Evaluation of the immature platelet fraction contribute to the differential diagnosis of hereditary, immune and other acquired thrombocytopenias

www.nature.com/scientificreports OPEN Received: 8 March 2017 Accepted: 3 May 2017 Published: xx xx xxxx Evaluation of the immature platelet fraction contribute to the differential diagnosis of hereditary, immune and other acquired thrombocytopenias F. L. B. Ferreira, M. P. Colella, S. S. Medina, C. Costa-Lima, M. M. L. Fiusa, L. N. G. Costa, F. A. Orsi, J. M. Annichino-Bizzacchi, K. Y. Fertrin, M. F. P. Gilberti, M. C. Ozelo & E. V. De Paula The differential diagnosis of immune (ITP) and hereditary macrothrombocytopenia (HM) is key to patient management. The immature platelet fraction (IPF) represents the subset of circulating platelets with higher RNA content, and has been shown to distinguish hypo- from hyperproliferative thrombocytopenias. Here we evaluated the diagnostic accuracy of IPF in the differential diagnosis between HM and other thrombocytopenias in a population of patients with post-chemotherapy thrombocytopenia (n = 56), bone marrow failure (n = 22), ITP (n = 105) and HM (n = 27). TPO levels were also measured in HM and ITP matched for platelet counts. Platelet counts were similar in all patient groups. Higher IPF values were observed in both ITP (12.3%; 2.4–65.6%) and HM (29.8%; 4.6–65.9%) compared to hypoproliferative thrombocytopenias. IPF values were also higher in HM compared to ITP, yielding a diagnostic accuracy of 0.80 (95%CI 0.70–0.90; P < 0.0001) to distinguish these two conditions. Intra- and inter-assays reproducibility of IPF in HM patients revealed that this is a stable parameter. In conclusion, IPF is increased in HM compared to both ITP and other thrombocytopenias and contributes to the differentiation between ITP and HM. Further studies are warranted to understand the biological rationale of these findings and to its incorporation in diagnostic algorithms of HM. The differential diagnosis of thrombocytopenias includes a variety of conditions such as hematologic malignancies, bone marrow failure (BMF), hypersplenism, immune thrombocytopenia (ITP), microangiopathic hemolytic anemias and hereditary macrothrombocytopenia (HM). Among these, the differential diagnosis between ITP and HM can be challenging due to the absence of specific tests, particularly in patients with mild bleeding symptoms1–3. Recently, the feasibility of using parameters of the complete blood count (CBC) to support this differential diagnosis was illustrated by a series of studies which demonstrated and validated that the mean platelet volume (MPV) can help the segregation of patients with ITP and HM4, 5. In recent years, new parameters were incorporated to the CBC, including the immature platelet fraction (IPF), which represents a population of newly formed platelets containing a greater amount of residual RNA6. Initially, the IPF was measured by flow cytometry, and described as reticulated platelets7. Recently, studies have reported the clinical utility of measuring immature platelets in clinical settings using automated hematology analyzers8–10. The correlation between flow cytometry and hematology analyzers in quantifying this population has been previously demonstrated11. Although the utility of the IPF in the differential diagnosis between hypo- and hyperproliferative thrombocytopenias has been already reported8, 12, less information is available about its use in the differential diagnosis between ITP and HM. In 2000, Fabris et al. evaluated reticulated platelets by flow cytometry in a population of 29 patients with HM, observing reduced IPF values when compared to ITP13. More recently, Miyazaki et al.14, in a study with 15 patients with HM in which the IPF was measured in an automated hematology analyzer observed Faculty of Medical Sciences/Hematology and Hemotherapy Center, University of Campinas, Campinas, SP, Brazil. Correspondence and requests for materials should be addressed to E.V.D. (email: ) Scientific Reports | 7: 3355 | DOI:10.1038/s41598-017-03668-y 1 www.nature.com/scientificreports/ significantly higher IPF values in HM compared to ITP. To further elucidate the value of IPF determination in the diagnosis of thrombocytopenias, with focus on the differential diagnosis between ITP and HM, we investigated the diagnostic accuracy and the precision of IPF measurements in a population of patients with different causes of thrombocytopenia, including a well-characterized cohort of patients with HM. In addition, we also evaluated whether thrombopoietin (TPO) levels could further facilitate this diagnosis. Methods Study design and patient population. This was a cross-sectional diagnostic accuracy study, designed according to STARD (Standards for the Reporting of Diagnostic accuracy studies) guidelines15. The study population consisted of patients with thrombocytopenia confirmed in two independent samples and microscopic analysis, in regular clinical follow-up in the Hemostasis outpatient clinic of University of Campinas, or admitted to the hematology ward of the same institution. The inclusion criteria was a confirmed diagnosis of any of the following: (i) ITP (based on previously established guidelines)16; (ii) bone marrow failure (including aplastic anemia and myelodisplatic syndromes with a platelet count below 150 × 109/L)17, 18; (ii) post-chemotherapy (Ctx) thrombocytopenia (in admitted patients with hematological malignancies) with a platelet count below 150 × 109/L; and (iv) HM, with platelet counts below 150 × 109/L. Diagnosis of HM was established by clinical and laboratory criteria as determined by international guidelines19–21. These included the exclusion of all other causes of thrombocytopenia, objective confirmation of thrombocytopenia in first-degree relatives, platelet aggregation studies, molecular analyses and specific tests such as platelet glycoprotein studies and electron microscopy in selected cases. Exclusion criteria included: a platelet count above 150 × 109/L in ITP patients at the day of enrollment; (ii) the presence of conditions known to influence IPF values such as sepsis and other inflammatory diseases22, or (iii) the use of antiplatelet agents. The study was approved by the Institutional Review Board of University of Campinas (certificate number 411.620/2013) and performed in accordance with the Declaration of Helsinki. All patients provided written informed consent prior to enrollment. Recruitment occurred between July 2013 and February 2015. ITP, HM and BMF patients were included consecutively, aiming for an enrollment of 100 patients with ITP, 20 with BMF and all patients with HM. Post-Ctx patients were included using a convenience sampling strategy, which consisted in weekly visits to the hematology ward, with a target of 50 patients. Sample collection and processing. Samples were collected by venipuncture by the same nursing team responsible for the collection of routine clinical samples, and using the same standard operating procedures in both clinical sites. Blood was drawn in Vacutainer EDTA K2 tubes (Becton Dick (...truncated)