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\begin{document}

\title{Lung Clearance Index in Children with Sickle Cell Disease}



\author[1]{Evans Machogu}%
\author[1]{Monica Khurana}%
\author[1]{Jennifer Kaericher}%
\author[2]{Charles Clem}%
\author[1]{James Slaven}%
\author[3]{Joseph Hatch}%
\author[4]{Stephanie Davis}%
\author[5]{Stacey Peterson-Carmichael}%
\affil[1]{Indiana University School of Medicine}%
\affil[2]{James Whitcomb Riley Hospital for Children}%
\affil[3]{University of North Carolina System}%
\affil[4]{University of North Carolina at Chapel Hill School of Medicine}%
\affil[5]{Wake Forest University}%


\vspace{-1em}



  
  \date{June 13, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Introduction: The Lung Clearance Index (LCI) derived from the
multiple-breath washout test (MBW), is both feasible and sensitive to
early lung disease detection in young children with cystic fibrosis and
asthma. The utility of LCI has not been studied in children with sickle
cell disease (SCD). We hypothesized that children with SCD, with or
without asthma or airway hyper reactivity (AHR), would have an elevated
LCI compared to healthy controls. Methods: Children with SCD from a
single center between the ages of 6-18 years were studied at baseline
health and completed MBW, spirometry, plethysmography and blood draws
for serum markers. Results were compared to healthy controls of similar
race, age and gender. Results: Control subjects (n=35) had a
significantly higher daytime oxygen saturation level, weight and body
mass index (BMI) but not height compared to subjects with SCD (n=34).
Total Lung Capacity(TLC) z-scores were significantly higher in the
healthy controls compared to those with SCD (0.87 (1.13), 0.02 (1.27),
p=0.005) while differences in Forced Expiratory Volume in 1 second
(FEV1) z-scores approached significance (0.26 (0.97), -0.22 (1.09),
p=0.055). There was no significant difference in LCI among the
comparison groups (7.29 (0.72), 7.40 (0.69), p=0.514). Conclusion: LCI
did not differentiate SCD from healthy controls in children between the
ages of 6 and 18 years at baseline health. TLC may be an important
pulmonary function measure to follow longitudinally in the pediatric SCD
population.%
\end{abstract}%



\sloppy




\emph{INTRODUCTION}

Sickle Cell Disease (SCD) affects about one in every 400
African-Americans in the United States\textsuperscript{1} with
significant complications including recurrent severe pain
episodes\textsuperscript{2} and acute chest syndrome
(ACS)\textsuperscript{3}for which each episode carries a 4\% chance of
death\textsuperscript{4}. Pulmonary complications are the cause of death
in over 50\% of autopsies in SCD patients yet reliable and sensitive
tests to detect early lung disease are not available. An estimated
30-70\% of children with SCD have wheezing and airway hyper-reactivity
(AHR) and air trapping\textsuperscript{5-11} and those with asthma have
higher rates of pain and ACS as well as premature
death\textsuperscript{12-17}. Ultimately, patients with multiple
episodes of ACS and wheezing develop Sickle Cell Lung Disease (SCLD),
which is a restrictive lung disorder\textsuperscript{18}, often
associated with pulmonary hypertension.

Diagnosis of early lung disease in SCD is delayed as spirometry and
plethysmography, measures of obstructive or restrictive lung disease,
cannot often be reliably performed in children under the age of 5 years.
Additionally, spirometry is relatively insensitive in identifying and
assessing early lung disease in the smaller, more distal airways until
significant disease has developed\textsuperscript{19,20}. The multiple
breath washout (MBW) technique, which measures the Lung Clearance Index
(LCI), is a novel noninvasive measurement of ventilation inhomogeneity
that is simple, involves no risk or hazardous exposures and can be used
in children, including preschoolers. LCI has been shown to be a
sensitive tool in detecting lung disease in cystic fibrosis and asthma
before any changes are apparent on spirometry\textsuperscript{21,22}and
has been demonstrated to be abnormal in patients with normal lung
function on spirometry\textsuperscript{23,24}.

To date, MBW has not been studied in children with SCD. One study
explored the potential relationship between nitrogen single-breath
washout~(N2SBW) and exercise intolerance in 38 adults with SCD (median
age of 28 years) \textsuperscript{25}. Results suggested that phase III
slope (SIIIN2) values correlated with hemoglobin level, forced vital
capacity (FVC), diffusing capacity and SpO2 after exercise. Given this,
assessing the ability of MBW to detect potential ventilation
inhomogeneity may prove beneficial and allow for earlier intervention in
patients where the disease may go unrecognized. Since pulmonary
complications such as ACS, wheezing and airway hyper-reactivity (AHR)
often start early in SCD\textsuperscript{11,26}we hypothesized that
children with SCD (with or without asthma) would have an abnormal and
elevated LCI compared to healthy controls. Findings of abnormal LCI in
children with SCD during which time spirometry may be normal would be
useful in initiating early intervention and more in-depth diagnostic
screening.

\emph{METHODS}

In this case-control study, we recruited patients with SCD with or
without a physician diagnosis of asthma and healthy African American
controls between 6 and 18 years of age. \textbf{Inclusion/exclusion
criteria:} Inclusion criteria were a diagnosis of sickle cell disease
(HbSS, HbS\selectlanguage{greek}β-\selectlanguage{english}thalassemia\textsuperscript{+},
HbS\selectlanguage{greek}β-\selectlanguage{english}thalassemia\textsuperscript{0}, HbSC, HbSO Arab) confirmed by
hemoglobin analysis. Age criteria was 6 to 18 years of age. Exclusion
criteria were preterm birth (born at \textless{} 37-week gestation) or
an inability to complete study activities. Participants completed study
activities only if they were at baseline health without respiratory or
SCD related complications occurring within the previous 4 weeks. Healthy
African American controls between the ages of 6 to 18 years were also
recruited but were excluded from the study for a diagnosis of asthma,
any episodes of wheezing leading to shortness of breath in the previous
12 months or presence of a bronchodilator response ([?] 12\% increase in
FEV\textsubscript{1} or FVC post-bronchodilator).

This study was approved by our institutional IRB and informed consent
and assent, where applicable, were obtained for all participants. A
thorough history was obtained, and a physical examination completed at
the time of study participation. A review of the participants' medical
charts was conducted to obtain data regarding episodes of ACS, severe
pain crises requiring an inpatient stay, a physician-documented
diagnosis of asthma and other co-morbidities. All participants performed
pulmonary function tests (PFT) including MBW (Eco Medics Exhalyzer D,
software version 3.1.6) using nitrogen as the washout gas,
plethysmography (Morgan Scientific, ComPAS software version
1.10.2642.4440), and pre- and post- bronchodilator spirometry (Morgan
Scientific, ComPAS software version 1.10.2642.4440). Blood was obtained
from healthy controls at the time of the study visit as well as in those
SCD subjects with no laboratory studies in the previous 3 months

\textbf{Study protocol and procedures:}

The study participants first completed MBW according to American
Thoracic Society (ATS) guidelines\textsuperscript{27}. LCI was
determined according to standardized protocols\textsuperscript{28}.
Subsequently spirometry followed by lung volumes (plethysmography) were
performed according to ATS criteria and
standards\textsuperscript{29,30}. To measure airway
hyper-responsiveness\textsuperscript{27}(AHR), spirometry was repeated
20 minutes after administration of 4 puffs of albuterol via MDI. For
healthy controls and SCD patients without a recent blood draw (3
months), whole blood samples were collected for processing on the day of
testing.

\textbf{Definitions:} Obstructive lung disease was defined as 1 or more
lung function measures (FEV\textsubscript{1} or
FEV\textsubscript{1}/FVC) \textless{} lower limit of normal (LLN)
equivalent to a z-score of -1.645 based on Global Lung Initiative (GLI)
normative data for age, height, weight and race. Restrictive lung
disease was defined as Total Lung Capacity (TLC) or Forced Vital
Capacity (FVC) \textless{}LLN based on GLI normative data for age,
height, weight and race. \emph{AHR}was defined as having a positive
bronchodilator response after repeat spirometric measures with an
increase in FEV\textsubscript{1} and/or FVC of [?]12\% from
baseline\textsuperscript{31}.

\textbf{Statistical Analysis:} The sample size was based on results from
Zwitserloot et al.\textsuperscript{32}where the comparison of LCI
between patients with asthma and healthy children (controls) showed a
LCI difference of 0.27 with standard deviations of 0.48 and 0.38 for the
two groups respectively. There are no defined normative LCI values in
African American children. We assumed a similar LCI difference and
standard deviation between SCD and healthy control children. In order to
achieve 80\% power and a 5\% error rate, we needed to enroll 42 children
in each group. Demographics, clinical events, lab results, and pulmonary
function data were compared across groups using Student's t-tests for
continuous variables. Wilcoxon tests were performed for non-linear data.
Chi-Square tests were performed for categorical variables and Fisher's
Exact tests were used when cell counts were low. Correlation analyses
were performed to analyze the associations between clinical and
pulmonary function data. To check for possible moderating effects due to
participant age, interaction terms were assessed with age groups 6-12
years of age and 13-18 years, as well as performing analyses stratified
by age groups. Multivariable models were then used to determine if
associations with LCI would become attenuated in the larger model.
Variables in the multivariable model were chosen due to statistical
significance at the bivariate level and for clinical meaningfulness. All
analytic assumptions were verified, with non-parametric tests (i.e.
Wilcoxon tests and Spearman correlations) used when data were
non-linear. Collinearity was assessed in the multivariable model with
variance inflation factors. All analyses were performed using SAS v9.4
(SAS Institute, Cary, NC).

\emph{RESULTS}

Out of a total of 193 subjects assessed for study eligibility, 69
completed all study requirements and are included in our analysis

\textbf{(Figure 1).}

Baseline demographics are shown in \textbf{Table 1} . Thirty-four (34)
participants with SCD and 35 healthy controls met criteria for analysis.
We categorized the participants and controls into two age groups (6-12
years and 13-18 years). Control subjects had a significantly higher
weight and body mass index (BMI) but not height compared to patients
with SCD. Of the 34 participants with SCD, 27 had HbSS and
HBS\selectlanguage{greek}β\selectlanguage{english}\textsuperscript{0} as their genotype and thus eligible for
hydroxyurea at the time of study participation. Only 20 of the 27
patients eligible for hydroxyurea were on it at time of our study. Four
participants required a NICU stay for various reasons other than
respiratory distress though one was briefly placed on oxygen.

\textbf{Clinical and laboratory comparisons}

Four from the control group declined a blood draw (or had inadequate
sample for processing) and were presumed to be either carriers or have
HBAA genotype given no stigmata of SCD \textbf{(Table 2).} SCD subjects
demonstrated a significantly lower hemoglobin concentration, higher
white blood cell count and lower daytime oxygen saturation level.

A comparison of pulmonary function data is shown in \textbf{table 3} .
TLC and FEV\textsubscript{1} were significantly higher in the healthy
controls compared to those with SCD. There was no significant difference
in LCI among the comparison groups. In our patient cohort, we saw no
changes in LCI with increasing age (\textbf{Figure 2} )

Amongst the subjects with SCD, a normal pattern of lung function was
noted in 31 patients (91\%), an obstructive pattern in 3 patients (9\%),
and a mixed obstructive/restrictive pattern in only 1 patient (3\%).
Four (12\%) of the SCD participants demonstrated bronchodilator
responsiveness. However, nineteen (56\%) had a physician diagnosis of
asthma. There was no statistically significant difference in pulmonary
function indices between patients with SCD (n=18) and healthy controls
(n=17) ages 6-12 years. However, a comparison between participants with
SCD and healthy controls ages 13-18 years revealed that TLC and
FEV\textsubscript{1} z-score values were significantly higher in the
healthy control group (0.86 +/-1.25 vs -0.22 +/-1.37; P=0.02) and (0.28
+/-1.11 vs -0.53 +/-1.01; p=0.03).

SCD subjects of all ages without a history of asthma had significantly
higher TLC z-score values vs those with asthma (0.74 +/-1.21 vs -0.32
+/- 1.11 with asthma; p=0.002) but there were no differences in LCI
(7.31 +/-0.65-without asthma vs 7.44 +/-0.83 with asthma; p= 0.5085).

A history of ACS was associated with a lower TLC z-score (-0.74 +/- 0.96
vs 0.41 +/- 1.30, p=0.015) and FEV\textsubscript{1} (-0.76 +/- 0.54,
0.08 +/- 1.25; P=0.013). However, a history of ACS was not associated
with higher LCI ((7.25 (0.48) for history of ACS vs 7.47 (0.78);
p=0.411)). A history of asthma or response to bronchodilator was not
significantly associated with LCI

\emph{Correlations between LCI, spirometry and plethysmography}

Amongst all study participants, FVC, FEV\textsubscript{1}, RV and TLC
are highly correlated with LCI, although these associations lose
statistical significance when looking at only the sickle cell group with
the only significant correlation being with TLC \textbf{(table 4).}

\emph{Multivariate analysis}

In the adjusted multivariable model for the SCD group only\textbf{(table
5)} hemoglobin was independently associated with LCI when all the
variables are included in the model.

\emph{DISCUSSION}

To our knowledge, this is the first study to evaluate LCI in the
childhood sickle cell population. In this study, LCI did not
differentiate SCD from healthy controls in children between the ages of
6 and 18 years of age at baseline health. Furthermore, we saw no
cross-sectional differences in LCI with increasing age. Additionally,
neither a history of asthma nor ACS was associated with a significantly
higher LCI among the SCD participants compared to healthy controls.
There was a high correlation between
FEV\textsubscript{1}\textsuperscript{,} FVC, RV and TLC with LCI amongst
all subjects, although some of these associations lose statistical
significance when looking at only the sickle cell group likely due to
small sample size.

In the SCD only group, the only significant association with LCI was
TLC. There was no correlation between LCI values and past episodes of
ACS or diagnosis of asthma in our study. TLC was significantly higher in
healthy controls compared to SCD subjects and was highly correlated with
LCI in the SCD group only. This may suggest that TLC is a much more
sensitive measure of early lung disease in the adolescent group (ages
13-18yrs) before any obvious obstructive or restrictive patterns emerge.
Future studies focusing on plethysmography may bring better
understanding to the potential use of LCI for detecting lung disease in
this population.

Our data support previous studies\textsuperscript{33,34}that have
reported decreased baseline oxygen saturation in the SCD population. In
children between the ages of 13 and 18 years of age, TLC and
FEV\textsubscript{1} were significantly higher in the healthy subjects
compared to those with SCD. This would further support existing
literature that lung disease in sickle cell patients worsens over time,
and that identifying an early screening technique to differentiate the
higher risk SCD populations before they have recurrent ACS and recurrent
VOC is critical.

Three (9\%) of our patients demonstrated an obstructive disease pattern
on spirometry and only 1 (3) \% had a mixed obstructive/restrictive
disease pattern. This is similar to previous studies\textsuperscript{35}
with the majority of SCD patients having normal lung function or some
type of mixed picture. The number of SCD patients with asthma based on
physician diagnosis was 19 (56\%). There was likely some recruitment
bias as subjects who were approached for the study and agreed to
participate were often patients seen in Pediatric Pulmonology clinic.
LDH values differed significantly with a mean of 447 in the ``asthma''
group versus 306 in the ``no asthma'' group. These values may reflect
treatment and adherence to hydroxyurea. Our data revealed that 80\% of
the SCD ``asthma'' group verbalized having hydroxyurea as a current
prescription medication versus 6 \% in the ``no asthma'' group. This was
not part of our aims or hypotheses but interesting for future research
into potential protective effects of hydroxyurea in SCD patients with
pulmonary diagnoses other than ACS such as asthma, wheezing or chronic
hypoxia.

Study limitations include the inability to reach our goal enrollment of
42 participants in each of the SCD and control study groups. We were
able to report data on 69 total participants, and due to the lack of
normative LCI data for African American participants, our power
calculation was performed based on LCI in pediatric asthma. However, our
research allowed the opportunity to collect LCI data in an African
American normative cohort. Pritchard et al\textsuperscript{36} studied
the basic biologic processes behind lung disease in SCD animal models,
however, the in-vivo pathophysiology of lung disease in SCD, especially
in the ``healthy'' SCD pediatric population is still not well
understood. The current understanding of pathophysiology in SCD led to
the proposed hypothesis to test LCI where perhaps using other
non-invasive methods such as FOT (forced oscillation technique) would be
more appropriate. The other limitation is the nature of the study design
such that the patients were not followed longitudinally to assess future
frequency of ACS, and other SCD complications. Future studies in the use
of LCI in the SCD population might focus on LCI values during and after
acute complications such as ACS, wheezing episode, asthma exacerbation.
The other population of interest would be the adult SCD group where the
incidence of respiratory disease and abnormal PFT's is much higher.

In conclusion, there was no significant difference in LCI measures
between SCD and healthy controls at baseline health. Furthermore, TLC
may be an important pulmonary function measure to follow longitudinally
in the pediatric SCD population. Future studies at time of ACS recovery,
or in the adult population may bring better understanding to the
potential use of LCI for detecting lung disease in this population.

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