Neonatal platelet count trends during inhaled nitric oxide therapy

Introduction

Platelets derive from precursor megakaryocytes in bone marrow, as well as megakaryocytes that have migrated to the lung (Lefrançais et al, 2017; Zucker-Franklin & Philipp, 2011). The clinical significance of lung-derived platelets remains a matter of debate.

Platelet transfusions are commonly administered in the neonatal intensive care unit (NICU) to prevent bleeding in the setting of thrombocytopenia (platelet count below 150 ×10⁹ per L). Given that platelet transfusions increase morbidity and mortality in preterm infants (Curley et al, 2018), considerable debate surrounds clinical transfusion guidelines (Sparger et al, 2015).

Pulmonary arterial hypertension (PH) diminishes pulmonary blood flow. PH is frequently encountered in NICU from reduced pulmonary vascular relaxation after birth and/or abnormal lung development. PH is associated with thrombocytopenia in adults (Mojadidi et al, 2014; Le et al, 2019). Among modalities, PH can be treated with mechanical ventilation and direct pulmonary vasodilation (e.g., inhaled nitric oxide, iNO). Trials demonstrating efficacy for iNO in treating infants with PH have not focused on platelet count trends (Barrington et al, 2017).

Here, we analyzed platelet count trends in infants receiving iNO treatment for PH. Bone marrow-derived platelets take about 7 days to form, whereas pre-formed megakaryocytes in the lung might be expected to yield platelets substantially faster. We hypothesized that rapid increases in platelets related to iNO initiation would reflect output from pre-formed lung-resident megakaryocytes, and reasoned that rapid iNO effects would permit temporal correlations distinct from other thrombocytopenia risks in these subjects (e.g., sepsis (Guida et al, 2003)).

Methods

Subjects admitted to our neonatal intensive care unit (NICU) from 2010-2018 at <28 d of age who received iNO therapy were identified retrospectively. These subjects were expected to have clinically significant PH. We excluded subjects who received platelet transfusions or required extracorporeal membrane oxygenation (ECMO), as these confound endogenous platelet quantification. Subjects with abnormal lung development from congenital diaphragmatic hernia (CDH), pulmonary hypoplasia or omphalocele were excluded from our primary cohort. Those with CDH were considered separately.

We considered subjects for whom complete blood counts (CBCs) were available within 48 h prior to iNO initiation and within 48 h following iNO discontinuation. We also analyzed CBC results within 48h after starting iNO and within 48 h before stopping iNO. We grouped up to 5 laboratory values to estimate average blood count values within each time frame.

Respiratory severity score (RSS) correlates well with oxygenation index in neonates (Iyer & Mhanna, 2013). RSS, calculated by multiplying the fraction of inspired oxygen by mean airway pressure in mechanically ventilated subjects, was used to mark pulmonary response to iNO. In many cases, the arterial partial pressure of oxygen was unknown.

Results

Table 1 shows characteristics for 138 subjects meeting inclusion criteria. Average iNO therapy duration was 22.7 days. Many subjects (54%) received platelet transfusions, demonstrating the heightened risk of transfusion in this population.

Table 1.

Clinical characteristics of patients that met inclusion criteria for this study. Thrombocytopenia is defined as platelet count <150 ×10⁹ per L. Some patients met multiple exclusion criteria, including need for ECMO or platelet transfusion. Those with ‘other pulmonary hypoplasia’ include diagnosis of omphalocele or idiopathic pulmonary hypoplasia.

Characteristic	Quantity
Infants meeting inclusion criteria	138
Female gender (%)	63 (46%)
Birth gestational age (mean±SD)	35.7±3.8 weeks
Birth weight (mean±SD)	2.69±0.9 kg
Avg age at iNO start	4.8 d
PPHN (# included)	101 (44)
CDH (# included)	24 (11)
Other pulmonary hypoplasia diagnosis	13
Required ECMO	27
Received ≥1 platelet transfusion	75

After applying exclusion criteria, 44 subjects comprised our primary analysis cohort. Platelet count did not change in the setting of iNO initiation (Fig. 1A, ‘Pre’ vs ‘Start’), despite a significant decrease in respiratory severity scores (Fig. 1B). Platelet counts remained stable yet mildly depressed for the duration of iNO therapy. Unexpectedly, platelet counts increased 50% upon iNO discontinuation (Fig. 1A, ‘Post’), by an average of 114 ×10⁹ per L compared to pre-iNO treatment levels (239±89 pre-iNO vs 353±220 ×10⁹ per L post-iNO initiation, mean±SD). White blood cell (WBC) and red blood cell (RBC) counts remained stable during all analyzed time periods (Fig. 1C–D).

Figure 1. Platelet count in infants with PPHN (excluding CDH or pulmonary hypoplasia) is consistent before and during iNO therapy, but increase after stopping iNO.

(A) Platelet counts are relatively consistent before (Pre) and during iNO therapy (Start and End), but acutely increase in the 48 h after stopping iNO (Post). (B) Respiratory severity score significantly decreases following iNO initiation. (C) Red blood cell (RBC) and (D) white blood cell (WBC) counts are consistent in the time periods before, during and after iNO therapy. *p<0.05, **p<0.01. ns, not statistically significant.

The observed trends in cell counts and respiratory severity scores were generally consistent in a separate analysis of 11 subjects with CDH (Supplementary Fig. 1).

Temporal analyses confirmed that there were no clinically meaningful changes in platelet count in the first 7 d of iNO treatment for our patient cohorts (Supplementary Fig. 2). At minimum, these analyses confirm that platelet count does not acutely increase at the onset of pulmonary vascular dilation in the setting of iNO initiation.

Discussion

In summary, our PH cohort initially had relative thrombocytopenia that improved once iNO therapy was discontinued. This association mirrors the risk of thrombocytopenia in adults with PH (Mojadidi et al, 2014; Le et al, 2019). The lack of correlation between pulmonary blood flow and platelet counts suggests that lung-resident megakaryocytes are not a significant untapped reservoir for platelets for this population.

Platelet counts did not significantly change following iNO initiation. Even minimal pulmonary blood flow in PH patients may be sufficient to promote platelet elaboration from lung megakaryocytes prior to iNO initiation. These findings may be confounded by dilutional changes related to fluid resuscitation, although unchanged RBC and WBC counts argue against this scenario. Overall, bone marrow megakaryo-thrombopoiesis and/or resolution of critical illness seem primarily responsible for platelet count trends seen in our cohort.

Our findings suggest platelets might increase following iNO discontinuation for some infants. We were surprised by the magnitude of this increase, which was similar to increases following 10-15 ml/kg platelet transfusions (Kline et al, 2008). This could help inform clinical transfusion decisions, although it will be important to investigate and confirm these trends in larger cohorts. Mechanistically, the observed platelet increase is unlikely to be due to consumption or bleeding during iNO therapy. Despite historical concern for iNO reducing platelet function, there has been no correlation between iNO therapy and bleeding (Barrington et al, 2017). Biochemical studies suggest that nitric oxide may acutely increase platelet production by promoting megakaryocyte apoptotic mechanisms (Battinelli et al, 2002). Over time, this might be expected to cause an overall decrease in megakaryopoiesis and/or thrombopoiesis. Finally, it is also possible that this change reflects a reactive thrombocytosis following resolution of lung injury, as seen in patients with sickle cell disease (Villagra et al, 2007). Further studies are needed to address specific iNO effects that could alter thrombopoiesis in this patient cohort.