Sickle Cell Disease

Pathophysiology and
R e l a t e d Mo l e c u l a r a n d
Biophysical Biomarkers
Elna Saah, MDa, Payam Fadaei, MS
b
, Umut A. Gurkan, PhD
b,c,d,
*,
Vivien Sheehan, MD, PhDa,*

KEYWORDS
 Sickle cell disease  Biomarkers  Red blood cells  Rheology
 Clinical trial endpoints

KEY POINTS
 Although a monogenic disease, individuals with sickle cell disease (SCD) can have vastly
different clinical phenotypes. Early interest in biomarkers stemmed from a desire to be
able to predict the clinical course.
 Many biomarkers for diagnosing or predicting the risk of SCD complications have been
proposed, but few have been validated.
 Devices that measure the biophysical properties of blood cells are a promising source of
biomarkers for SCD.
 In this new era of multiple therapies for SCD, biomarkers are needed to choose the best
second-line therapies for the individual and as endpoints for clinical trials.

INTRODUCTION

Sickle cell anemia (SCA) refers to the homozygous state; a2bS2 (HbSS) and HbSb0
(HbSb0-thal), and they are clinically similar. Sickle cell disease (SCD) is an umbrella
term that includes other genotypes such as hemoglobin SC and sickle b1 thalassemia
(Sb1 thalassemia), which are generally clinically milder than SCA. The effect of SCD
genotype, alpha thalassemia coinheritance, and other genetic modifiers of SCD on

a
Department of Pediatrics, Emory University School of Medicine, Children’s Healthcare of
Atlanta, Atlanta, GA, USA; b Department of Mechanical and Aerospace Engineering, Case
Western Reserve University, Cleveland, OH, USA; c Department of Biomedical Engineering,
Case Western Reserve University, Cleveland, OH, USA; d Case Comprehensive Cancer Center,
Case Western Reserve University School of Medicine, Case Western Reserve University, Cleve-
land, OH, USA
* Corresponding authors.
E-mail addresses: umut@case.edu (U.A.G.); vivien.sheehan@emory.edu (V.S.)

Hematol Oncol Clin N Am 36 (2022) 1077–1095

https://doi.org/10.1016/j.hoc.2022.06.005 hemonc.theclinics.com
0889-8588/22/ª 2022 Elsevier Inc. All rights reserved.
Descargado para Anonymous User (n/a) en Cayetano Heredia Pervuvian University de
ClinicalKey.es por Elsevier en diciembre 27, 2022. Para uso personal exclusivamente. No se
permiten otros usos sin autorización. Copyright ©2022. Elsevier Inc. Todos los derechos
reservados.
1078 Saah et al

the clinical course are discussed elsewhere.1 Sickle hemoglobin (HbS) is created by a
point mutation that results in the replacement of a hydrophilic glutamic acid by the hy-
drophobic valine (b 6Glu/Val). Deoxygenated HbS polymerizes, increasing red cell
fragility and decreasing red cell survival. The pathophysiology of SCD can be divided
into four categories (Fig. 1): (i) polymerization of the deoxygenated HbS, (ii) cellular
adhesion to the endothelium, (iii) endothelial dysfunction from exposure to products
of hemolysis, and (iv) sterile inflammation.2 The complex interplay of these factors
set in motion a vicious cycle causing acute vaso-occlusive episodes (painful crisis
and acute chest syndrome [ACS]) and end-organ damage (cerebral infarcts, nephrop-
athy, retinopathy, and pulmonary hypertension [PH]).
Biomarkers are defined as identifiable and measurable markers of normal, patho-
physiologic processes, or biologic responses to exposures or interventions. There
are three main types of biomarkers: (i) susceptibility, risk, or predictive biomarkers;
(ii) response and monitoring biomarkers; and (iii) prognostic and surrogate end point
markers.3 In SCD, some candidate biomarkers were selected because they represent
a measurable aspect of SCD pathophysiology; others arose from devices designed to
measure biophysical properties of blood components known to be abnormal in SCD.
Many biomarkers have been proposed for SCD; few have been analytically or clinically
validated.

Established Sickle Cell Disease Biomarkers

The most commonly used and widely accepted biomarkers in SCD are derived from
conventional laboratory testing (Tables 1–3). The impact of fetal hemoglobin (HbF)
levels on disease severity has been extensively studied; addition of HbF to the growing

Fig. 1. Sickle cell disease pathophysiology. A point mutation produces an abnormal hemo-
globin that polymerizes under hypoxia; the downstream effects impact other blood cells,
cause inflammation, damage the vasculature, and produce severe clinical complications. Dis-
ease severity is ameliorated by higher levels of fetal hemoglobin, as it prevents further sickle
hemoglobin addition to the growing polymer.
Descargado para Anonymous User (n/a) en Cayetano Heredia Pervuvian University de
ClinicalKey.es por Elsevier en diciembre 27, 2022. Para uso personal exclusivamente. No se
permiten otros usos sin autorización. Copyright ©2022. Elsevier Inc. Todos los derechos
reservados.
 SCD Pathophysiology-Based Biomakers 1079

Table 1
Established sickle cell disease (SCD) biomarkers reflecting SCD pathophysiology based on
conventional laboratory tests

SCD
Pathophysiology Biomarker Current Use
Hemolysis/RBC Total Hb Indication for more aggressive
production/ disease-modifying therapies,
sequestration including HU, voxelotor, and transfusion
Reticulocytes Reflect RBC production; evidence
of therapy-induced myelosuppression
Hemolysis LDH Distinguish cause of anemia;
Bilirubin RBC production vs. destruction;
post-transfusion complications
Anti-sickling HbF Rationale for initiation of HbF induction therapies
Inflammation CRP Used serially in individual patients to assist in
diagnosis and monitoring of infections
such as osteomyelitis

HbS polymer prevents further polymerization (see Fig. 1). Individuals with SCD with
higher-than-average HbF levels, either endogenously or therapeutically, typically
have a milder course. However, HbF has limitations as a biomarker; some patients
with high %HbF still experience significant clinical complications,4,5 either because
of heterogenous distribution of HbF across the red cell population (heterocellular vs
pancellular HbF), or other factors counteracting the benefit of reduced polymerization.
Higher HbF reduces sickling, prolonging red cell survival and reducing hemolysis.
Individuals with lower total hemoglobin levels,6 lactate dehydrogenase (LDH) bilirubin,
and higher absolute reticulocyte count (ARC) are considered to have a hemolytic
phenotype (see Fig. 1). Hemolysis increases free heme and scavenges nitric oxide,
which damages the endothelium and contributes to vascular damage (see Fig. 1).

Table 2
Commonly used biomarkers associated with risk of acute sickle cell disease complications

SCD
Complication Biomarker Level of Validation Current Use
Pain event High WBC, High Hb, None Predisposition to
low %HbF, high whole frequent pain episodes.
blood viscosity, and
poor RBC
deformability
Acute chest High WBC, low Hb, Expert opinion: Aggressive management
syndrome high ARC, and LDH evaluate for of airway
ACS if Hb 2 g/dL hyperreactivity,
below baseline early institution
of incentive
spirometry, prompt
red cell transfusion
Overt stroke TCD (increased risk High level of evidence Determination of
of stroke) (ref NHLBI guidelines) increased stroke risk
Splenic Hb, platelets35 Expert opinion Evaluate for splenic
sequestration sequestration
if Hb 2 g/dL
below baseline

Descargado para Anonymous User (n/a) en Cayetano Heredia Pervuvian University de

ClinicalKey.es por Elsevier en diciembre 27, 2022. Para uso personal exclusivamente. No se
permiten otros usos sin autorización. Copyright ©2022. Elsevier Inc. Todos los derechos
reservados.
1080 Saah et al

Table 3
Commonly used biomarkers associated with chronic sickle cell disease complications

Level of
SCD Complication Biomarker Validation Current Use
Sickle cell Microalbuminuria Consensus Annual screen
nephropathy (SCN) expert beginning at age 10
opinion
Silent cerebral MRI Standard Not widely
infarcts (SCI) of care implemented
for diagnosis due to cost and
sedation requirements
for young children.
Pulmonary Screening echocardiogram: Consensus Refer for cardiac
hypertension (PH) Tricuspid Regurgitation expert catheterization
Velocity (TRV) 2.5 m/s opinion and definitive
diagnosis of PH
Leg ulcers Hb and LDH Single studies Not widely used

Individuals with high levels of hemolysis are more likely to have leg ulcers, strokes, pri-
apism, ACS, and early mortality.6,7 Patients with higher hemoglobin levels have a high
viscosity phenotype, and may be more likely to experience pain events, ACS, avas-
cular necrosis (AVN), and proliferative retinopathy.
Markers of Inflammation
SCD is marked by chronic inflammation from ischemia from vaso-occlusion and he-
molysis. Erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) may be
chronically elevated, and increase further during acute events, particularly infection.
CRP correlates with high baseline white blood cell (WBC) and in some studies predicts
increased vaso-occlusive crisis (VOC).8 ESR and CRP levels vary among individuals,
but may be useful for serial monitoring, or distinguishing between pain events and in-
fections, for example, osteomyelitis.9

BIOMARKERS OF ACUTE CLINICAL COMPLICATIONS

One of the nuances of SCD is that despite having the same causative mutation, indi-
viduals vary greatly in their disease severity and in the types of complications they
experience.1 Rather than seeking a biomarker of global severity, it may be more help-
ful to develop biomarkers that assess an individual’s risk for a specific clinical compli-
cations. Below we describe the pathophysiology of common SCD complications, and
the biomarkers associated with the complications (see Table 2).
Acute Pain Events
The acute painful vaso-occlusive event (VOE) is a hallmark of SCD, accounting for
most of the acute care utilization, hospitalizations, and reduced quality of life. The
sentinel event is often cell adhesion, followed by deoxygenation, polymerization of
HbS, RBC deformation, blockage of blood flow, and ischemia (see Fig. 1). Adhesion
of the red cell to the endothelium can be potentiated by increased circulating neutro-
phils, platelets, and reticulocytes. The sickled erythrocyte itself is more adhesive due
to perturbations in surface proteins, such as phosphatidylserine, and phosphatidyleth-
anolamine. Reticulocytes are particularly adherent due to increased expression of
membrane proteins CD36 and a4b1 integrin, which bind to vascular cell adhesion
molecule-1 (VCAM-1) expressed on the endothelium. P-selectin, expressed both on
Descargado para Anonymous User (n/a) en Cayetano Heredia Pervuvian University de
ClinicalKey.es por Elsevier en diciembre 27, 2022. Para uso personal exclusivamente. No se
permiten otros usos sin autorización. Copyright ©2022. Elsevier Inc. Todos los derechos
reservados.
 SCD Pathophysiology-Based Biomakers 1081

the vascular endothelium and on platelets, is increased in patients with frequent VOE
(see Table 2). The development and The United States Food and Drug Administration
(FDA) approval of Crizanlizumab, a monoclonal antibody against the adhesion mole-
cule P-selectin used to prevent VOE,10 is a success story of the clinical targeting of
an aspect of SCD pathophysiology associated with pain events in SCD.
Pro-inflammatory cytokines from the interleukin (IL) family, including IL-4 and IL-8,
facilitate neutrophil chemotaxis, perpetuating the ischemia–reperfusion cycle (see
Fig. 1).11 Several studies have shown correlations between pro-inflammatory circu-
lating interleukins (IL-4,8), associated with increased incidence of VOE; and anti-
inflammatory modulatory interleukins, IL-10, associated with reduced incidence of
VOE.9 CRP, ESR, and WBC levels are associated with frequent VOE.8 Serial measure-
ments would be needed to detect trends, however, as individual variability prevents
the use of diagnostic cutoff values.

Acute Chest Syndrome

ACS is characterized by fever or hypoxia, and new infiltrates on a chest X-ray. It is a
major cause of morbidity and mortality in children12 and adults with all subtypes of
SCD.13 Early inflammation in the alveolar bed, irrespective of trigger, infectious or
otherwise, leads to cytokine release, recruitment of neutrophils and macrophages,
capillary leak, and ventilation-perfusion mismatch. The mechanisms that predispose
and ultimately lead to the development of ACS include infection, antecedent painful
VOE, hypoventilation from aggressive pain control, hemolysis,14 thrombosis,15 and
micro- and macro-fat emboli.2 Treatment of ACS includes antibiotics, often transfu-
sions, and supportive care. Biomarkers that permit early identification of ACS in pa-
tients could identify those needing red cell transfusion before clinical deterioration.16
Biomarkers that associate with a predisposition to ACS include an elevated WBC, low
hemoglobin, and low HbF.17 Secretory phospholipase A2 (sPLA2) is an inflammatory
mediator proposed as a biomarker of ACS, but had a positive predictive value of only
27% when tested in a large multi-site study.16,18–25 Other inflammatory markers may
be potential biomarkers for ACS, including thrombospondin1 (TSP-1)26 and CD40
ligand (CD40 L), a pro-inflammatory modulator of endothelial cell activation (see
Table 2).27 Both TSP-1 and CD40 L are increased in individuals with a history of multiple
episodes of ACS. Their role as predictive biomarkers needs validation in larger trials.

Stroke and Silent Cerebral Infarcts

Before the institution of transfusion for primary stroke prevention, stroke occurred in
approximately 11% of individuals with SCD under the age of 20. Large-vessel SCD
stroke is the result of progressive vascular damage that causes stenosis.28 Large-
vessel stenosis is visible on cerebral angiography and can be inferred by elevated trans-
cranial Doppler ultrasound (TCD) velocities (see Table 2). Hb<9 g/dL is associated with
elevated stroke risk. In young children, acutely low hemoglobin in the setting of acute
febrile illness, or transient bone marrow aplasia, as seen with parvo-B-19 infections
carries an increased stroke risk. In high-resource countries, TCD screening and initia-
tion of chronic blood transfusions for those found to be at high risk are now part of
routine patient care, reducing incidence from 11% to approximately 1%. However,
some children identified as conditional TCDs do not progress29 and many children
who would not go on to have a stroke are identified as high risk. The number needed
to treat (chronically transfuse) to prevent one stroke is approximately 10.30; better pre-
dictive biomarkers are still needed. Clusters of biomarker signatures derived from the
analysis of the Cooperative Study of sickle cell disease (CSSCD) stroke risk have
been proposed.31 Several candidate single-nucleotide polymorphisms (SNPs) are
Descargado para Anonymous User (n/a) en Cayetano Heredia Pervuvian University de
ClinicalKey.es por Elsevier en diciembre 27, 2022. Para uso personal exclusivamente. No se
permiten otros usos sin autorización. Copyright ©2022. Elsevier Inc. Todos los derechos
reservados.
1082 Saah et al

associated with higher stroke risk, as well as plasma proteins a-2 antiplasmin,
thrombospondin-4, a-2 macroglobulin, apolipoprotein B-100, gelsolin, and retinol-
binding protein.32 All require further validation before they can be considered for use
in patient care.
Silent cerebral infarct (SCI) is the most common neurologic complication of SCD. Up
to a quarter of children with SCD under the age of 6 experience SCI, and SCI can be
detected in over a third of older adolescents under 18 years. As silent infarcts are only
identified by MRI (see Table 3),32,33 and imaging is not routinely performed, these
numbers are likely underestimated. Other associated biomarkers for increased SCI
risk include male gender, stenosis of the internal carotid artery, history of overt stroke,
low baseline hemoglobin, and elevated systolic blood pressures. Proteomic and multi-
omics studies have reported associations between silent infarcts and genes or pro-
teins involved in hypercoagulability, inflammation, and atherosclerosis.34

BIOMARKERS OF SICKLE CELL DISEASE CHRONIC COMPLICATIONS

SCD is a chronic progressive inflammatory disease resulting in end-organ damage.

Below, we will discuss affected organs for which biomarkers have been proposed
(see Table 3).

Sickle Cell Nephropathy

The kidney is extremely susceptible to injury in patients with SCD.13 The microcirculation
of the renal medulla is hypoxic, with low pH and hyperosmolality, increasing Hb polymer-
ization and sickling within the renal medulla, leading to repeated cycles of vaso-
occlusion, infarction, and vascular injury. In response to the ischemic environment,
vasodilatory substances are released, causing hyper-perfusion. Endothelial dysfunction,
progressive proteinuria, and progressive sickle-related nephropathy culminate in chronic
kidney disease or end-stage renal disease (ESRD) in up to 30% of adults with SCD.36
Biomarkers of sickle nephropathy include proteinuria and urine microalbuminuria/
creatinine ratio.35 The presence of microalbuminuria is seen more frequently in pa-
tients with a hemolytic phenotype37 and correlates with other markers of hemolysis:
low baseline hemoglobin, high reticulocyte counts, and increased LDH (see
Table 3). Microalbuminuria is a late manifestation of sickle nephropathy; biomarkers
that allow earlier detection of nephropathy are urgently needed.

Pulmonary Hypertension
PH may arise independently from repeated direct damage to the pulmonary vascular
bed, or be secondary to left ventricular dysfunction in SCD. Increased pulmonary
vascular resistance (PVR) occurs due to progressive destruction of pulmonary arteri-
oles over time, from repeated microinfarcts and thrombosis. Increased PVR perpetu-
ates increased afterload, left-sided cardiovascular dysfunction, and ultimately results
in cor pulmonale and early mortality.38 PH incidence correlates with markers of hemo-
lysis. Tricuspid regurgitant jet velocity (TRV) obtained by echocardiography has been
used as a biomarker for PH, but overestimates PH compared with the gold standard
diagnostic, right heart catheterization.2,7 With further validation, blood plasma levels of
N-terminal pro-brain natriuretic peptide (NT-proBNP) may be used in conjunction with
TRV to screen for PH in SCD (see Table 3).39

Avascular Necrosis
AVN typically occurs in the femoral and humeral heads; blood flows at an acute angle
through a single vessel, increasing the risk of ischemia.40 The high viscosity, high
Descargado para Anonymous User (n/a) en Cayetano Heredia Pervuvian University de
ClinicalKey.es por Elsevier en diciembre 27, 2022. Para uso personal exclusivamente. No se
permiten otros usos sin autorización. Copyright ©2022. Elsevier Inc. Todos los derechos
reservados.
 SCD Pathophysiology-Based Biomakers 1083

hemoglobin phenotype is particularly vulnerable to developing AVN. Platelet-derived

microparticles and endothelial microparticles may play a role in pathophysiologically
related hypercoagulability and microvascular thrombosis associated with osteonecro-
sis of the femoral head. Circulating plasma microparticle levels are about 2$5-fold
higher in patients with SCD than in healthy controls, and higher in individuals with
SCD with AVN compared with those without, suggesting that with validation micropar-
ticles may be a clinically useful biomarker of AVN.41

BIOMARKERS IN CLINICAL TRIALS AND PRECISION MEDICINE

There is an urgent need for objective, quantitative biomarkers of a pain event for use in
clinical trials and in patient care. Current SCD clinical trials typically have subjective
endpoints such as the number of VOE per year.32,42 Patients may struggle to differen-
tiate acute pain events from chronic pain exacerbations,11 and there are no biomarkers
that objectively diagnose a pain event.32 Several gene-based therapies are now in clin-
ical trials, but as with drug therapies, reduction in pain events is the most common study
endpoints.43–45 Diagnostic tests that determine if blood components are functionally
normal are urgently needed. Furthermore, the readouts should not just provide an aver-
aged value but should show the functionality of subpopulations or single cells in the
samples. Abnormal blood cells may be reduced in number or improved by editing,
but still be capable of causing long-term organ damage, morbidity, and early mortality.
We must perform detailed, single-cell level functional evaluations of blood components
from patients who have undergone gene-based therapy.
Biomarkers that provide a functional assessment of blood components are espe-
cially valuable in this new landscape of SCD therapies. Conventional lab tests are pri-
marily quantitative, but SCD is a qualitative blood disorder. For example, a normal
individual and an individual with HbSS after gene-based therapy may both have total
hemoglobin of 12 g/dL. However, there is concern that if the RBCs still damage the
vascular endothelium, and a population of poorly deformable RBCs is identified by
a cell deformability cytometry system, then these results suggest that cells have not
been fully normalized after gene treatment, and the disease cannot be considered
cured to the level of a successful full chimerism allogeneic hematopoietic stem cell
transplant (alloHSCT).46
In addition to hydroxyurea, there are now three new FDA-approved therapies for
SCD,47–49 and more novel therapies in clinical trials. These second-line agents target
specific aspects of SCD pathophysiology. Currently, there is no systematic way to
determine which of these agents should be added to hydroxyurea for an individual pa-
tient. These novel agents are expensive at $40,000 to 100,000 per patient per year,50
and not without risks and side effects.42,51,52 Consistent with the principles of preci-
sion medicine, we should choose the right drug for the individual based on functional
biomarkers that predict response to that drug.42 To do this, we need robust functional
testing that can collectively capture all aspects of SCD pathophysiology. These tests
should be evaluated not just for association with complications, but for evidence that
modifying the biomarker changes clinical outcomes. Functional biomarkers will allow
us to identify blood defects uncorrected by current therapy, then select the appro-
priate second agent based on our testing of the functional benefits individuals on
each therapy experienced, a perfect pairing of abnormality and correction.

RED CELL FUNCTION ASSAYS IN DEVELOPMENT AND CLINICAL VALIDATION

Sickle RBCs are poorly deformable and adhesive in the microcirculation,53–56 and
blood viscosity is elevated for the level of hemoglobin.53–55,57–60 Emerging
Descargado para Anonymous User (n/a) en Cayetano Heredia Pervuvian University de
ClinicalKey.es por Elsevier en diciembre 27, 2022. Para uso personal exclusivamente. No se
permiten otros usos sin autorización. Copyright ©2022. Elsevier Inc. Todos los derechos
reservados.
1084 Saah et al

technologies for in vitro biomarkers of RBC biophysical properties therefore include

measures of adhesion, deformability, and whole blood viscosity (WBV).17,53,55,61–63
In this section, emerging technologies for measuring functional RBC characteristics
are discussed (Table 4).

Red Blood Cell Deformability

Ektacytometry exposes RBCs in a viscous medium to shear stress. A laser beam
passes through the sample, producing an elliptical pattern by diffraction called elon-
gation index (EI), which is an indicator of cell deformability. Oxygen gradient ektacy-
tometry measures EI under a range of oxygen concentrations, generating an EImin,
deformability of the deoxygenated RBC, EImax, deformability of the oxygenated
RBC, and point of sickling (PoS), the oxygen level at which the deformability drops
due to hypoxia. Recently, EI was assessed as a clinical endpoint in one of the first
SCD patients cured by gene therapy and was shown to be associated with the risk
of VOE.46 A limitation of ektacytometry is that the significant cellular heterogeneity
within SCD patient blood samples may not be fully captured,64–66 following a curative
genetic therapy for example,
Microfluidic technologies can also be used to measure RBC deformability in a wide
range of conditions, through channels of physiologic, microcapillary size,66,67 or capil-
lary size openings.68 Some devices permit single cell or subpopulation detection of
deformability and incorporate hypoxia.66 A limitation of microfluidic deformability
measurements is the dependence on peripheral equipment, such as high-resolution
optical microscopy, complex microfabrication steps of microfluidic devices, and tech-
nically skilled personnel. An emerging strategy is to measure deformability as an oc-
clusion index (OI) of an artificial capillary bed as detected by electrical impedance.68
OI represents percent occlusion of a microcapillary network by poorly deformable
RBCs and has been shown to associate with disease severity in SCD.69,70 Electronic
readouts and comprehensive prospective clinical validation are needed to advance
these technologies as primary biomarker endpoints for new therapies in SCD (see
Table 4).

Red Blood Cell Adhesion

RBC adhesion to endothelium is associated with disease activity,71 and may diminish
with treatment in SCD.72 Key studies have shown that RBC adhesion, WBC adhe-
sion,73 and aberrant endothelial activation contribute to the pathogenesis of
SCD74–77 and associate with disease severity.71,78,79 Measurement by flow cytometry
of aberrant surface molecule expression or activation has served as a surrogate for
directly measuring abnormal adhesion.80–82 Studies on cellular adhesion in SCD tradi-
tionally relied on open systems with static conditions, endothelialized parallel plate
flow chambers, or endothelialized microchannels.71,78,83–87 These complex and
labor-intensive techniques have not been successfully used in longitudinal analyses
or in large-scale multi-site clinical trials to date. Standardized microfluidic adhesion
assays have been developed, analytically, and clinically validated to measure
abnormal RBC adhesion in whole blood samples and have reported associations
with clinical phenotypes.55,56,61,88–97 For example, RBC adhesion to subendothelial
proteins is significantly greater in SCD than in nonanemic controls. Following in vitro
exposure to hypoxia, sickle RBCs show greater adhesion, whereas HbF-containing
sRBCs (>8% HbF) show lesser adhesion. Sickle RBC adhesion changes heteroge-
neously with acute pain,98 WBC adhesion increases during painful episodes,99 and
decreases following anti-adhesive therapy.100 Endothelium-on-a-chip microfluidic
systems can be activated with patient-specific plasma, to better recapitulate real-
Descargado para Anonymous User (n/a) en Cayetano Heredia Pervuvian University de
ClinicalKey.es por Elsevier en diciembre 27, 2022. Para uso personal exclusivamente. No se
permiten otros usos sin autorización. Copyright ©2022. Elsevier Inc. Todos los derechos
reservados.
 Table 4
Biophysical biomarkers and devices

SCD Analytical Clinical Limitation

Biomarker Device Pathophysiology Validation Validation Current Use and Need Future Use
RBC LORRCA EI as an Analytically Clinical Exploratory RBC Unable to Primary endpoint
ClinicalKey.es por Elsevier en diciembre 27, 2022. Para uso personal exclusivamente. No se
permiten otros usos sin autorización. Copyright ©2022. Elsevier Inc. Todos los derechos

Elongation Oxygen gradient indicator of validated associations biomarker in resolve in RBC modifying
Descargado para Anonymous User (n/a) en Cayetano Heredia Pervuvian University de

Index (EI) ektacytometry RBC stiffness and laboratory reported clinical trials single-cell therapy clinical trials
Point of sickling under method in the level RBC
Sickling (PoS) hypoxia literature heterogeneity
Technical
complexity
and cost
reduction
are needed
RBC Occlusion Microfluidic RBC-mediated Analytical Clinical Exploratory RBC New Primary endpoint
Index (OI) OcclusionChip microcapillary validation, associations biomarker in microtechnology in RBC deformability
Assay occlusion in reproducibility reported clinical studies works with optical modifying therapy
reservados.

Microfluidic normoxia results in the microscopy clinical trials

Impedance and hypoxia presented literature Rapid electronic

SCD Pathophysiology-Based Biomakers

Red Cell Assay in the readout and
(MIRCA) literature more clinical
validation
are needed
RBC Adhesion SCD BioChip Abnormal cellular Analytically Clinical Identifying Dependent on Primary endpoint in
Index Adhesion Assay adhesion validated associations subpopulations high-resolution blood cell adhesion
Off-the-shelf to endothelium- reported of adherent optical modifying therapy
Microfluidic associated in the RBCs and microscopy clinical trials
Cell Adhesion proteins or literature reporting the More clinical
Assays endothelial cells result as validation in
Endothelium- an adhesion prospective
on-a-Chip index studies is needed
Adhesion
Assay

(continued on next page)

1085
 1086
Saah et al
Table 4
(continued )
SCD Analytical Clinical Limitation
ClinicalKey.es por Elsevier en diciembre 27, 2022. Para uso personal exclusivamente. No se
permiten otros usos sin autorización. Copyright ©2022. Elsevier Inc. Todos los derechos

Descargado para Anonymous User (n/a) en Cayetano Heredia Pervuvian University de

Biomarker Device Pathophysiology Validation Validation Current Use and Need Future Use
RBC Density ADVIA RBC density Analytically Clinical Exploratory use in Single-cell Measure effects
(g/dL) MagDense measurement validated associations clinical studies resolution RBC of therapies
Magnetic levitation reported density on red cell
microfluidic assay in the measurement hydration
literature is needed;
hypoxia needed
RBC and Cone and plate Plasma and Analytically Clinical Exploratory Viscosity Primary endpoint
Whole viscometer whole blood validated associations use in measurement in RBC and blood
Blood SCD BioChip viscosity reported clinical studies in normoxia and viscosity modifying
Viscosity Microfluidic measurement in the hypoxia therapy clinical trials
reservados.

(centipoise, Viscosity Assay RBC and literature are needed

cP) blood viscosity Need more clinical
measurement in validation in
hypoxia prospective studies
and normoxia
 SCD Pathophysiology-Based Biomakers 1087

world physiology.61 Assessing whether a therapeutic can reduce an adhesive interac-

tion between blood cells and activated endothelium is a key feature of these in vitro
biomarker assays.94,95 Prospective clinical studies are needed to comprehensively
validate these technologies as clinical biomarkers for SCD (see Table 4).

Red Blood Cell Density

HbS polymerization may result in dehydration of RBCs and subsequent RBC density
increase, via increased cation permeability due to activation of transport channels
such as the Na1/K1/2Cl-cotransporter 1 ([NKCC1]), Gardos channels, and Psickle
channels.96,101–103 Rapid HbS polymerization is inversely proportional to the intracel-
lular concentration of HbS;38 therefore, increased cell density due to dehydration can
catalyze the HbS polymerization and sRBC sickling. Sickle RBCs have a significantly
higher density (>1.11 g/mL) compared with HbA-containing RBCs.104,105 Elevated
RBC density in SCD has been associated with hemolysis and comorbidities such as
skin ulcers, renal failure, and priapism.106 In addition, animal studies have shown
that vascular occlusion may be initiated by a small number of highly dense
RBCs,107 which is consistent with reports of increased presence of dense RBCs in in-
dividuals with SCD during painful VOCs.108 RBC density can be measured by con-
structing a density gradient and allowing the RBC to equilibrate.106,108,109 However,
this method is time-consuming and does not allow accurate density measurements
or assessment of the heterogeneity of RBCs at the single-cell level. The ADVIA hema-
tology analyzer can make a binary classification of red blood cells by density.110 A
magnetic levitation-based microfluidic assay was developed to produce a continuous
density gradient with a minimum resolvable density change of 0.0001 g/mL.111 Micro-
scope images of the capillary channel can be post-processed to obtain additional in-
formation about the RBCs (ie, size). RBC density needs to be further validated in
clinical studies as a biomarker of SCD.

Viscosity
Polymerization of HbS leads to impaired RBC deformability and altered RBC
morphology, particularly in hypoxia, contributing to significant changes in blood
rheology and WBV.62,65,112,113 RBC deformability and hematocrit (Hct) together deter-
mine WBV.114–116 For example, low WBV because of low Hct in normoxia could lead to
reduced endothelial shear stress and endothelial activation.117 Hypoxia acutely in-
creases WBV in HbSS, which could contribute to local occlusion and ischemia. A
change in blood viscosity is considered a key clinical parameter in evaluating treat-
ment outcomes in many hematologic disorders.62,118–120 High fibrinogen levels have
been shown to associate with high blood plasma viscosity and may play a role in blood
rheology abnormalities in SCD and SCT.120,121 Cone and plate viscometers are typi-
cally used to measure plasma viscosity and WBV, which only allows measurements
in normoxia, as the blood sample is in contact with room air. A new microfluidic assay
was developed based on the micro-particle imaging velocimetry technique to mea-
sure RBC velocities under physiologic pressure gradients and normoxic/hypoxic con-
ditions.122 Under normoxia, an inverse association between WBV and sickle RBC
adhesion was shown, and an acute rise in viscosity under hypoxia (SpO2 of 83%)
was reported, which reflects acute-on-chronic pathophysiology.122 Using microfluidic
biomarker assays, it was shown that sickle RBC adhesion to ICAM-1 is mediated by
fibrinogen and associated inversely with %HbF, and directly with a history of right-to-
left shunts.122 Inclusion of RBC and WBV measurement before and after therapy as a
primary outcome measure could objectively show the impact of blood rheology modi-
fying therapies. Future work could focus on including novel emerging biomarkers as
Descargado para Anonymous User (n/a) en Cayetano Heredia Pervuvian University de
ClinicalKey.es por Elsevier en diciembre 27, 2022. Para uso personal exclusivamente. No se
permiten otros usos sin autorización. Copyright ©2022. Elsevier Inc. Todos los derechos
reservados.
1088 Saah et al

part of clinical studies and clinical trials to validate and determine the associations be-
tween the disease state and treatment response.

SUMMARY

One of the challenges in SCD is its clinical variability. Our ability to identify the compli-
cations that a patient is at risk for is limited by a lack of validated diagnostic and prog-
nostic biomarkers. Clinical care is limited by a lack of diagnostics to capture the
biological variability needed to precisely direct patient care. Many biomarkers have
been proposed, but few validated. We must make a concerted effort as a field to rigor-
ously test proposed biomarkers to improve outcomes for our patients.

CLINICS CARE POINTS

 SCD biomarkers are needed to serve as endpoints in clinical trials, select optimal therapies,
and monitor efficacy.
 Many molecular and biophysical biomarkers have been proposed, but few have been
clinically validated.
 In order to advance the translation of biomarkers to clinical use, we must rigorously test
proposed biomakers for their ability to predict clinical outcomes.

REFERENCES

1. Lebrun-Harris LA, McManus MA, Ilango SM, et al. Transition Planning Among
US Youth With and Without Special Health Care Needs. Pediatrics 2018;
142(4). https://doi.org/10.1542/peds.2018-0194.
2. Kalpatthi R, Novelli EM. Measuring success: utility of biomarkers in sickle cell
disease clinical trials and care. Hematol Am Soc Hematol Educ Program
2018;2018(1):482–92.
3. BEST (Biomarkers, EndpointS, and other Tools) Resource. 2016. Available at:
https://www.fdanews.com/ext/resources/files/2020/11-24-20-BEST.pdf?
1606261388.
4. Odenheimer DJ, Sarnaik SA, Whitten CF, Rucknagel DL, Sing CF. The relation-
ship between fetal hemoglobin and disease severity in children with sickle
cell anemia. Am J Med Genet 1987;27(3):525–35.
5. Akinsheye I, Alsultan A, Solovieff N, et al. Fetal hemoglobin in sickle cell anemia.
Blood 2011;118(1):19–27.
6. Aliyu ZY, Kato GJ, Taylor Jt, et al. Sickle cell disease and pulmonary hyperten-
sion in Africa: a global perspective and review of epidemiology, pathophysi-
ology, and management. Am J Hematol 2008;83(1):63–70.
7. Gladwin MT, Sachdev V, Jison ML, et al. Pulmonary hypertension as a risk factor
for death in patients with sickle cell disease. N Engl J Med 2004;350(9):886–95.
8. Krishnan S, Setty Y, Betal SG, et al. Increased levels of the inflammatory
biomarker C-reactive protein at baseline are associated with childhood sickle
cell vasocclusive crises. Br J Haematol 2010;148(5):797–804.
9. Rees DC, Gibson JS. Biomarkers in sickle cell disease. Br J Haematol 2012;
156(4):433–45.
10. Ataga KI, Kutlar A, Kanter J, et al. Crizanlizumab for the Prevention of Pain Cri-
ses in Sickle Cell Disease. N Engl J Med 2017;376(5):429–39.
Descargado para Anonymous User (n/a) en Cayetano Heredia Pervuvian University de
ClinicalKey.es por Elsevier en diciembre 27, 2022. Para uso personal exclusivamente. No se
permiten otros usos sin autorización. Copyright ©2022. Elsevier Inc. Todos los derechos
reservados.
 SCD Pathophysiology-Based Biomakers 1089

11. Darbari DS, Sheehan VA, Ballas SK. The vaso-occlusive pain crisis in sickle cell
disease: Definition, pathophysiology, and management. Eur J Haematol 2020;
105(3):237–46.
12. Vichinsky EP, Styles LA, Colangelo LH, Wright EC, Castro O, Nickerson B. Acute
chest syndrome in sickle cell disease: clinical presentation and course. Cooper-
ative Study of Sickle Cell Disease. Blood 1997;89(5):1787–92.
13. Platt OS, Brambilla DJ, Rosse WF, et al. Mortality in sickle cell disease. Life ex-
pectancy and risk factors for early death. Research Support, U.S. Gov’t, P.H.S.
N Engl J Med 1994;330(23):1639–44.
14. Adisa OA, Hu Y, Ghosh S, Aryee D, Osunkwo I, Ofori-Acquah SF. Association
between plasma free haem and incidence of vaso-occlusive episodes and
acute chest syndrome in children with sickle cell disease. Br J Haematol
2013;162(5):702–5.
15. Ataga KI, Brittain JE, Desai P, et al. Association of coagulation activation with
clinical complications in sickle cell disease. PLoS One 2012;7(1):e29786.
16. Styles LA, Abboud M, Larkin S, Lo M, Kuypers FA. Transfusion prevents acute
chest syndrome predicted by elevated secretory phospholipase A2. Br J Hae-
matol 2007;136(2):343–4.
17. Alkindi S, Al-Busaidi I, Al-Salami B, Raniga S, Pathare A, Ballas SK. Predictors of
impending acute chest syndrome in patients with sickle cell anaemia. Sci Rep
2020;10(1):2470.
18. Styles LA, Aarsman AJ, Vichinsky EP, Kuypers FA. Secretory phospholipase
A(2) predicts impending acute chest syndrome in sickle cell disease. Blood
2000;96(9):3276–8.
19. Kuypers FA, Styles LA. The role of secretory phospholipase A2 in acute chest
syndrome. Cell Mol Biol (Noisy-le-grand) 2004;50(1):87–94.
20. Naprawa JT, Bonsu BK, Goodman DG, Ranalli MA. Serum biomarkers for iden-
tifying acute chest syndrome among patients who have sickle cell disease and
present to the emergency department. Pediatrics 2005;116(3):e420–5.
21. Bargoma EM, Mitsuyoshi JK, Larkin SK, Styles LA, Kuypers FA, Test ST. Serum
C-reactive protein parallels secretory phospholipase A2 in sickle cell disease
patients with vasoocclusive crisis or acute chest syndrome. Blood 2005;
105(8):3384–5.
22. Ballas SK, Files B, Luchtman-Jones L, et al. Secretory phospholipase A2 levels
in patients with sickle cell disease and acute chest syndrome. Hemoglobin
2006;30(2):165–70.
23. Styles L, Wager CG, Labotka RJ, et al. Refining the value of secretory phospho-
lipase A2 as a predictor of acute chest syndrome in sickle cell disease: results
of a feasibility study (PROACTIVE). Br J Haematol 2012;157(5):627–36.
24. Miller ST, Kim HY, Weiner D, et al. Inpatient management of sickle cell pain: a
’snapshot’ of current practice. Am J Hematol 2012;87(3):333–6.
25. Ball JB, Khan SY, McLaughlin NJ, et al. A two-event in vitro model of acute chest
syndrome: the role of secretory phospholipase A2 and neutrophils. Pediatr
Blood Cancer 2012;58(3):399–405.
26. Novelli EM, Kato GJ, Hildesheim ME, et al. Thrombospondin-1 inhibits
ADAMTS13 activity in sickle cell disease. Haematologica 2013;98(11):e132–4.
27. Garrido VT, Sonzogni L, Mtatiro SN, Costa FF, Conran N, Thein SL. Association
of plasma CD40L with acute chest syndrome in sickle cell anemia. Cytokine
2017;97:104–7.
28. Hebbel RP, Belcher JD, Vercellotti GM. The multifaceted role of ischemia/reper-
fusion in sickle cell anemia. J Clin Invest 2020;130(3):1062–72.
Descargado para Anonymous User (n/a) en Cayetano Heredia Pervuvian University de
ClinicalKey.es por Elsevier en diciembre 27, 2022. Para uso personal exclusivamente. No se
permiten otros usos sin autorización. Copyright ©2022. Elsevier Inc. Todos los derechos
reservados.
1090 Saah et al

29. Hankins JS, McCarville MB, Rankine-Mullings A, et al. Prevention of conversion

to abnormal transcranial Doppler with hydroxyurea in sickle cell anemia: A
Phase III international randomized clinical trial. Am J Hematol 2015;90(12):
1099–105.
30. Buchanan GR, DeBaun MR, Quinn CT, Steinberg MH. Sickle cell disease. Hem-
atol Am Soc Hematol Educ Program 2004;35–47.
31. Du M, Van Ness S, Gordeuk V, et al. Biomarker signatures of sickle cell disease
severity. Blood Cells Mol Dis 2018;72:1–9.
32. Farrell AT, Panepinto J, Carroll CP, et al. End points for sickle cell disease clinical
trials: patient-reported outcomes, pain, and the brain. Blood Adv 2019;3(23):
3982–4001.
33. DeBaun MR, Gordon M, McKinstry RC, et al. Controlled trial of transfusions for
silent cerebral infarcts in sickle cell anemia. N Engl J Med 2014;371(8):699–710.
34. Goodman SR, Pace BS, Hansen KC, et al. Minireview: Multiomic candidate bio-
markers for clinical manifestations of sickle cell severity: Early steps to precision
medicine. Exp Biol Med (Maywood) 2016;241(7):772–81.
35. Yawn BP, Buchanan GR, Afenyi-Annan AN, et al. Management of sickle cell dis-
ease: summary of the 2014 evidence-based report by expert panel members.
Jama 2014;312(10):1033–48.
36. Naik RP, Derebail VK. The spectrum of sickle hemoglobin-related nephropathy:
from sickle cell disease to sickle trait. Expert Rev Hematol 2017;10(12):1087–94.
37. Ofori-Acquah SF. Sickle cell disease as a vascular disorder. Expert Rev Hematol
2020;13(6):645–53.
38. Sundd P, Gladwin MT, Novelli EM. Pathophysiology of Sickle Cell Disease. Annu
Rev Pathol 2019;14:263–92.
39. Garadah T, Mandeel F, Jaradat A, Bin Thani K. The Effects of Hydroxyurea Ther-
apy on the Six-Minute Walk Distance in Patients with Adult Sickle Cell Anemia:
An Echocardiographic Study. J Blood Med 2019;10:443–52.
40. Naseer ZA, Bachabi M, Jones LC, Sterling RS, Khanuja HS. Osteonecrosis in
Sickle Cell Disease. South Med J 2016;109(9):525–30.
41. Marsh A, Schiffelers R, Kuypers F, et al. Microparticles as biomarkers of osteo-
necrosis of the hip in sickle cell disease. Br J Haematol 2015;168(1):135–8.
42. Dela-Pena JC, King MA, Brown J, Nachar VR. Incorporation of novel therapies
for the management of sickle cell disease: A pharmacist’s perspective.
J Oncol Pharm Pract 2022. https://doi.org/10.1177/10781552211072468.
10781552211072468.
43. Kanter J, Walters MC, Krishnamurti L, et al. Biologic and Clinical Efficacy of Len-
tiGlobin for Sickle Cell Disease. N Engl J Med 2021. https://doi.org/10.1056/
NEJMoa2117175.
44. Esrick EB, Lehmann LE, Biffi A, et al. Post-Transcriptional Genetic Silencing of
BCL11A to Treat Sickle Cell Disease. N Engl J Med 2021;384(3):205–15.
45. Frangoul H, Altshuler D, Cappellini MD, et al. CRISPR-Cas9 Gene Editing for
Sickle Cell Disease and beta-Thalassemia. N Engl J Med 2021;384(3):252–60.
46. Rab MAE, Kanne CK, Bos J, et al. Oxygen gradient ektacytometry-derived bio-
markers are associated with vaso-occlusive crises and correlate with treatment
response in sickle cell disease. Am J Hematol 2021;96(1):E29–32.
47. Vichinsky E, Hoppe CC, Ataga KI, et al. A Phase 3 Randomized Trial of Voxelotor
in Sickle Cell Disease. N Engl J Med 2019;381(6):509–19.
48. Niihara Y, Miller ST, Kanter J, et al. A Phase 3 Trial of l-Glutamine in Sickle Cell
Disease. N Engl J Med 2018;379(3):226–35.
Descargado para Anonymous User (n/a) en Cayetano Heredia Pervuvian University de
ClinicalKey.es por Elsevier en diciembre 27, 2022. Para uso personal exclusivamente. No se
permiten otros usos sin autorización. Copyright ©2022. Elsevier Inc. Todos los derechos
reservados.
 SCD Pathophysiology-Based Biomakers 1091

49. Field JJ, Majerus E, Ataga KI, et al. NNKTT120, an anti-iNKT cell monoclonal
antibody, produces rapid and sustained iNKT cell depletion in adults with sickle
cell disease. PLoS One 2017;12(2):e0171067.
50. Langley PC. More Unnecessary Imaginary Worlds - Part 4: The ICER Evidence
Report for Crizanlizumab, Voxelotor and L-Glutamine for Sickle Cell Disease. In-
nov Pharm 2020;11(2). https://doi.org/10.24926/iip.v11i2.3123.
51. Karkoska K, Quinn CT, Clapp K, McGann PT. Severe infusion-related reaction to
crizanlizumab in an adolescent with sickle cell disease. Am J Hematol 2020;
95(12):E338–9.
52. Lee M, Stringer T, Jacob J, et al. First case of DRESS (drug reaction with eosin-
ophilia and systemic symptoms) associated with voxelotor. Am J Hematol 2021;
96(11):E436–9.
53. Moreno-Smith M, Lakoma A, Chen Z, et al. p53 Nongenotoxic Activation and
mTORC1 Inhibition Lead to Effective Combination for Neuroblastoma Therapy.
Clin Cancer Res 2017;23(21):6629–39.
54. Barabino GA, Platt MO, Kaul DK. Sickle cell biomechanics. Annu Rev Biomed
Eng 2010;12:345–67.
55. Alapan Y, Fraiwan A, Kucukal E, et al. Emerging point-of-care technologies for
sickle cell disease screening and monitoring. Expert Rev Med Devices 2016;
13(12):1073–93.
56. Alapan Y, Little JA, Gurkan UA. Heterogeneous red blood cell adhesion and de-
formability in sickle cell disease. Sci Rep 2014;4:7173.
57. Brittenham GM, Schechter AN, Noguchi CT. Hemoglobin S polymerization: pri-
mary determinant of the hemolytic and clinical severity of the sickling syn-
dromes. Blood 1985;65(1):183–9.
58. Dong C, Chadwick RS, Schechter AN. Influence of sickle hemoglobin polymer-
ization and membrane properties on deformability of sickle erythrocytes in the
microcirculation. Biophysical J 1992;63(3):774–83.
59. Manwani D, Frenette PS. Vaso-occlusion in sickle cell disease: pathophysiology
and novel targeted therapies. Blood 2013;122(24):3892–8.
60. Sandor B, Marin M, Lapoumeroulie C, et al. Effects of Poloxamer 188 on red
blood cell membrane properties in sickle cell anaemia. Br J Haematol 2016;
173(1):145–9.
61. Kucukal E, Ilich A, Key NS, Little JA, Gurkan UA. Red Blood Cell Adhesion to
Heme-Activated Endothelial Cells Reflects Clinical Phenotype in Sickle Cell Dis-
ease. Am J Hematol 2018. https://doi.org/10.1002/ajh.25159.
62. Yuan C, Quinn E, Kapoor S, et al. Hypoxia Enhanced Red Cell Adhesion in Vitro
May Identify Patients at Risk for Vasculopathy. Blood 2018;132. https://doi.org/
10.1182/blood-2018-99-120207.
63. Nader E, Romana M, Guillot N, et al. Association Between Nitric Oxide, Oxida-
tive Stress, Eryptosis, Red Blood Cell Microparticles, and Vascular Function in
Sickle Cell Anemia. Front Immunol 2020;11:551441.
64. Sosa JM, Nielsen ND, Vignes SM, Chen TG, Shevkoplyas SS. The relationship
between red blood cell deformability metrics and perfusion of an artificial micro-
vascular network. Clin Hemorheol Microcirc 2014;57(3):275–89.
65. Li X, Dao M, Lykotrafitis G, Karniadakis GE. Biomechanics and biorheology of
red blood cells in sickle cell anemia. J Biomech 2017;50:34–41.
66. Guruprasad P, Mannino RG, Caruso C, et al. Integrated automated particle
tracking microfluidic enables high-throughput cell deformability cytometry for
red cell disorders. Am J Hematol 2019;94(2):189–99.
Descargado para Anonymous User (n/a) en Cayetano Heredia Pervuvian University de
ClinicalKey.es por Elsevier en diciembre 27, 2022. Para uso personal exclusivamente. No se
permiten otros usos sin autorización. Copyright ©2022. Elsevier Inc. Todos los derechos
reservados.
1092 Saah et al

67. Lu M, Kanne CK, Reddington RC, Lezzar DL, Sheehan VA, Shevkoplyas SS.
Concurrent Assessment of Deformability and Adhesiveness of Sickle Red Blood
Cells by Measuring Perfusion of an Adhesive Artificial Microvascular Network.
Front Physiol 2021;12:633080.
68. Man Y, Maji D, An R, et al. Microfluidic electrical impedance assessment of red
blood cell-mediated microvascular occlusion. 10.1039/D0LC01133A. Lab A
Chip 2021;21(6):1036–48.
69. Man Y, Kucukal E, An R, et al. Microfluidic assessment of red blood cell medi-
ated microvascular occlusion. Lab Chip 2020;20(12):2086–99.
70. Man Y, Kucukal E, An R, Bode A, Little JA, Gurkan UA. Standardized microflui-
dic assessment of red blood cell mediated microcapillary occlusion: association
with clinical phenotype and hydroxyurea responsiveness in sickle cell disease.
Microcirculation 2020;e12662. https://doi.org/10.1111/micc.12662.
71. Hebbel RP, Boogaerts MA, Eaton JW, Steinberg MH. Erythrocyte adherence to
endothelium in sickle-cell anemia. A possible determinant of disease severity.
N Engl J Med 1980;302(18):992–5.
72. Hillery CA, Du MC, Wang WC, Scott JP. Hydroxyurea therapy decreases the
in vitro adhesion of sickle erythrocytes to thrombospondin and laminin. Br J
Haematol 2000;109(2):322–7.
73. Turhan A, Weiss LA, Mohandas N, Coller BS, Frenette PS. Primary role for
adherent leukocytes in sickle cell vascular occlusion: a new paradigm. Proc
Natl Acad Sci U S A 2002;99(5):3047–51.
74. Kaul DK, Fabry ME, Nagel RL. Microvascular sites and characteristics of sickle
cell adhesion to vascular endothelium in shear flow conditions: pathophysiolog-
ical implications. Proc Natl Acad Sci U S A 1989;86(9):3356–60.
75. Kaul DK, Finnegan E, Barabino GA. Sickle Red Cell-Endothelium Interactions.
Microcirculation 2009;16(1):97–111.
76. Matsui NM, Borsig L, Rosen SD, Yaghmai M, Varki A, Embury SH. P-selectin me-
diates the adhesion of sickle erythrocytes to the endothelium. Blood 2001;98(6):
1955–62.
77. Matsui NM, Varki A, Embury SH. Heparin inhibits the flow adhesion of sickle red
blood cells to P-selectin. Blood 2002;100(10):3790–6.
78. Hebbel RP. Adhesive interactions of sickle erythrocytes with endothelium. J Clin
Invest 1997;100(11):S83–6.
79. Ballas SK, Smith ED. Red blood cell changes during the evolution of the sickle
cell painful crisis. Blood 1992;79(8):2154–63.
80. Styles LA, Lubin B, Vichinsky E, et al. Decrease of very late activation antigen-4
and CD36 on reticulocytes in sickle cell patients treated with hydroxyurea. Blood
1997;89(7):2554–9.
81. Manwani D, Chen G, Carullo V, et al. Single-dose intravenous gammaglobulin
can stabilize neutrophil Mac-1 activation in sickle cell pain crisis. Am J Hematol
2015;90(5):381–5.
82. Picot J, Goudot C, Berkenou J, et al. Flow cytometry analyses reveal association
between Lu/BCAM adhesion molecule and osteonecrosis in sickle cell disease.
Am J Hematol 2014;89(1):115–7.
83. Barabino GA, McIntire LV, Eskin SG, Sears DA, Udden M. Endothelial cell inter-
actions with sickle cell, sickle trait, mechanically injured, and normal erythro-
cytes under controlled flow. Research Support, Non-U.S. Gov’t Research
Support, U.S. Gov’t, P.H.S. Blood 1987;70(1):152–7.
Descargado para Anonymous User (n/a) en Cayetano Heredia Pervuvian University de
ClinicalKey.es por Elsevier en diciembre 27, 2022. Para uso personal exclusivamente. No se
permiten otros usos sin autorización. Copyright ©2022. Elsevier Inc. Todos los derechos
reservados.
 SCD Pathophysiology-Based Biomakers 1093

84. Tsai M, Kita A, Leach J, et al. In vitro modeling of the microvascular occlusion
and thrombosis that occur in hematologic diseases using microfluidic technol-
ogy. J Clin Invest 2012;122(1):408–18.
85. White J, Lancelot M, Sarnaik S, Hines P. Increased erythrocyte adhesion to
VCAM-1 during pulsatile flow: Application of a microfluidic flow adhesion
bioassay. Clin Hemorheol Microcirc 2015;60(2):201–13.
86. White J, Krishnamoorthy S, Gupta D, et al. VLA-4 blockade by natalizumab in-
hibits sickle reticulocyte and leucocyte adhesion during simulated blood flow.
Br J Haematol 2016;174(6):970–82.
87. Lancelot M, White J, Sarnaik S, Hines P. Low molecular weight heparin inhibits
sickle erythrocyte adhesion to VCAM-1 through VLA-4 blockade in a standard-
ized microfluidic flow adhesion assay. Br J Haematol 2017;178(3):479.
88. Gurkan UA. Biophysical and rheological biomarkers of red blood cell physiology
and pathophysiology. Curr Opin Hematol 2021;28(3):138–49.
89. Alapan Y, Kim C, Adhikari A, et al. Sickle cell disease biochip: a functional red
blood cell adhesion assay for monitoring sickle cell disease. Transl Res 2016;
173:74–91.e8.
90. Alapan Y, Matsuyama Y, Little JA, Gurkan UA. Dynamic deformability of sickle
red blood cells in microphysiological flow. Technology 2016;4(2):71–9.
91. Kim M, Alapan Y, Adhikari A, Little JA, Gurkan UA. Hypoxia-enhanced adhesion
of red blood cells in microscale flow. Microcirculation 2017;24(5). https://doi.org/
10.1111/micc.12374.
92. Kucukal E, Little JA, Gurkan UA. Shear dependent red blood cell adhesion in
microscale flow. Integr Biol 2018;10(4):194–206.
93. Kucukal E, Man Y, Hill A, et al. Whole blood viscosity and red blood cell adhe-
sion: Potential biomarkers for targeted and curative therapies in sickle cell dis-
ease. Am J Hematol 2020;95:1246–56.
94. Kucukal E, Man Y, Quinn E, et al. Red blood cell adhesion to ICAM-1 is mediated
by fibrinogen and is associated with right-to-left shunts in sickle cell disease.
Blood Adv 2020;4(15):3688–98.
95. Man Y, Goreke U, Kucukal E, et al. Leukocyte adhesion to P-selectin and the
inhibitory role of Crizanlizumab in sickle cell disease: A standardized microflui-
dic assessment. Blood Cells Mol Dis 2020;83:102424.
96. Noomuna P, Risinger M, Zhou S, et al. Inhibition of Band 3 tyrosine phosphory-
lation: a new mechanism for treatment of sickle cell disease. Br J Haematol
2020;190(4):599–609.
97. Yuan C, Quinn E, Kucukal E, Kapoor S, Gurkan UA, Little JA. Priapism, hemo-
globin desaturation, and red blood cell adhesion in men with sickle cell anemia.
Blood Cells Mol Dis 2019;79:102350.
98. Boye-Doe A, Little J. Red Blood Cell Adhesion in Adult Patients with Sickle Cell
Disease, at Baseline and with Pain, Measured on SCD Biochip Microfluidic
Assay. Blood 2018;132(Supplement 1):2386.
99. Pittman DD, Hines PC, Beidler D, et al. Evaluation of Longitudinal Pain Study in
Sickle Cell Disease (ELIPSIS) by patient-reported outcomes, actigraphy, and
biomarkers. Blood 2021;137(15):2010–20.
100. Albo C, Kumar S, Pope M, et al. Characteristics and potential biomarkers of
adult sickle cell patients with chronic pain. Eur J Haematol 2020;105(4):419–25.
101. Lew VL, Bookchin RM. Ion transport pathology in the mechanism of sickle cell
dehydration. Physiol Rev 2005;85(1):179–200.
Descargado para Anonymous User (n/a) en Cayetano Heredia Pervuvian University de
ClinicalKey.es por Elsevier en diciembre 27, 2022. Para uso personal exclusivamente. No se
permiten otros usos sin autorización. Copyright ©2022. Elsevier Inc. Todos los derechos
reservados.
1094 Saah et al

102. Lew VL, Tiffert T. On the Mechanism of Human Red Blood Cell Longevity: Roles
of Calcium, the Sodium Pump, PIEZO1, and Gardos Channels. Mini Review.
Front Physiol 2017;8(977). https://doi.org/10.3389/fphys.2017.00977.
103. Zheng S, Krump NA, McKenna MM, et al. Regulation of erythrocyte Na(1)/K(1)/
2Cl(-) cotransport by an oxygen-switched kinase cascade. J Biol Chem 2019;
294(7):2519–28.
104. Horiuchi K, Stephens MJ, Adachi K, Asakura T, Schwartz E, Ohene-
Frempong K. Image analysis studies of the degree of irreversible deformation
of sickle cells in relation to cell density and Hb F level. Br J Haematol 1993;
85(2):356–64.
105. Rakotoson MG, Di Liberto G, Audureau E, et al. Biological parameters predictive
of percent dense red blood cell decrease under hydroxyurea. Orphanet J rare
Dis 2015;10:57.
106. Bartolucci P, Brugnara C, Teixeira-Pinto A, et al. Erythrocyte density in sickle cell
syndromes is associated with specific clinical manifestations and hemolysis.
Blood 2012;120(15):3136–41.
107. Kaul DK, Fabry ME. In vivo studies of sickle red blood cells. Microcirculation
2004;11(2):153–65.
108. Warth JA, Rucknagel DL. Density ultracentrifugation of sickle cells during and
after pain crisis: increased dense echinocytes in crisis. Blood 1984;64(2):
507–15.
109. Kumar AA, Patton MR, Hennek JW, et al. Density-based separation in multi-
phase systems provides a simple method to identify sickle cell disease. Proc
Natl Acad Sci 2014;111(41):14864–9.
110. Sadaf A, Quinn CT, Korpik JB, et al. Rapid and automated quantitation of dense
red blood cells: A robust biomarker of hydroxyurea treatment response. Blood
Cell Mol Dis 2021;90:102576.
111. Goreke U, Bode A, Yaman S, Gurkan UA, Durmus NG. Size and density mea-
surements of single sickle red blood cells using microfluidic magnetic levitation.
Lab Chip 2022;22(4):683–96.
112. Dufu K, Patel M, Oksenberg D, Cabrales P. GBT440 improves red blood cell de-
formability and reduces viscosity of sickle cell blood under deoxygenated con-
ditions. Clin Hemorheol Microcirc 2018;70(1):95–105.
113. Chien S, Usami S, Bertles JF. Abnormal rheology of oxygenated blood in sickle
cell anemia. J Clin Invest 1970;49(4):623–34.
114. Chen CF, Jia HY, Ma HW, Wang DY, Guo SS, Qu S. Rheologic determinant
changes of erythrocytes in Binswanger’s disease. Zhonghua Yi Xue Za Zhi 5
Chin Med J Free China ed 1999;62(2):76–85.
115. Pop GA, Duncker DJ, Gardien M, et al. The clinical significance of whole blood
viscosity in (cardio)vascular medicine. Neth Heart J 2002;10(12):512–6.
116. Weidman J, Sloop G, St Cyr JA. Validated formulae for estimation of whole blood
viscosity underestimate the influence of erythrocyte aggregation and deform-
ability. Ther Adv Cardiovasc Dis 2016;10(4):271–3.
117. Montes RA, Eckman JR, Hsu LL, Wick TM. Sickle erythrocyte adherence to
endothelium at low shear: role of shear stress in propagation of vaso-occlusion.
Am J Hematol 2002;70(3):216–27.
118. Quinn CT. Minireview: Clinical severity in sickle cell disease: the challenges of
definition and prognostication. Exp Biol Med (Maywood) 2016;241(7):679–88.
119. Connes P, Renoux C, Romana M, et al. Blood rheological abnormalities in sickle
cell anemia. Clin Hemorheol Microcirc 2018;68(2–3):165–72.
Descargado para Anonymous User (n/a) en Cayetano Heredia Pervuvian University de
ClinicalKey.es por Elsevier en diciembre 27, 2022. Para uso personal exclusivamente. No se
permiten otros usos sin autorización. Copyright ©2022. Elsevier Inc. Todos los derechos
reservados.
 SCD Pathophysiology-Based Biomakers 1095

120. Nader E, Skinner S, Romana M, et al. Blood Rheology: Key Parameters, Impact
on Blood Flow, Role in Sickle Cell Disease and Effects of Exercise. Front Physiol
2019;10:1329.
121. Connes P, Hue O, Tripette J, Hardy-Dessources MD. Blood rheology abnormal-
ities and vascular cell adhesion mechanisms in sickle cell trait carriers during
exercise. Clin Hemorheol Microcirc 2008;39(1–4):179–84.
122. Kucukal E, Man Y, Hill A, et al. Whole blood viscosity and red blood cell adhe-
sion: Potential biomarkers for targeted and curative therapies in sickle cell dis-
ease. Am J Hematol 2020;95(11):1246–56.

Descargado para Anonymous User (n/a) en Cayetano Heredia Pervuvian University de

ClinicalKey.es por Elsevier en diciembre 27, 2022. Para uso personal exclusivamente. No se
permiten otros usos sin autorización. Copyright ©2022. Elsevier Inc. Todos los derechos
reservados.