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

Probing single-cell oxygen reserve in sickled erythrocytes via in

vivo photoacoustic microscopy

Andria L. Ford

1,2,†

Hsun-Chia Hsu

3,4,†

Michael M. Binkley

Stephen Rogers

Toru Imai

Konstantin Maslov

Allan Doctor

Lihong V. Wang

3,*

Jin-Moo Lee

1,2,6,*

Department of Neurology, Washington University School of Medicine, St. Louis, MO

Mallinckrodt Institute of Radiology, Washington University School 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 School of Medicine, St. Louis,

Missouri

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

. The

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 polymerization, the latter which has been linked to early

erythrocyte deformation or “reversible” sickling

. Eventually, severe polymerization

weakens the cell membrane, 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 single capillaries and

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; leejm@wustl.edu, Lihong Wang, PhD, 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; lihong@caltech.edu.

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

†

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

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.

HHS Public Access

Author manuscript

Am J Hematol

. Author manuscript; available in PMC 2023 January 01.

Published in final edited form as:

Am J Hematol

. 2022 January 01; 97(1): E11–E14. doi:10.1002/ajh.26387.

Author Manuscript

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. Number 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 included: blood 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 statistical methods are described in the

Supplementary Methods.

Ten adults with SCD (HbSS) and healthy controls (HbAA) underwent PAM cuticle imaging

sessions, totaling 97 capillaries and 180 erythrocytes imaged (Table S1). Hemoglobin

and hematocrit measured from peripheral blood correlated with cuticle hemoglobin and

hematocrit using PAM (Hb:

=0.825,

=0.002; Hct:

=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. Erythrocyte 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

Ford et al.

Page 2

Am J Hematol

. Author manuscript; available in PMC 2023 January 01.

Author Manuscript

duration was inversely associated with blood velocity (

=0.009), but not associated with

OEF or capillary diameter. We also observed a direct association between pause duration

and capillary tortuosity that approached significance (

=0.09) (Figure S4).

PAM imaging can resolve individual erythocytes, permitting a metric of 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 1A). While

the SCD cohort demonstrated a wider dynamic range of EI values, substantial overlap

was seen across lower EI between the two cohorts, suggesting that SCD patients have

a population of normal or near-normal shaped erythrocytes, 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 velocity 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 cohorts were higher than the

velocities measured with the time-averaged method for capillaries.

Arteriolar oxygen saturation (sO

2 in

) was lower in SCD vs. controls (

=0.014, Figure 1C)

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 velocity 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 was associated with decreased

2 in

(

=−17.3,

=0.038). Erythrocyte velocity was also modelled 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 microstructural and physiological measures

within capillaries and single-cells to advance our understanding of the pathophysiology

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

with SCD demonstrated reduced bulk flow velocity, increased OEF, more frequent

erythrocyte pauses, and prolonged pause duration compared to controls. In single-cell

Ford et al.

Page 3

Am J Hematol

. Author manuscript; available in PMC 2023 January 01.

Author Manuscript

measurements, we found that erythrocyte elongation (EI) was much higher in SCD vs.

controls. Moreover, EI distribution in patients with SCD was much broader, suggesting that

a large subset of cells are elongated due to HbS polymerization (Figure 1A, above red

dashed line). Previous studies have noted a spectrum of erythrocyte morphology 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 sO

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

2 in

and increased OEF across the capillary loop, thus severe reductions in blood flow or

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 conducted 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 of tissue infarction and could be identified for early

intervention using PAM technology at the bedside.

The current 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 hemostasis

. 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.

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.

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).

Competing interests:

L.W. has a financial interest in Microphotoacoustics, Inc., CalPACT, LLC, and Union Photoacoustic Technologies,

Ltd., which, however, did not support this work. The remaining authors do not have any competing interests.

Ford et al.

Page 4

Am J Hematol

. Author manuscript; available in PMC 2023 January 01.

Author Manuscript

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

1. Piel FB, Steinberg MH, Rees DC. Sickle Cell Disease. N Engl J Med 2017; 376(16): 1561–73.

[PubMed: 28423290]

2. Eaton WA, Hofrichter J. Hemoglobin S gelation and sickle cell disease. Blood 1987; 70(5): 1245–

66. [PubMed: 3311198]

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

with single-cell resolution. J Biomed Opt 2016; 21(5): 56004. [PubMed: 27207113]

4. Nahavandi M, Nichols JP, Hassan M, Gandjbakhche A, Kato GJ. Near-infrared spectra absorbance

of blood from sickle cell patients and normal individuals. Hematology 2009; 14(1): 46–8. [PubMed:

19154664]

5. Alvarez O, Montague NS, Marin M, O’Brien R, Rodriguez MM. Quantification of sickle cells in

the peripheral smear as a marker of disease severity. Fetal Pediatr Pathol 2015; 34(3): 149–54.

[PubMed: 25517885]

6. Gillespie AH, Doctor A. Red Blood Cell Contribution to Hemostasis. Front Pediatr 2021; 9: 629824.

[PubMed: 33869111]

Ford et al.

Page 5

Am J Hematol

. Author manuscript; available in PMC 2023 January 01.

Author Manuscript

Figure 1. Single cell hemo-metabolic parameters and ellipticity index (EI) as a predictor of

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 erythrocytes (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

Ford et al.

Page 6

Am J Hematol

. Author manuscript; available in PMC 2023 January 01.

Author Manuscript

of pauses is unaccounted for.

(C)

Arteriolar oxygen 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 (sO

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

2 in

(

=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, erythrocyte 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, statistical significance was achieved,

indicated as * P < 0.05 or ** P < 0.01.

Ford et al.

Page 7

Am J Hematol

. Author manuscript; available in PMC 2023 January 01.

Author Manuscript