Probing single‐cell oxygen reserve in sickled erythrocytes via in vivo photoacoustic microscopy

Ford Andria (Orcid ID: 0000-

0003-

0605

-8943)

Probing single

-cell oxygen reserve in sickled erythrocytes via in vivo

photoacoustic microscopy

Authors:

Andria L. Ford

1, 2†

, Hsun-

Chia Hsu

3, 4

†

, Michael M. Binkley

, Stephen Rogers

, Toru

Imai

, Konstantin Maslov

, Allan Doctor

, Lihong V. Wang

, Jin

-Moo Lee

1, 2, 6

Affiliations:

Department of Neurology, Washington University Sc

hool of Medicine, St. Louis, MO

Mallinckrodt Institute of Radiology, Washington University Sc

hool of Medicine, St. Louis, MO

Caltech Optical Imaging Laboratory, Andrew and Peggy Cherng Department of Medical

Engineering, Department of Electrical Engineering, Pasadena, CA

Currently at Center for Devices and Radiological Health,

U.S. Food and Drug

Administration, Silver Spring, Maryland

Center for Blood Oxygen Transport and Hemostasis &

Department of Pediatrics,

University of Maryland, Baltimore, Maryland

Department of Biomedical Engineering, Washington University S

chool of Medicine, St. Louis,

Missouri

†Drs. Ford and Hsu contributed equally, and are designated as co-

first authors.

*Drs. Lee and Wang contributed equally, and are designated as co-

senior authors.

Running

Title:

Impaired oxygen

reserve in sickle

erythrocytes

* Materials & Correspondence:

Jin

-Moo Lee, MD, PhD

Department of Neurology; Washington University School of Medicine; 600 South Euclid Avenue,

Campus Box 8111; Saint Louis, Missouri 63110;

Email:

leejm@wustl.edu

Lihong Wang, PhD

This article has been accepted for publication and undergone full peer review but has not been

through the copyediting, typesetting, pagination and proofreading process which may lead to

differences between this version and the Version of Record. Please cite this article as doi:

10.1002/ajh.26387

Caltech Optical Imaging Laboratory; Andrew and Peggy Cherng Department of Medical

Engineering ; California Institute of Technology; 1200 E. California Blvd., MC 138-

78 Pasadena,

California 91125; Email: lihong@

caltech.edu

To the Editor:

Individuals

with sickle cell disease

(SCD)

face

ongoing risk of multi

-organ ischemia

resulting in

chronic

disability

, frequent hospitalizations

, and early mortality

. T

he relationship

between

hemoglobin (Hb) S

polymerization,

erythrocyte

sickling,

and

tissue

ischemia

has been

of great interest

. Oxygen off

-loading and increasing deoxy

-hemoglobin

concentration

promote

HbS

polym

erization, the latter which has been linked to early

erythrocyte

deformation

“reversible” sickling

. Eventually,

severe

polymerization

weakens

the cell

mbrane, leading to

“irreversibly

” sickled

cells

. Whether

degree of

polymerization

and

the cell’s

morphologic state

, in

turn,

influence

oxygen binding

and thus

, tissue oxygen availability

has been

of interest

, but

technically challenging

to study

in patients

. Over

two decades,

Wang

and colleagues

developed

an imaging platform, photoacoustic microscopy (PAM), which offers two unique aspects

compared to other intravital microscopy systems: (1)

high resolution

in vivo human

imaging using

the cuticle as the window to a highly organized vascular bed capable of imaging

single capillary

loops, and (2) measurements of oxy-

and

deoxy

-hemoglobin levels within sin

gle capillar

ies

and

single erythrocytes

. In this study

, we aimed to: (

1) characterize capillary morphology

and

hemodynamic

/oxygen

metabolic properties in the cuticle

nailbed

of individuals with SCD

compared

to healthy

controls

; and, (2)

track single erythrocytes

along the capillary loop to obtain

measurements of

erythrocyte

elongation

(ellipticity index, EI)

and

oxygen saturation

before and

after tissue oxygen exchange

We hypothesized that

erythrocyte

EI, as

an index of

HbS

polymerization, would be

associated with

decreased

arteriolar

oxygen saturation and/or

increased

oxygen extraction

fraction (OEF) across capillaries

—representing compromised “oxygen

reserve”

Adult participants with SCD (HbSS) and controls (HbAA), were prospectively enrolled

and

excluded

for

recent

hospitalization, chronic transfusion therapy,

and

history of stem cell

transplant. Controls were excluded

for

any chronic medical disorder.

Hemoglobin type was

confirmed

by peripheral blood

electrophoresis

. Written informed consent was obtained from all

participants.

PAM

is a dual

-wavelength

optical resolution system

with 3μm lateral and 15μm axial

resolution

(Figure

S1). The

blood

absorption spectra from individuals with SCD have previously

been

found to be similar to that of healthy controls

. The

nailbed cuticle

imaging procedure

consisted of both wide-

field and high-

speed dynamic imaging.

Capillary measurements

included

density

, diameter

, and tortuosity

. N

umber and duration of erythrocyte pauses were measured

from the spatiotemporal image and its frequency domain image. Multiple hemo-

metabolic

parameters were calculated from both time

-averaged, capillary measurements and single

erythrocytes,

and

includ

ed: b

lood velocity

, oxygen saturation (sO

), OEF, and relative metabolic

rate of oxygen utilization (MRO

). Single

-cell

PAM additionally yielded measurements of arteriolar

(sO

2 in

) and venular (sO

2 out

) oxygen saturation, from which single-

cell OEF was calculated.

Elongation of single-

cells

, termed “ellipticity index” (EI),

was measured

as the mean EI of six

frames for

each

flowing

erythrocyte (Figure

S2).

Details of PAM imaging and s

tatistical methods

are described in the Supplementary Methods.

Ten adults with SCD (HbSS)

and healthy controls (HbAA)

underwent PAM cuticle imaging

sessions

, total

ing 97 capillaries and 180 erythrocytes imaged

(Table

). Hemoglobin and

hematocrit

measured

from

peripheral blood correlated with cuticle hemoglobin and hematocrit

using PAM

(Hb: ρ=

0.825,

=0.002; Hct: ρ=

0.800,

0.003).

Capillary diameter, density, and

tortuosity, were

statistically

increased in SCD

vs.

healthy controls

. Time

-averaged

capillary

blood

velocity was decreased in

SCD

vs.

controls:

62.5 μm/sec [51.0, 74.3] vs. 69.8 μm/sec [63.6, 77.4],

respectively (

0.013); capillary OEF was increased in SCD vs.

controls:

0.205 [0.150, 0.246] vs.

0.147 [0.121, 0.188], respectively (

0.049)

; and

capillary

MRO

was similar in SCD vs. controls

49.9 arbitrary units (a.u.) [21.3, 60.9] vs. 39.0 a.u. [26.1, 50.6], respectively (

0.394) (

Figure

S3).

Animal models of SCD have demonstrated the presence of red blood cell pauses,

although the etiologies of pauses such as mechanical obstruction or endothelial adhesion are

incompletely understood. E

rythrocyte pause count was higher in SCD vs.

controls:

2 [2, 3] vs. 1.5

[1, 2],

respectively,

(

<0.0001).

Pause duration was also higher in SCD

vs.

controls

: 14.6 sec

[10.3, 19.9] vs. 7.0 sec [4.5, 9.0]

, respectively,

(

<0.0001)

. Using a linear mixed

-model to account

for repeated

measures within individuals, we

evaluated the relationship between pause duration

and capillary morphological

and

hemo-

metabolic properties. Pause duration was inversely

associat

ed with blood velocity

(

0.009)

, but not associated with OEF or capillar

y diameter. We

also observed a dire

association between pause duration and capillary tortuosity that

approached

significance (

0.09)

(Figure

S4).

PAM

imaging can

resolve individual

erythocytes

, permitting

a metric

elongated

erythrocytes using “

ellipticity index

” (EI,

Figure

S2). EI was increased in

SCD

vs.

controls:

0.201

[0.133, 0.245] vs. 0.112 [0.075, 0.153], respectively

(

<0.0001) (Figure

). While the SCD

cohort demonstrated a wider dynamic range of EI values, substantial overlap was seen across

lower EI between the t

wo cohorts, suggesting that SCD patients have a population of normal or

near

-normal shaped er

ythrocytes, consistent with the literature on peripheral blood

. Moreover,

the population of cells in the SCD cohort with increased EI extended beyond the EI distribution in

controls, an observation consistent with HbS polymerization and sickling.

In contrast to velocity measurements using the time-

averaged approach, mean single-

cell

blood veloci

ty was higher in SCD vs.

controls:

79.8 μm/sec [69.4, 90.4] vs. 68.9 μm/sec [64.7,

79.7],

respectively (

0.003)

(Figure 1B).

During single

-cell image acquisition, stalled

erythrocytes were not included; therefore, this result likely represents an increase

in cell

velocity

without accounting for the effect of pauses. Consistent with this, the measured velocities from

the single

-cell method for both control and SCD co

horts were higher than the velocities measured

with the time

-averaged method

for capillarie

Arteriolar

oxygen saturation (sO

2 in

) was lower in SCD vs.

controls (

0.014, Figure 1

suggesting decreased oxygen availability

to the capillary bed in SCD.

Consistent

with time

averaged results, single-

cell OEF was increased in the SCD cohort

vs.

controls:

0.101 [0.077,

0.133] vs. 0.067 [0.051, 0.091],

respectively (

<0.0001) (Figure 1D

). The finding of higher single-

cell OEF is consistent with the observed anemia, diminished erythrocyte velocities

, and reduced

arteriolar

oxygen saturation in

patients

with SCD, which suggests that greater oxygen extraction

from individual erythrocytes is required to meet oxygen metabolic demand.

To examine the relationship between

EI, as an index of Hb polymerization and sickling,

and measures of erythrocyte ve

locity

and oxygen metabolism, we performed mixed

-model linear

regression

to examine

EI as a predictor of velocity, sO

2 in

, and OEF

(Figure 1E

-H). In SCD, but

not in controls, an increase in EI was associated with slower erythrocyte velocity (

-98.0,

0.001)

. Consistent with this, erythrocytes with greater EI were associated with increased OEF

(

=21.5

0.013)

. Further, increased EI w

as associated with decreased sO

2 in

(

-17.3,

0.038

). Erythrocyte velocity was

also

model

led in relation to OEF.

Both in controls and SCD,

individual erythrocyte OEF was inversely proportional to velocity indicating that erythrocyte OEF

was increased with prolonged capillary transit time.

In this study,

we used cuticle

PAM to examine

micro

structural and physiological

measures

within

capillar

ies

and single-

cells

to advance

our

understanding of the pathophysiology underlying

tissue ischemia in SCD. In addition to altered capillary architecture, i

ndividuals

with SCD

demonstrated

reduced bulk flow velocity, increased OEF, more frequent

erythrocyte pauses

, and

prolonged

pause

duration compared t

o controls. In single

-cell

measurements

, we

found that

erythrocyte elongation (EI) was much higher in SCD vs. controls. Moreover,

EI distr

ibution

patients

with SCD

was much broader, suggesting

that

a large subset of cells are

elongated

due

HbS

polymerization (Figure 1

A, above

red

dashed line). Previous studies

have noted a

spectrum of erythrocyte morpholog

y and wide variation in the proportion of sickled cells (

29%

43%

)

. Indeed, we found higher EI in the SCD cohort predicted slower erythrocyte velocity, lower

2 in

, and higher OEF, while these relationships were absent in controls. These

findings suggest

that sickled erythrocytes exhibit lower oxygen reserve than normally shaped erythrocytes, as

indicated by both lower sO

2 in

and increased OEF across the capillary loop, thus severe reductions

in blood flow or sO

2 in

would potentially place

the tissue at risk of ischemia.

In summary, we found that increased EI, as a

proposed

index of polymerized HbS, was

associated with distinct single-

cell characteristics (velocity, arteriolar oxygen binding, and oxygen

off -loading). This study was conduct

ed in SCD patients who were not actively symptomatic,

suggesting that baseline oxygen reserve (decreased sO

2 in

and increased OEF) in patients with

SCD may be compromised in a subset of erythrocytes. Increased OEF resulted in preserved

MRO

, suggesting overall, a well

-compensated metabolic state. Future PAM studies examining

patients during vaso-

occlusive crises may reveal an elevated proportion of “sickled” erythrocytes

with elevated EI and decreased oxygen reserve. Such patients may be on the precipice o

f tissue

infarction and could be identified for early intervention using PAM technology at the bedside.

The cur

rent work represents a proof

-of-concept study suggesting that PAM technology

may

improve our understanding of the relationship between HbS polymerization, erythrocyte

morphology, and tissue oxygen transport in SCD. Larger studies will be required to confirm our

findings and examine

covariates which could further define these

relationships. While our results

demonstrate a link between EI and

tissue oxygen availability, these relationships do not prove

causality. Finally, we did not measure

erythrocyte deformability

, blood viscosity

, or shear rates

which

are known to be altered in

patient with

SCD and likely play a role in thrombosis and

hemos

tasis

. The potential

influence

of erythrocyte rheology

on oxygen availability

, however,

should not impact the

accuracy of hemo

-metabolic

measurements, nor

minimize

the cohort

differences identified between SCD and controls

Data Availability

The data that support the findings of this study are available on request from the corresponding

author. The data are not publicly available due to privacy or ethical restrictions.

References

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Hsu HC, Wang L, Wang LV. In vivo photoacoustic microscopy of human cuticle

microvasculature with single-

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J Biomed Opt

2016;

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Alvarez O, Montague NS, Marin M, O'Brien R, Rodriguez MM. Quantification of sickle

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Acknowledgements:

This work was supported by National Institutes of Health R01HL129241

and RF1NS116565 (A.L.F.);

R01NS094692,

R37NS110699, U24NS107230 (J

-M.L.), and

R01GM113838 (A.D)

Author

information:

A.L.F and H.H. designed the experiment, collected the data, analyzed and

interpreted data, and prepared the manuscript. M.M.B., S.R., T.I., and K.M. analyzed and

interpreted the

data. A.D., L.W., and J.M.L designed the experiment and analyzed and

interpreted data. All authors critically reviewed and approved the final version of the manuscript.

Competing interests:

L.W. has a financial interest in Microphotoacoustics, Inc., C

alPACT,

LLC, and Union Photoacoustic Technologies, Ltd., which, however, did not support this work.

The remaining authors do not have any competing interests.

Figure Legend

Figure

1. Single cell hemo

-metabolic parameters and

ellipticity index (EI) as a

predictor o

single

-cell blood flow and oxygenation

in adults with SCD compared to controls

.

(A)

Using

PAM to measure the shape of single

erythrocytes

, EI, a metric of cellular anisotropy

and

deformation, was increased in the SCD cohort compared to controls (

<0.0001). While the SCD

cohort demonstrated a wider dynamic range of EI values, substantial overlap is seen across lower

EI between the two cohorts, suggesting that SCD patients have a population of normal or near

normal shaped erythrocytes (

below the

red dashed line) as well as a subpopulation of

elongated/sickled erythroc

ytes (above the red dashed line,

indicating EI

greater than

two standard

deviations above the mean EI from the control cohort

(B)

Single

-cell blood velocity was higher

in SCD compared to controls (

=0.003). During single

cell image acquisition, stalled erythrocytes

were not included; therefore, this result represents an increase in velocity of red blood cells when

the effect of pauses is unaccounted for.

(C)

Arteriolar oxy

gen saturation (sO

2 in

) was lower in SCD

compared to controls (

=0.014) suggesting decreased oxygen delivery to the capillary bed in

SCD.

(D)

In line with a decreased oxygen saturation in the arteriolar capillary bed, single-

cell

oxygen extraction fraction (

OEF

) was increased in the SCD cohort compared to controls (

<0.0001). To evaluate the effect of erythrocyte ellipticity, as an index of Hb polymerization and

sickling, on cellular flow and oxygen metabolism, mixed-

model linear regression evaluated EI as

a predictor of erythrocyte velocity, arteriolar

oxygen saturation (

2 in

), and

oxygen extraction

fraction (

OEF

(E)

Increasing EI predicted a decrease in blood velocity (

= 0.001) in SCD, but

not in controls.

(F)

Consistent with this, increasing EI predicted an increase in OEF in SCD, but

not in controls (

=0.013).

(G)

Further, increasing EI

non-

significantly

predicted a decrease in sO

(

=0.038) suggesting erythrocytes with greater ellipticity may have a lower oxygen content when

entering the capillary.

(H)

Both in controls and SCD, OEF was inversely proportional to velocity

suggesting that as the tissue’s oxygen demands increase, erythro

cyte velocity decreases allowing

additional time for oxygen exchange, regardless of HbS polymerization and sickling.

a.u. indicates

arbitrary units.

Raw p

-values are reported. After adjusting for multiple testing with Benjamini

Hochberg procedure, statist

ical significance was achieved, indicated as * P < 0.05 or ** P

< 0.01

AJH_26387_FIgure.tif