Immature platelet fraction as novel laboratory parameter predicting the course of neonatal thrombocytopenia

Abstract

Thrombocytopenia occurs in up to 30% of neonates admitted to neonatal intensive care units (NICUs). In preterm neonates, severe thrombocytopenia is associated with increased risk of intracerebral bleeding. Thus, diagnosis of thrombocytopenia often leads to repeated subsequent analysis of blood cell counts and transfusion of platelets. However, no evidence has been provided that current practice for platelet transfusion prevents bleeding complications and improves clinical outcome. A rapid automated method to assess reticulated platelets, the immature platelet fraction (IPF), is available (Briggs et al, 2000). The aim of this study was to investigate the diagnostic value of IPF as a marker for megakaryopoietic activity in neonates. All 612 neonates admitted to our NICUs over a 6-month period were included in the study. Written parental consent and organisational approval were obtained. During the first postnatal week, blood samples were obtained as clinically indicated. To generate reference values for the IPF, patients were divided in two groups: The control group (n = 456) always displayed normal platelet counts (150–450 × 109/l). In the thrombocytopenic group (n = 156), platelet counts dropped below 150 × 109/l at least once. Blood samples obtained after platelet transfusion were excluded from analysis. Platelet counts were optically measured with the automated analyser XE-2100 (Sysmex, Kobe, Japan) equipped with the software xe-ipf-master. Determination of IPF is based on fluorescence flow cytometry using a nucleic acid specific dye that stains RNA in erythrocytic and platelet reticulocytes. The computed algorithm discriminates mature and immature platelet populations applying a preset gate (fluorescent intensity = RNA-content; forward scatter = cell volume). The IPF is normally expressed as a percentage of the platelet count to indicate the rate of platelet production; additionally, absolute values may be used to differentiate between insufficient platelet production and increased consumption as cause of thrombocytopenia. Regression analysis was used to calculate the correlation between platelet counts and IPF. Student’s t-test was applied to compare group means. Statistical analysis was performed using the software statgraphics (Manugistics Inc., Rockville, MD, USA). A P-value < 0·05 was considered to indicate statistical significance. In 1045 out of 1339 blood specimens IPF was routinely determined in addition to platelet counts. Four hundred and fifty-six patients (838 specimens) were assigned to the control group, 25·5% of patients (n = 156; 501 specimens) to the thrombocytopenic group. The mean [standard deviation (SD)] birth weight in the control group was significantly higher than in the thrombocytopenic group [2717 g, (SD ± 849) vs. 2188 g, (SD ± 105), P < 0·001]; this also refers to mean gestational age [36·3 weeks, (SD ± 3·7) vs. 34·2 weeks, (SD ± 5·0), P < 0·001]. In controls, the mean IPF value was 4·3% [95% confidence interval (CI) 0·7–7·9%] during the first postnatal week. In the thrombocytopenic group, the mean IPF (SD) during the first postnatal week was always significantly higher than in the control group [e.g. day 1: IPF 8·7% (9·3) vs. 4·53% (1·8); P < 0·001; Fig 1). 36% of thrombocytopenic neonates displayed an IPF above 7·9%. We found no significant correlation between gestational age and IPF. Using simultaneous IPF and platelet measurements of both groups, a significant negative correlation between IPF and platelet counts was found with an exponential decay (r = −0·62, P < 0·001). Increasing platelet counts were anticipated by an increased IPF, reflecting enhanced platelet production (Fig 1). Longitudinal analysis of platelet count (mean ± standard error of mean) and IPF percentages (mean ± SEM) during first postnatal week. The control group (platelet counts >150 × 109/l, black symbols) and the thrombocytopenic group (platelet counts once <150 × 109/l, red symbols) are shown. Triangles represent IPF, circles represent platelet counts. According to the study aim, we analysed whether the platelet counts on the following day were somehow predicted by means of previous IPF values. In patients whose blood samples were analysed on two subsequent days, the difference in platelet counts was plotted against the corresponding IPF value (Fig 2). In this scatter plot, specimens (n = 398) were divided in to four quadrants according to IPF (>8%, which is outside CI > 95% of controls) and difference in platelet counts (severe decrease >50 × 109/l) on the following day. In only five of 99 samples (5%) platelet counts dropped, even though the production rate exceeded an IPF of 8%, whereas in 71 of 299 samples (24%) the platelet count dropped if IPF was <8%. On the basis of this distribution, we calculated that the relative risk to exhibit a decrease in platelet counts more than 50 × 109/l on the following day was 4·7 times higher (95%CI = 1·9–11·8) in patients with IPF < 8%. If platelet counts decreased more than 50 × 109/l, the mean (SD) absolute number of IPF was significantly lower compared to neonates who maintained stable platelet counts [7·8 × 109/l (2·9) vs. 9·5 × 109/l (3·8), P < 0·05]. Analysis of the predictive value of IPF on consecutive severe decrease (more than 50 × 109/l) in neonatal platelet counts within 24 h. In patients who had blood counts on two subsequent days the difference of their platelet counts (y-axis, delta platelet count) was plotted against corresponding IPF% (x-axis). Black symbols represent controls, red symbols represent neonates in the thrombocytopenic group (platelet count once <150 × 109/l). Platelet counts dropped in only five samples, even at IPF values >8%. Reticulated platelets have been previously considered to evaluate neonatal thrombocytopenia (Peterec et al, 1996), but their detection is time consuming and yet not standardised. Herein, we tested the hypothesis that fully automated standardised determination of IPF is helpful in predicting the course of neonatal thrombocytopenia. The present study provides the first evidence that non-thrombocytopenic neonates had IPF values that were higher than reported in adults (mean 3·4%, range 1·1–6·1) (Briggs et al, 2004), most likely reflecting higher megakaryopoietic activity linked to higher circulating thrombopoietin concentrations in the foetal and neonatal period (Dame, 2002). We also showed that overall IPF-%-values negatively correlate with platelet counts, similar as reported in children and adults (Abe et al, 2006; Saigo et al, 2008). In 36% of thrombocytopenic neonates IPF was elevated implying increased platelet production. However, in some patients, stimulation of megakaryopoiesis seemed to be inadequate low and platelet consumption might have exceeded platelet production. These patients displayed IPF values <8%, although high demand of platelets was required, resulting in a severe decrease (>50 × 109/l platelet counts) on the following day. Since only a slight or no increase in IPF values occurs in patients with disorders in megakaryopoiesis due to bone marrow failure or inappropriate thrombopoietin production, these patients may reflect the small proportion of neonates that suffer from inadequate megakaryopoiesis with or without increased platelet destruction (Sola et al, 1999). If absolute IPF numbers are taken into consideration, neonates could be identified who did not compensate platelet consumption by increased platelet production. In conclusion, IPF measurement in neonates is feasible and reflects the platelet production rate. The IPF is also helpful in predicting the course of thrombocytopenia and identifies patients with a risk for rapid severe drop in platelet counts nearly five times higher. Thus, the analysis of IPF may lead to more reasonable indications for platelet transfusions in neonates. The authors thank Prof. G. Gaedicke, Dr H. Schulze, Dr G. Strauss, Dr S. Ziemer, Dr A. Lun and Dr O. Meyer for intensive discussion of the data. Malte Cremer has been elected to participate in the competition for the Sysmex Outstanding Science Award 2009.