bonan JL, Anandan S, Rinder CS, Rodrigues PA, Smith BR. Noninvasive measurement of platelet kinetics in normal and hypertensive pregnancies. Am J Obstet Gynecol 1994;170:117–22. [43] Everett TR, Garner SF, Lees C, Goodall AH. Immature platelet fraction analysis demonstrates a difference in thrombopoiesis between normotensive and preeclamptic pregnancies. Thromb Haemost 2014;111. [44] Moles-Moreau MP, Ternisien C, Tanguy-Schmidt A, et al. Flow cytometry-evaluated platelet CD36 expression, reticulated platelets and platelet microparticles in essential thrombocythaemia and secondary thrombocytosis. Thromb Res 2010: e394–6. [45] Perl L, Lerman-Shivek H, Rechavia E, et al. Response to prasugrel and levels of circulating reticulated platelets in patients with ST-elevation myocardial infarction. J Am Coll Cardiol 2014;63(6):513–7. [46] Grove EL, Hvas AM, Kristensen SD. Immature platelets in patients with acute coronary syndromes. Thromb Haemost 2009;101:151–6. [47] Gabriel EA, Locali RF, Matsuoka PK, Cherbo T, Buffolo E. On-pump coronary artery bypass graft surgery: biochemical, hormonal and cellular features. Rev Bras Cir Cardiovasc 2011;26:525–31. [48] Chen YF, Tsai WC, Lin CC, et al. Effect of leukocyte depletion on endothelial cell activation and transendothelial migration of leukocytes during cardiopulmonary bypass. Ann Thorac Surg 2004;78:634–42 [discussion 642–633]. [49] Gu YJ, de Vries AJ, Boonstra PW, van Oeveren W. Leukocyte depletion results in improved lung function and reduced inflammatory response after cardiac surgery. J Thorac Cardiovasc Surg 1996;112:494–500. [50] Marguerite S, Levy F, Quessard A, Dupeyron JP, Gros C, Steib A. Impact of a phosphorylcholine-coated cardiac bypass circuit on blood loss and platelet function: a prospective, randomized study. J Extra Corpor Technol 2012;44:5–9. [51] Goncalo AP, Barbosa IL, Campilho F, Campos A, Mendes C. Predictive value of immature reticulocyte and platelet fractions in hematopoietic recovery of allograft patients. Transplant Proc 2011;43:241–3. [52] Michur H, Maslanka K, Szczepinski A, Marianska B. Reticulated platelets as a marker of platelet recovery after allogeneic stem cell transplantation. Int J Lab Hematol 2008;30:519–25.
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[53] Takami A, Shibayama M, Orito M, et al. Immature platelet fraction for prediction of platelet engraftment after allogeneic stem cell transplantation. Bone Marrow Transplant 2007;39:501–7. [54] Mitani N, Yujiri T, Tanaka Y, et al. Hematopoietic progenitor cell count, but not immature platelet fraction value, predicts successful harvest of autologous peripheral blood stem cells. J Clin Apher 2011;26:105–10. [55] Noronha JF, Costa FF, Saad ST, Lorand-Metze IG, Grotto HZ. Evaluation of reticulated platelets in patients with sickle cell diseases. Thromb Res 2007;121:259–67. [56] Himmelfarb J, Holbrook D, McMonagle E, Ault K. Increased reticulated platelets in dialysis patients. Kidney Int 1997;51:834–9. [57] Würtz M, Wulff LN, Grove EL, Kristensen SD, Hvas AM. Influence of renal function and platelet turnover on the antiplatelet effect of aspirin. Thromb Res 2012;129: 434–40. [58] Cesari F, Marcucci R, Gori AM, et al. High platelet turnover and reactivity in renal transplant recipients patients. Thromb Haemost 2010;104:804–10. [59] De Blasi RA, Cardelli P, Costante A, Sandri M, Mercieri M, Arcioni R. Immature platelet fraction in predicting sepsis in critically ill patients. Intensive Care Med 2013;39:636–43. [60] Pankraz A, Ledieu D, Pralet D, Provencher-Bolliger A. Detection of reticulated platelets in whole blood of rats using flow cytometry. Exp Toxicol Pathol 2008;60:443–8. [61] Hanahachi A, Moritomo T, Kano R, Watari T, Tsujimoto H, Hasegawa A. Thiazole orange-positive platelets in healthy and thrombocytopenic dogs. Vet Rec 2001; 149:122–3. [62] Maruyama H, Yamagami H, Watari T, et al. Reticulated platelet levels in whole blood and platelet-rich plasma of dogs with various platelet counts measured by flow cytometry. J Vet Med Sci 2009;71:195–7. [63] Katsman Y, Foo AH, Leontyev D, Branch DR. Improved mouse models for the study of treatment modalities for immune-mediated platelet destruction. Transfusion 2010; 50:1285–94. [64] Sayed D, Abd Elwanis ME, Abd Elhameed SY, Galal H. Does occupational exposure to low-dose ionizing radiation affect bone marrow thrombopoiesis? Int Arch Med 2011;4:8. [65] Freitas LG, Carvalho M, Dusse LM. Reticulated platelets: how to assess them? Clin Chim Acta 2013;422:40–1.
Contents lists available at ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Invited critical review
Clinical applicability of reticulated platelets Luci Maria SantAna Dusse ⁎, Letícia Gonçalves Freitas Department of Clinical and Toxicological Analysis, Faculty of Pharmacy-Universidade Federal de Minas Gerais, Brazil
a r t i c l e
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Article history: Received 1 April 2014 Received in revised form 7 October 2014 Accepted 15 October 2014 Available online 23 October 2014 Keywords: Immature platelet Reticulated platelets Thrombopoiesis Thrombocytopenia
a b s t r a c t Background: Reticulated platelets (RPs), immature platelets newly released from the bone marrow into the circulation, have a high content of ribonucleic acid and are larger and more active in thrombus formation. Objective: This review compiles articles that evaluated RP in order to establish their clinical significance. Discussion: RPs increase when platelet production rises and decrease when production falls. As such, the measurement of circulating RPs allows the assessment of thrombocytopenia, i.e., bone marrow production or peripheral destruction. Conclusion: RPs are a promising laboratory tool for evaluation of idiopathic thrombocytopenia (differentiating hypoproduction from accelerated platelet destruction), chemotherapy and after stem cell transplantation (predicting platelet recovery) and thrombocytosis (estimating platelet turnover). Additional randomized and well controlled clinical studies are required to clearly establish the significance of circulating RPs in other clinical conditions. © 2014 Elsevier B.V. All rights reserved.
Contents 1. 2.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Properties of reticulated platelets . . . . . . . . . . . . . . . . . . . 2.1. Clinical applicability of RPs in thrombopoietic disorders . . . . . . 2.1.1. Diagnosis of thrombocytopenias . . . . . . . . . . . . 2.1.2. Thrombocytopenia management in pregnancy . . . . . . 2.1.3. Thrombocytopenia in preeclampsia . . . . . . . . . . . 2.1.4. Thrombocytosis and essential thrombocythemia . . . . . 2.2. Clinical applicability of reticulated platelets in other conditions . . 2.2.1. Coronary artery disease . . . . . . . . . . . . . . . . 2.2.2. Myocardial revascularization with cardiopulmonary bypass 2.2.3. Recovery hematopoietic after marrow transplantation . . 2.2.4. Vaso-occlusive crisis in sickle cell anemia . . . . . . . . 2.2.5. Kidney dysfunction . . . . . . . . . . . . . . . . . . 2.2.6. Predicting sepsis in critically ill patients . . . . . . . . . 2.2.7. Veterinary medicine . . . . . . . . . . . . . . . . . . 2.2.8. Monitoring exposure to ionizing radiation . . . . . . . . 3. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction ⁎ Correspondent author at: Departamento de Análises Clínicas e Toxicológicas, Faculdade de Farmácia, Universidade Federal de Federal Minas Gerais, Av. Antônio Carlos, 6627, Pampulha CEP: 31270-901, Belo Horizonte, Minas Gerais, Brazil. Tel.: +55 31 3409 6880/6900; fax: +55 31 3409 6985. E-mail address: [email protected] (L.M.S. Dusse).
http://dx.doi.org/10.1016/j.cca.2014.10.024 0009-8981/© 2014 Elsevier B.V. All rights reserved.
Reticulated platelets (RPs) or immature platelets are similar to reticulocytes newly released by the bone marrow into the circulation [1,2]. RPs were identified in 1969 by Ingram and Coopersmith who observed increased circulating immature platelets in dogs after acute
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blood loss [3,4]. These platelets exhibited residual RNA when stained with methylene blue. During maturation, cellular RNA is unstable and degrades within 24 h [5,6]. Our understanding of the mechanisms associated with platelet production, ie, thrombopoiesis, has improved recently. Identification of the growth factor thrombopoietin (a critical hematopoietic cytokine) and its receptor c-Mpl (mainly expressed on megakaryocytes) represented an important advance in the field [7]. Further, two erythroid transcription factors, nuclear factor erythroid-2 (NF-E2) and GATA1, were found to be essential for megakaryocytes to complete maturation and platelet release [8,9]. These findings and the availability of recombinant thrombopoietin have encouraged other studies aiming to clarify fundamental aspects of thrombopoiesis [10]. Thrombopoietin is produced by the liver and it is the primary regulator of megakaryocyte progenitor expansion and differentiation. Thrombopoietin, through cMpl, activates several signaling pathways, promoting cellular survival and proliferation. Due to its central role in hematopoiesis, alterations of thrombopoietin or its receptor contribute to the occurrence of diseases; as congenital and acquired thrombocytosis, thrombocytopenia and aplastic anemia [11]. Circulating platelets depends on thrombopoiesis stimulation and platelet removal from the bloodstream. It is known that activationdependent mechanisms enhance platelet turnover and contribute to the presence of RPs in circulation. Therefore, assessment of RP could differentiate peripheral destruction vs suppression of bone marrow thrombopoiesis [12]. Although described in 1969, RPs were not clinically assessed for many years. Lack of appropriate methods and inability to compare results complicated these studies. Many factors contributed to this problem analytically, ie, types and concentration of fluorescent dyes, incubation time and temperature, fixation, RNAse treatment and the flow cytometric data analysis, including gating and threshold settings [13]. A major technical problem was that platelets show non-RNA specific binding to fluorescent dye resulting in background staining that is size-dependent [14]. 2. Properties of reticulated platelets The number of circulating RPs reflect the rate of thrombopoiesis, increasing with increased synthesis and decreasing with decreased production. RP exhibits a greater mass and higher prothrombotic potential compared to smaller platelets. Their count might reflect increased platelet consumption during the evolution of thrombosis or a prelude to the thrombus development [15]. Recently, McBane et al. [16], reported that newly formed platelets synthesized various granule and membrane glycoproteins, including glycoprotein (GP) Ib-IX-V and GP IIb/IIIa (integrin αIIbβ3) following stimulation. These observations suggest that young platelets are preferentially recruited for thrombus participation vs older ones. These researchers have postulated that younger RPs have an increased propensity for thrombus participation under high shear conditions compared to mature platelets. An increased receptor density of integrin β3 in younger platelets may probably justify this finding. RP aggregates faster with collagen and have increased P-selectin and thromboxane A2 [17]. P-selectin has an essential role in the recruitment of leukocytes to the inflammatory focus [18]. It has been suggested that RPs are present in inflammation and interact with leukocytes and endothelial cells [19]. Moreover, RPs have reduced response to antiplatelet drugs, ie, aspirin, possibly due to their greater reactivity and impaired cyclooxygenase-1 and -2 inhibition [20]. Although analysis of peripheral RPs have emerged as a powerful tool to estimate bone marrow production, this approach has some inherent limitations. Poor analytic standardization as well as the lack of appropriate internal and external quality control makes it difficult to compare inter-laboratory results. Currently, two Hematology analyzers, Sysmex (XE- and XN-series) and Abbott (CELL-DYN Sapphire),
are commercially available to measure RPs. Despite different methodologies, both have showed clinical utility [15]. Two prospective observational studies assessed RPs count for diagnosis of thrombocytopenia and concluded that this method showed high sensitivity (93%) and specificity (85%) [21,22]. In-house methods have established the normal range of RPs in healthy subjects to be 8.6 ± 2.8% [23] and 1.1–6.1% [4]. In a recent review, Hoffmann [15], reported that the normal reference range of 1– 15%. Unfortunately, poor standardization makes it difficult to establish a well-defined normal range [23]. Recently, several studies have assessed RPs normal ranges using Hematology analyzers. Comparable results were obtained. For example, the Abbott (CELL-DYN Sapphire) range was 0.4–2.8% [24], or 0.4–6.0% [25,26] whereas the Sysmex (XE- and XN-series) range was 0.8–6.3% [27] or 0.70–5.50% for males and 0.90–5.30% for females [28]. Mangalpally et al. [29], investigated platelet activation patterns relative to size and compared the inhibitory effects of aspirin. These researchers found that a higher proportion of large platelets (density, N1.055; mean volume, 12 μ3) bound fibrinogen and von Willebrand factor following stimulation. These large platelets also expressed Pselectin and integrin αIIbβ3 in absence or presence of aspirin. Similarly, Guthikonda et al. [30,31], demonstrated that in healthy subjects and patients with coronary artery disease, RPs were associated with impaired aspirin effectiveness ex vivo. These researchers concluded that this finding could be due to RPs increased activity vs senescent platelets. Hoffmann [15], using a Sysmex Hematology analyzer, demonstrated a strong positive correlation between RP count and fraction of large platelets, except in aplastic anemia. Although the large platelet fraction appeared highly correlated to mean platelet volume (MPV), data was not provided. On the other hand, Meintker et al. [32], using Abbott CELL-DYN found that RPs and MPV were poorly correlated in relatively small study groups. They concluded, however, that these parameters reflect different aspects of thrombopoiesis and are not interchangeable. In fact, in a large group of subjects with normal platelet counts, they found a significant negative correlation between RPs and MPV effectively demonstrating that RPs are not necessarily large. 2.1. Clinical applicability of RPs in thrombopoietic disorders 2.1.1. Diagnosis of thrombocytopenias Because thrombocytopenia can be caused by decreased bone marrow production, excessive peripheral destruction or abnormal storage, it is not always simple to define etiology. Methods currently used to evaluate thrombopoiesis are complex, expensive and invasive. These include bone marrow aspiration, quantification of IgG-bound platelets and platelet survival studies [23]. RPs count seems to be a promising alternative to assess bone marrow production [21]. The percentage of RPs in patients with idiopathic thrombocytopenic purpura (ITP) was significantly higher (8.41 ± 5.35%) than healthy subjects (1.92 ± 1.27%) [23]. Thomas-Kaskel et al. [33], evaluated RPs in ITP with respect to treatment and its usefulness in identifying complex causes of thrombocytopenia in allogeneic stem cell transplantation. They concluded that the RPs were a viable tool to improve the diagnosis and prognosis. Thrombocytopenia frequently occurs in liver disease and even more frequently in hepatitis. Possible mechanisms of thrombocytopenia in hepatitis are immune-mediated platelet destruction (due to antiplatelet antibodies or immune complexes), hypersplenism and decreased production by the bone marrow (due to reduced hepatic thrombopoietin synthesis or viral effect on megakaryocytes). Interferon therapy for hepatitis may also induce thrombocytopenia and finding which can justify suspension of antiviral therapy. Mechanisms of thrombocytopenia vary and some have multiple causes [34]. A new thrombopoietin receptor agonist has recently been reported to increase platelet counts in hepatitis C virus related cirrhosis, thereby allowing a subset of
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untreatable patients to receive antiviral therapy. Peripheral destruction and sequestration are major mechanisms of thrombocytopenia in chronic hepatitis. Hypersplenism is an obvious possible cause for peripheral sequestration in these patients. Splenomegaly with cirrhosis and portal hypertension was significantly more common in thrombocytopenic subjects than in those with normal platelet counts [35]. Using an automated method, Koike et al. [36], evaluated RP count and reported that, despite some limitations, RPs were a promising and reliable clinical laboratory parameter. 2.1.2. Thrombocytopenia management in pregnancy Suitable thrombocytopenia management in pregnancy is essential for the well-being of both mother and fetus. The healthy-appearing mother with mild thrombocytopenia may have either gestational benign thrombocytopenia, which does not produce fetal thrombocytopenia, or immune-mediated thrombocytopenia, which can produce fetal thrombocytopenia. The etiology of pregnancy-associated thrombocytopenia must be differentiated to facilitate clinical decision making [37]. Uhrynowska et al. [38], evaluated RP count in the second and third trimester and at delivery to discriminate pregnancy-related thrombocytopenia, immunologic thrombocytopenia and hereditary thrombocytopenia. They concluded that this method was useful for preliminary differentiation of pregnancy-related and immunologic thrombocytopenia in the second trimester. Higher RP count was informative as to the probability of immunologic thrombocytopenia in pregnancy and in newborns at delivery [38]. 2.1.3. Thrombocytopenia in preeclampsia Preeclampsia (PE) is a complex disorder characterized by hypertension and proteinuria occurring after the 20th week of pregnancy with no previous symptoms. PE is associated with increased platelet activation, which may be involved in its pathogenesis not only by promoting coagulation, but also as an important inflammatory mediator [39,40]. PE, frequently associated with thrombocytopenia, is probably due to consumption because activated platelets would be trapped in the platelet plug [41]. In response, young platelets would be released into the peripheral circulation. An early study suggested that RPs were useful for monitoring PE and may reflect increased platelet consumption during the evolution of placenta thrombosis or contribute to PE [42]. Everett et al. [43], recently demonstrated direct evidence that increased circulating RPs in PE was associated with increased consumption. 2.1.4. Thrombocytosis and essential thrombocythemia Thrombocytosis is defined as a platelet count greater than 400 × 109/L in peripheral blood. It can be secondary, ie, due to inflammation, infection, neoplastic or stressful processes (reactive thrombocytosis) or primary, ie, myeloproliferative disorder, caused by a clonal cellular defect (essential thrombocythemia). Although it is crucial to distinguish etiology to initiate clinical intervention, this is generally very difficult to achieve. For example, Moles-Moreau et al. [44], evaluated RP count, CD36 expression and platelet microparticles in differentiation of reactive thrombocytosis and essential thrombocythemia. They concluded that these markers, even when used in combination, were unable to discriminate these conditions. 2.2. Clinical applicability of reticulated platelets in other conditions 2.2.1. Coronary artery disease Guthikonda et al. [31], evaluated the relationship among RPs, platelet volume and function in coronary artery disease treated with antiplatelet agents, aspirin and clopidogrel. They concluded that RP was strongly correlated to antiplatelet therapy response. Larger platelets showed increased reactivity even in patients under antiplatelet therapy. Similarly, high RP number was associated with increased platelet reactivity despite prasugrel treatment [45]. A rise of RPs in patients
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with thrombotic disease without thrombocytosis and acute coronary syndromes, especially in the acute phase of myocardial infarction, has been reported [46]. 2.2.2. Myocardial revascularization with cardiopulmonary bypass McCabe et al. [47], assessed the biochemical, hormonal and cellular changes resulting from myocardial revascularization in cardiopulmonary bypass (CPB) surgery. The evaluation was performed pre-operatively, immediately at the end of CPB, 24 and 48 h postoperatively. Decreased platelet count was observed immediately after the end of CPB. Platelet trans-endothelial migration probably resulted from endothelial dysfunction during CPB, mainly from the interaction between endothelial cells and neutrophils activated by inflammatory molecules. This can result in adhesion molecule activation, which promotes migration and displacement of platelets to various tissues, favoring thromboembolic events. Inflammatory effects associated with CPB should be evaluated to achieve the best post-operative outcome. Thus, RP count could be an important and promising noninvasive laboratory parameter to assess platelet turnover in these patients [48, 49]. Recently, Marguerite et al. [50], evaluated platelets and bleeding risk in patients undergoing coronary artery bypass graft surgery or mitral valve repair using CPB. They concluded that RP count was a powerful tool to assess bone marrow recovery post-operatively. 2.2.3. Recovery hematopoietic after marrow transplantation Currently, the evaluation criteria for hematopoietic recovery after allogeneic marrow transplantation include a neutrophil count greater than 0.5 × 109/L (three consecutive days) and a platelet count above 20 × 109/L (seven consecutive days) [51]. Michur et al. [52], observed that increased RP paralleled these parameters suggesting its usefulness post-transplantation. Studies aiming to evaluate the RP kinetics in patients who received allogeneic progenitor cells have shown an association between increased circulating RPs and transplantation success [53]. As such, RP count may be useful to assess early marrow function following transplantation. Early estimation of bone marrow platelet regeneration is important to avoid prophylactic platelet transfusion thus minimizing the possibility of infection and other associated risks [1,54]. The immature reticulocyte fraction (IRF) and the immature platelet fraction (IPF) predict early hematopoietic recovery. Neutrophil count and platelet recovery were significantly correlated, suggesting that serial IRF and IPF measurement be used to trace hematopoietic restoration after bone marrow transplantation [46]. Mitani et al. [54], investigated whether RP count before apheresis could predict the number of peripheral blood stem cells that could potentially be collected from patients with hematologic malignancies or as an alternative to CD34+ cell counts. Unfortunately, they concluded that RP count was not reliable in predicting CD34+ cell harvest-time. 2.2.4. Vaso-occlusive crisis in sickle cell anemia Noronha et al. [55], demonstrated that patients with sickle cell vasoocclusive crisis had higher circulating RP vs patients in stable phase. 2.2.5. Kidney dysfunction Himmelfarb et al. [56], revealed that patients undergoing peritoneal or hemodialysis have markedly increased circulating RPs, indicating accelerated platelet turnover. Increased platelet activation and turnover may, however, contribute to platelet dysfunction in these patients. Recently, Würtz et al. [57], evaluated the antiplatelet effect of aspirin in patients with moderately reduced renal function and concluded that the reduced effect of aspirin could be explained by the increased RPs. Several mechanisms can be responsible for cardiovascular complications, i.e., hyperlipidemia and hypertension, in renal transplantation. Increased circulating RPs has been proposed as an important mechanism
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associated with cardiovascular risk in these patients [20]. A significant association between RP count and platelet function supports the role for platelets in cardiovascular complications in kidney transplantation [58]. 2.2.6. Predicting sepsis in critically ill patients It has recently been demonstrated that critically ill ICU patients have increased circulating RPs prior to the onset of sepsis [59]. 2.2.7. Veterinary medicine RP count has been widely studied in veterinary medicine. It was shown useful in assessing toxic myeloproliferation or drug induced myelosuppression in rats [60]. The percentage of RPs has also been used for differential diagnosis of thrombocytopenia in dogs thus avoiding bone marrow aspiration [61,62]. RPs count can help optimize platelet transfusion in murine models, particularly in immunemediated platelet destruction [63]. 2.2.8. Monitoring exposure to ionizing radiation The biologic effect of high level radiation exposure is fairly well known, but the effect of low level exposure is difficult to define given the lack of clinical findings. RPs count was measured in subjects exposed to a low level radiation to assess bone marrow thrombopoiesis [64]. Results demonstrated that RPs may be used as an early indicator of early bone marrow exposure. 3. Conclusions Current evidence indicates that RPs evaluation is a promising laboratory parameter to estimate bone marrow production and assess etiology of thrombocytopenia. Despite some limitations in standardization [65], RPs count has emerged as a rapid, non-invasive approach to investigate the megakaryopoietic activity. RPs can be of diagnostic or prognostic value in idiopathic thrombocytopenia (differentiating hypoproduction from accelerated destruction), chemotherapy and after stem cell transplantation (predicting platelet recovery) and thrombocytosis (estimating platelet turnover). Certainly, the routine delivery of this parameter for hematology analyzers will bring greater knowledge about their utility in other clinical conditions. Despite these promising findings, it is clear that additional, randomized and well-controlled clinical studies involving larger groups are required to effectively establish the clinical significance of circulating RPs. Acknowledgment The authors thank FAPEMIG and CNPq/Brazil. LMSD is grateful for CNPq Research Fellowship (PQ). References [1] Briggs C, Hart D, Kunka S, Oguni S, Machin SJ. Immature platelet fraction measurement: a future guide to platelet transfusion requirement after haematopoietic stem cell transplantation. Transfus Med 2006;16:101–9. [2] Briggs C, Harrison P, Machin SJ. Continuing developments with the automated platelet count. Int J Lab Hematol 2007;29:77–91. [3] Ingram M, Coopersmith A. Reticulated platelets following acute blood loss. Br J Haematol 1969;17:225–9. [4] Briggs C, Kunka S, Hart D, Oguni S, Machin SJ. Assessment of an immature platelet fraction (IPF) in peripheral thrombocytopenia. Br J Haematol 2004;126:93–9. [5] Ault KA, Knowles C. In vivo biotinylation demonstrates that reticulated platelets are the youngest platelets in circulation. Exp Hematol 1995;23:996–1001. [6] Harrison P, Robinson MS, Mackie IJ, Machin SJ. Reticulated platelets. Platelets 1997; 8:379–83. [7] Kaushansky K. Thrombopoietin: the primary regulator of platelet production. Blood 1995;86:419–31. [8] Shivdasani RA, Rosenblatt MF, Zucker-Franklin D, et al. Transcription factor NF-E2 is required for platelet formation independent of the actions of thrombopoietin/MGDF in megakaryocyte development. Cell 1995;81:695–704.
[9] Shivdasani RA, Fujiwara Y, McDevitt MA, Orkin SH. A lineage-selective knockout establishes the critical role of transcription factor GATA-1 in megakaryocyte growth and platelet development. EMBO J 1997;16:3965–73. [10] Schulze H, Shivdasani RA. Mechanisms of thrombopoiesis. J Thromb Haemost 2005; 3:1717–24. [11] Hitchcock IS, Kaushansky K. Thrombopoietin from beginning to end. Br J Haematol 2014;165:259–68. [12] Martinelli G, Merlo P, Fantasia R, Gioia F, Crovetti G. Reticulated platelet monitoring after autologous peripheral haematopoietic progenitor cell transplantation. Transfus Apher Sci 2009;40:175–81. [13] Matic GB, Chapman ES, Zaiss M, Rothe G, Schmitz G. Whole blood analysis of reticulated platelets: improvements of detection and assay stability. Cytometry 1998;34:229–34. [14] Robinson MS, Mackie IJ, Khair K, et al. Flow cytometric analysis of reticulated platelets: evidence for a large proportion of non-specific labelling of dense granules by fluorescent dyes. Br J Haematol 1998;100:351–7. [15] Hoffmann J. Reticulated platelets: analytical aspects and clinical utility. Clin Chem Lab Med 2014;52:1107–17. [16] McBane RD, Gonzalez C, Hodge DO, Wysokinski WE. Propensity for young reticulated platelet recruitment into arterial thrombi. J Thromb Thrombolysis 2014;37:148–54. [17] Tschoepe D, Roesen P, Kaufmann L, et al. Evidence for abnormal platelet glycoprotein expression in diabetes mellitus. Eur J Clin Invest 1990;20:166–70. [18] Lowenberg EC, Meijers JC, Levi M. Platelet-vessel wall interaction in health and disease. Neth J Med 2010;68:242–51. [19] Hartwig JH, Italiano-Jr JE. Life of the blood platelet. In: HR I, LS E, ST P, editors. Blood: principles, practice of hematology. Philadelphia: J. B. Lippincott; 1995. p. 959–1080. [20] Zanazzi M, Cesari F, Rosso G, et al. Reticulated platelets and platelet reactivity in renal transplant recipients receiving antiplatelet therapy. Transplant Proc 2010;42: 1156–7. [21] Abe Y, Wada H, Tomatsu H, et al. A simple technique to determine thrombopoiesis level using immature platelet fraction (IPF). Thromb Res 2006;118:463–9. [22] Pons I, Monteagudo M, Lucchetti G, et al. Correlation between immature platelet fraction and reticulated platelets. Usefulness in the etiology diagnosis of thrombocytopenia. Eur J Haematol 2010;85:158–63. [23] Ogata H. Measurement of reticulated platelets in thrombocytopenia—methodology and clinical utility. Kurume Med J 1998;45:165–70. [24] Costa O, Van Moer G, Jochmans K, Jonckheer J, Damiaens S, De Waele M. Reference values for new red blood cell and platelet parameters on the Abbott Diagnostics CellDyn Sapphire. Clin Chem Lab Med 2012;50:967–9. [25] dlIS Gordillo M, Lemes A, Lopez Brito J, Garcia Bello M, Molero T. Reticulated platelets (RP) counts by Cell-Dyn Sapphire (Abbott) method. Preliminary data. Int J Lab Hematol 2011;33(Suppl. 1):118. [26] Lang SDR, Cohen J. Reticulated platelet normal ranges on CELL-DYN Sapphire. J Lab Hematol 2013;35(Suppl. 1):112. [27] Pekelharing JM HO, De Jonge R, Lokhoff J, Sodikromo J, Spaans M, Brouwer R, De Lathouder S, Hinzmann R. Haematology reference intervals for established and novel parameters in healthy adults. Sysmex diagnostic perspectives, 1; 2010 1–11. [28] Hoffmann JJ, van den Broek NM, Curvers J. Reference intervals of reticulated platelets and other platelet parameters and their associations. Arch Pathol Lab Med 2013; 137:1635–40. [29] Mangalpally KK, Siqueiros-Garcia A, Vaduganathan M, Dong JF, Kleiman NS, Guthikonda S. Platelet activation patterns in platelet size sub-populations: differential responses to aspirin in vitro. J Thromb Thrombolysis 2010;30:251–62. [30] Guthikonda S, Lev EI, Patel R, et al. Reticulated platelets and uninhibited COX-1 and COX-2 decrease the antiplatelet effects of aspirin. J Thromb Haemost 2007; 5:490–6. [31] Guthikonda S, Alviar CL, Vaduganathan M, et al. Role of reticulated platelets and platelet size heterogeneity on platelet activity after dual antiplatelet therapy with aspirin and clopidogrel in patients with stable coronary artery disease. J Am Coll Cardiol 2008;52:743–9. [32] Meintker L, Haimerl M, Ringwald J, Krause SW. Measurement of immature platelets with Abbott CD-Sapphire and Sysmex XE-5000 in haematology and oncology patients. Clin Chem Lab Med 2013;51:2125–31. [33] Thomas-Kaskel AK, Mattern D, Kohler G, Finke J, Behringer D. Reticulated platelet counts correlate with treatment response in patients with idiopathic thrombocytopenic purpura and help identify the complex causes of thrombocytopenia in patients after allogeneic hematopoietic stem cell transplantation. Cytometry B Clin Cytom 2007;72:241–8. [34] Dou J, Lou Y, Wu J, Lu Y, Jin Y. Thrombocytopenia in patients with hepatitis B virusrelated chronic hepatitis: evaluation of the immature platelet fraction. Platelets 2014;25(6):399–404. [35] Zucker ML, Hagedorn CH, Murphy CA, Stanley S, Reid KJ, Skikne BS. Mechanism of thrombocytopenia in chronic hepatitis C as evaluated by the immature platelet fraction. Int J Lab Hematol 2012;34:525–32. [36] Koike Y, Miyazaki K, Higashihara M, et al. Clinical significance of detection of immature platelets: comparison between percentage of reticulated platelets as detected by flow cytometry and immature platelet fraction as detected by automated measurement. Eur J Haematol England 2010:183–4. [37] Schwartz KA. Gestational thrombocytopenia and immune thrombocytopenias in pregnancy. Hematol Oncol Clin North Am 2000;14:1101–16. [38] Uhrynowska M, Maslanka K, Kopec I, et al. Reticulated platelet count used for differentiation of the type of thrombocytopenia in pregnant women. Ginekol Pol 2012;83:265–9. [39] Szarka A, Rigo Jr J, Lazar L, Beko G, Molvarec A. Circulating cytokines, chemokines and adhesion molecules in normal pregnancy and preeclampsia determined by multiplex suspension array. BMC Immunol 2010;11:59.
L.M.S. Dusse, L.G. Freitas / Clinica Chimica Acta 439 (2015) 143–147 [40] Pennington KA, Schlitt JM, Jackson DL, Schulz LC, Schust DJ. Preeclampsia: multiple approaches for a multifactorial disease. Dis Model Mech 2012;5:9–18. [41] Holthe MR, Staff AC, Berge LN, Lyberg T. Different levels of platelet activation in preeclamptic, normotensive pregnant, and nonpregnant women. Am J Obstet Gynecol 2004;190:1128–34. [42] Rinder HM, Bonan JL, Anandan S, Rinder CS, Rodrigues PA, Smith BR. Noninvasive measurement of platelet kinetics in normal and hypertensive pregnancies. Am J Obstet Gynecol 1994;170:117–22. [43] Everett TR, Garner SF, Lees C, Goodall AH. Immature platelet fraction analysis demonstrates a difference in thrombopoiesis between normotensive and preeclamptic pregnancies. Thromb Haemost 2014;111. [44] Moles-Moreau MP, Ternisien C, Tanguy-Schmidt A, et al. Flow cytometry-evaluated platelet CD36 expression, reticulated platelets and platelet microparticles in essential thrombocythaemia and secondary thrombocytosis. Thromb Res 2010: e394–6. [45] Perl L, Lerman-Shivek H, Rechavia E, et al. Response to prasugrel and levels of circulating reticulated platelets in patients with ST-elevation myocardial infarction. J Am Coll Cardiol 2014;63(6):513–7. [46] Grove EL, Hvas AM, Kristensen SD. Immature platelets in patients with acute coronary syndromes. Thromb Haemost 2009;101:151–6. [47] Gabriel EA, Locali RF, Matsuoka PK, Cherbo T, Buffolo E. On-pump coronary artery bypass graft surgery: biochemical, hormonal and cellular features. Rev Bras Cir Cardiovasc 2011;26:525–31. [48] Chen YF, Tsai WC, Lin CC, et al. Effect of leukocyte depletion on endothelial cell activation and transendothelial migration of leukocytes during cardiopulmonary bypass. Ann Thorac Surg 2004;78:634–42 [discussion 642–633]. [49] Gu YJ, de Vries AJ, Boonstra PW, van Oeveren W. Leukocyte depletion results in improved lung function and reduced inflammatory response after cardiac surgery. J Thorac Cardiovasc Surg 1996;112:494–500. [50] Marguerite S, Levy F, Quessard A, Dupeyron JP, Gros C, Steib A. Impact of a phosphorylcholine-coated cardiac bypass circuit on blood loss and platelet function: a prospective, randomized study. J Extra Corpor Technol 2012;44:5–9. [51] Goncalo AP, Barbosa IL, Campilho F, Campos A, Mendes C. Predictive value of immature reticulocyte and platelet fractions in hematopoietic recovery of allograft patients. Transplant Proc 2011;43:241–3. [52] Michur H, Maslanka K, Szczepinski A, Marianska B. Reticulated platelets as a marker of platelet recovery after allogeneic stem cell transplantation. Int J Lab Hematol 2008;30:519–25.
147
[53] Takami A, Shibayama M, Orito M, et al. Immature platelet fraction for prediction of platelet engraftment after allogeneic stem cell transplantation. Bone Marrow Transplant 2007;39:501–7. [54] Mitani N, Yujiri T, Tanaka Y, et al. Hematopoietic progenitor cell count, but not immature platelet fraction value, predicts successful harvest of autologous peripheral blood stem cells. J Clin Apher 2011;26:105–10. [55] Noronha JF, Costa FF, Saad ST, Lorand-Metze IG, Grotto HZ. Evaluation of reticulated platelets in patients with sickle cell diseases. Thromb Res 2007;121:259–67. [56] Himmelfarb J, Holbrook D, McMonagle E, Ault K. Increased reticulated platelets in dialysis patients. Kidney Int 1997;51:834–9. [57] Würtz M, Wulff LN, Grove EL, Kristensen SD, Hvas AM. Influence of renal function and platelet turnover on the antiplatelet effect of aspirin. Thromb Res 2012;129: 434–40. [58] Cesari F, Marcucci R, Gori AM, et al. High platelet turnover and reactivity in renal transplant recipients patients. Thromb Haemost 2010;104:804–10. [59] De Blasi RA, Cardelli P, Costante A, Sandri M, Mercieri M, Arcioni R. Immature platelet fraction in predicting sepsis in critically ill patients. Intensive Care Med 2013;39:636–43. [60] Pankraz A, Ledieu D, Pralet D, Provencher-Bolliger A. Detection of reticulated platelets in whole blood of rats using flow cytometry. Exp Toxicol Pathol 2008;60:443–8. [61] Hanahachi A, Moritomo T, Kano R, Watari T, Tsujimoto H, Hasegawa A. Thiazole orange-positive platelets in healthy and thrombocytopenic dogs. Vet Rec 2001; 149:122–3. [62] Maruyama H, Yamagami H, Watari T, et al. Reticulated platelet levels in whole blood and platelet-rich plasma of dogs with various platelet counts measured by flow cytometry. J Vet Med Sci 2009;71:195–7. [63] Katsman Y, Foo AH, Leontyev D, Branch DR. Improved mouse models for the study of treatment modalities for immune-mediated platelet destruction. Transfusion 2010; 50:1285–94. [64] Sayed D, Abd Elwanis ME, Abd Elhameed SY, Galal H. Does occupational exposure to low-dose ionizing radiation affect bone marrow thrombopoiesis? Int Arch Med 2011;4:8. [65] Freitas LG, Carvalho M, Dusse LM. Reticulated platelets: how to assess them? Clin Chim Acta 2013;422:40–1.
