Pathophysiology of Acute Pain in Patients With Sickle Cell Disease

Episodic pain is the most common acute morbidity and the leading cause of hospitalization in
patients with sickle cell disease. It is caused by microvaso-occlusion induced by sickled red
blood cells, an outcome of the polymerization of deoxygenated hemoglobin S (HbS). Factors
that contribute to the acute sickle pain include the release of cell-free DNA (cfDNA) and
heme that initiate a downstream of events involving inflammation and thrombosis, and
ischemia-reperfusion injury.

Cell-free DNA has been shown to be present in plasma of healthy subjects, but elevated in
diseases and conditions that are characterized by increased cell death through necrosis or
apoptosis. Indeed, we have previously shown that cfDNA in patients with sickle cell disease
(SCD) increased dramatically during acute painful episodes. During acute sickle pain, marked
elevation of plasma hemoglobin has also been observed due to the acute increase in sickled
red blood cells and hemolysis. Both cfDNA and heme (break down product of hemoglobin), act as
damage-associated molecular pattern (DAMP) molecules, initiating endothelial inflammation,
stimulation of neutrophil extracellular trap (NET) formation, leukocyte recruitment, and
microvascular thrombosis.

Although there have been several studies of cytokines and chemokines in steady state and
acute sickle cell disease, there has been no comprehensive study of how the inflammatory
markers correlate with quantitative levels and profile of cfDNA. In this study, we would like
to apply next generation sequencing (NGS) to analyze cfDNA from the plasma of patients with
SCD in steadystate, and during painful crises to derive insights on the origin of tissue
damage. In parallel with the free plasma DNA, we propose to measure markers of hemolysis and
inflammation (cytokines, chemokines), and to investigate if interactions between these
circulating molecules and blood cells (e.g. neutrophils) have the potential to modulate the
progress and severity of the disease. In addition, we would also like to explore if there is
a distinctive cell-free DNA and inflammatory signature in SCD in steady-state and during
acute vaso-occlusive crises.

Overall, this study provides an opportunity to evaluate new biomarkers of sickle cell pain
crisis and to predict disease severity and prognosis. These measures may allow us to better
understand the role of vaso-occlusion, hemolysis, and inflammation-related events and
responses and serve as clinical endpoints in future studies of disease pathogenesis and/or
therapeutic intervention for sickle cell disease.

- SUBJECT INCLUSION CRITERIA (SCD):

1. Sickle cell disease (all genotypes) with a diagnosis of acute sickle cell pain
not related to other cause (if the patient also presents with any other sickle
related complication alongside acute sickle pain, including not limited to acute
chest syndrome, renal dysfunction, liver dysfunction, stroke and priapism can
also be included in the study)

2. Between 18 and 80 years of age

3. Ability to provide informed written consent

SUBJECT EXCLUSION CRITERIA (SCD):

1. <18 years of age

2. Pregnancy

3. Chronic inflammatory condition (e.g. SLE, Rheumatoid arthritis or any other infectious
process leading to chronic inflammation)

4. Failed stem cell transplantation for SCD

5. On active treatment with cytotoxic or immunosuppressive therapy

INCLUSION CRITERIA FOR HEALTHY CONTROL SUBJECTS:

1. Between 18 and 80 years of age

2. African, of African descent

3. Ability to provide informed written consent

EXCLUSION CRITERIA FOR HEALTHY CONTROL SUBJECTS:

1. Pregnancy

2. Diagnosis of with any of the following chronic disease or conditions:

1. Sickle cell disease or sickle cell trait

2. Uncontrolled high blood pressure (systolic blood pressure must not be greater
than 160 mmHg or diastolic pressure greater than 90 mmHg

3. Uncontrolled diabetes (must not have both a documented history of diabetes and
random blood glucose of greater than 200 mg/dL)

4. History of coronary artery disease

5. History of congestive heart failure

6. Chronic kidney disease (serum creatinine must not be greater than 2mg/dL)