Measurements of red cell deformability and hydration reflect HbF and HbA2 in blood from patients with sickle cell anemia

Accepted Manuscript Measurements of red cell deformability and hydration reflect HbF and HbA2 in blood from patients with sickle cell anemia

Nermi L. Parrow, Hongbin Tu, James Nichols, Pierre-Christian Violet, Corinne A. Pittman, Courtney Fitzhugh, Robert E. Fleming, Narla Mohandas, John F. Tisdale, Mark Levine PII: DOI: Reference:

S1079-9796(17)30150-X doi: 10.1016/j.bcmd.2017.04.005 YBCMD 2183

To appear in:

Blood Cells, Molecules and Diseases

Received date: Accepted date:

4 April 2017 14 April 2017

Please cite this article as: Nermi L. Parrow, Hongbin Tu, James Nichols, Pierre-Christian Violet, Corinne A. Pittman, Courtney Fitzhugh, Robert E. Fleming, Narla Mohandas, John F. Tisdale, Mark Levine , Measurements of red cell deformability and hydration reflect HbF and HbA2 in blood from patients with sickle cell anemia. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Ybcmd(2017), doi: 10.1016/j.bcmd.2017.04.005

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ACCEPTED MANUSCRIPT Measurements of red cell deformability and hydration reflect HbF and HbA2 in blood from patients with sickle cell anemia Nermi L. Parrow1, Hongbin Tu1, James Nichols2, Pierre- Christian Violet1, Corinne A. Pittman3, Courtney Fitzhugh3, Robert E. Fleming4,5, Narla Mohandas6, John F. Tisdale2 and Mark Levine1* Molecular and Clinical Nutrition Section, Digestive Diseases Branch, National Institute of

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Sickle Cell Branch, National Heart, Lung and Blood Institute, National Institutes of Health,

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Diabetes and Digestive and Kidney Diseases, 2 Molecular and Clinical Hematology Branch,

Bethesda, MD, USA, 4Department of Pediatrics, 5Edward A. Doisy Department of Biochemistry

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and Molecular Biology, Saint Louis University School of Medicine, St Louis, MO, USA, 6Red

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Cell Physiology Laboratory, New York Blood Center, New York, NY, USA *

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Corresponding author:

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Mark Levine, MD

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Chief, Molecular and Clinical Nutrition Section

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Digestive Diseases Branch

National Institute of Diabetes and Digestive and Kidney Diseases

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National Institutes of Health Building 10 Room 4D52 10 Center Drive Bethesda, MD 20892-1372 Phone: (301)-402-5588

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ACCEPTED MANUSCRIPT Fax: (301)-402-6436 e-mail: [email protected] Abstract word count: 197

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Figures: 7

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Supplementary Figures: 3

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Tables: 2 References: 49

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Keywords: fetal hemoglobin, sickle cell anemia, sickle cell disease, erythrocytes

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Running title: Effect of hemoglobin composition on cellular deformability

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Abbreviations

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EI: elongation index

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EI Max: maximum elongation index

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EI Min: minimum elongation index HbA: normal adult hemoglobin (22) HbA2: adult hemoglobin variant (22) HbF: fetal hemoglobin (22) HbS: sickle hemoglobin (2s2)

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ACCEPTED MANUSCRIPT HbSC: individuals with sickle cell disease resulting from the inheritance of one gene encoding HbS and the other encoding the abnormal hemoglobin variant, HbC Hct: hematocrit

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Hgb: hemoglobin

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HPFH: hereditary persistence of fetal hemoglobin HV: healthy volunteer

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LDH: lactate dehydrogenase

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LORRCA: laser assisted optical rotational red cell analyzer

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MCH: Mean corpuscular hemoglobin content

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MCHC: Mean corpuscular hemoglobin concentration

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MCV: Mean corpuscular volume

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O (EI Max): osmolality at maximum elongation at the iso-osmolar point

Pa: Pascal

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O Min: osmolality at minimum elongation in hypo-osmolar region of osmoscan

Pro-BNP: Pro brain natriuretic peptide RBC: red blood cell RDW: Red cell distribution width SCA: sickle cell anemia 3

ACCEPTED MANUSCRIPT SS ½: Shear stress value for half maximal elongation

Key point: Deformability and osmolality at the elongation index minimum in the hypotonic

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region, as well as diffraction pattern distortions may be useful for monitoring fetal hemoglobin in

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sickle cell anemia.

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Key point: Increased concentrations of the adult hemoglobin variant, HbA2, like HbS, are

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negatively correlated with red cell hydration and deformability parameters in SCA.

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Abstract

Decreased erythrocyte deformability, as measured by ektacytometry, may be associated with

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disease severity in sickle cell anemia (SCA). Heterogeneous populations of rigid and deformable

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cells in SCA blood result in distortions of diffraction pattern measurements that correlate with the concentration of hemoglobin S (HbS) and the percentage of irreversibly sickled cells. We

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hypothesize that red cell heterogeneity, as well as deformability, will also be influenced by the

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concentration of alternative hemoglobins such as fetal hemoglobin (HbF) and the adult variant, HbA2. To test this hypothesis, we investigate the relationship between diffraction pattern

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distortion, osmotic gradient ektacytometry parameters, and the hemoglobin composition of SCA blood. We observe a correlation between the extent of diffraction pattern distortions and percentage of HbF and HbA2. Osmotic gradient ektacytometry data indicate that minimum elongation in the hypotonic region is positively correlated with HbF, as is the osmolality at which it occurs. The osmolality at both minimum and maximum elongation is inversely correlated with HbS and HbA2. These data suggest that HbF may effectively improve surface-to-

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ACCEPTED MANUSCRIPT volume ratio and osmotic fragility in SCA erythrocytes. HbA2 may be relatively ineffective in improving these characteristics or cellular hydration at the levels found in this patient cohort. Introduction

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Sickle cell anemia arises from the inheritance of two copies of a mutated form of the gene

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encoding the beta-subunit of adult hemoglobin, resulting in the translation of valine rather than

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glutamic acid at position 6 of the protein 1,2. Subsequent incorporation into hemoglobin produces sickle hemoglobin (HbS), which, upon deoxygenation, forms polymers within erythrocytes that

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distort their normal shape and alter their deformability 3,4. Although alternatives are under

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development 5,6, hydroxyurea and blood transfusion remain cornerstones of treatment. Hydroxyurea treatment has been shown to improve erythrocyte deformability, survival and

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aggregation tendencies of red cells in SCA blood, without negatively influencing blood

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viscosity7,8. Hydroxyurea is effective, in part, through its ability to induce HbF, which decreases the intracellular concentration of HbS and is excluded from the formation of the deoxygenated

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HbS polymer 9-11. Hereditary persistence of fetal hemoglobin (HPFH), which results in the

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production of ~ 30% HbF in affected adults, is protective with respect to sickle cell disease complications in compound HbS/HPFH heterozygous patients, providing strong evidence for the

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protective properties of HbF at this concentration12. Although even low level induction of HbF is thought to be beneficial, higher doses of hydroxyurea are associated with better clinical outcomes 13. HbF per F cell is a more important indicator of therapeutic efficacy than overall percentage of HbF. Pancellular distribution of sufficient HbF to inhibit polymerization of HbS is more protective than heterocellular distribution even when the pancellular percentage of HbF is lower than the heterocellular

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ACCEPTED MANUSCRIPT concentration 14. The adult hemoglobin variant, HbA2 (), also inhibits polymerization of HbS and has the advantage of being natively pancellular 15,16. Although hemoglobin composition is a well-recognized determinant of the sickling phenomenon and subsequent disease severity 17, the influence of hemoglobins other than HbS on red cell deformability has not been previously

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investigated in SCA. Ektacytometry uses laser diffraction viscometry to provide a convenient measure of red cell

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deformability 18,19. Previous studies have shown that rigid red cells do not align or deform

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properly in response to increasing shear stress. Diamond-shaped diffraction patterns arise from the superposition of elliptical and spherical patterns generated by deformable and rigid cells,

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respectively 20-22. Rabai and colleagues initially showed that altering the ektacytometry

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diffraction pattern size strongly affects deformability values of SCA blood in response to shear stress. A heterogenous blood sample containing rigid and deformable cells so measured will

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demonstrate characteristic “degrees of distortion,” defined as the difference between the

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maximum deformability obtained from a smaller diffraction pattern size and the maximum deformability obtained from a larger diffraction pattern size divided by the smaller diffraction

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pattern size. Alternatively, differences in shear stress are defined as the difference between the

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shear stress required to achieve half maximal deformability from a smaller diffraction pattern size and the shear stress required to achieve half maximal deformability from a larger diffraction pattern size. The degree of distortion has been shown to correlate with the concentration of HbS and the percentage of irreversibly sickled cells 21,23. We have reported that the degree of distortion can be readily measured on the Lorrca ektacytometer by adjustments to the camera gain 23.

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ACCEPTED MANUSCRIPT Osmotic gradient ektacytometry (also referred to as osmoscan; the two terms will be used interchangeably) measures erythrocyte deformability as a continuous function of changes in the osmolality of the suspending medium. In addition to deformability, the osmotic gradient curve provides additional information regarding several other erythrocyte parameters, including surface

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to volume ratios, osmotic fragility, deformability, cellular hydration, and cytoplasmic viscosity.

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Specifically, the elongation index at minimum deformability corresponds to surface-to-volume

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ratio. The osmolality at minimum deformability, O Min, corresponds to the 50% lysis point in manual osmotic fragility assays. Maximum deformability (EI max) provides information about

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the deformability of the cell, and the osmolality at maximum deformability (O (EI Max) provides

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additional information about volume regulation and the state of cellular hydration. The hypertonic elongation index, EI hyper, mirrors cellular density and cytoplasmic viscosity as does

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the osmolality at which it occurs, O hyper 24,25.

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We hypothesize that the relative concentrations of HbF and HbA2 in erythrocytes from SCA

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patients will be reflected in diffraction distortion and/or differences in shear stress as measured by ecktacytometry. We tested this hypothesis using blood from 29 patients with SCA. We

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moreover analyzed the relationship between hemoglobin composition and certain additional

Methods

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erythrocyte parameters as measured by osmotic gradient ektacytometry.

Study design Study subjects comprised 29 individuals with SCA (HbSS), 8 of whom had a history of transfusion (i.e., had received one or more transfusions with residual HbA by hemoglobin electrophoresis). Blood from each of these individuals was analyzed for diffraction pattern 7

ACCEPTED MANUSCRIPT distortion. Additional measurements were made by osmotic gradient eckacytometry on blood from a subset of 21 of these individuals (5 of whom had a history of transfusion). For cellular density analyses, blood was collected from 6 HbSS patients either once (patient 6) or at a variable number of four month intervals (remaining 5 patients). All subjects gave written

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informed consent in accordance with Declaration of Helsinki and National Institutes of Health

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Institutional Review Board approved protocols. Blood for ektacytometry was collected into K2

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EDTA vacutainer tubes (BD vacutainer) and ektacytometry was initiated within 4 hours of blood draw. Complete blood counts, hemoglobin electrophoresis and other relevant clinical assays

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were performed by the Department of Laboratory Medicine in the Clinical Center at the National

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Institutes of Health.

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Ektacytometry

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Diffraction distortion analyses were performed basically as described 23. Briefly, 25L of blood was added to 5 mL Iso osmolar polyvinylpyrrolidone solution (Mechatronics, The Netherlands).

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One mL of the diluted blood sample was pipetted into the LORRCA MaxSis (Mechatronics, The

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Netherlands) and the camera gain was adjusted to obtain 3.8 cm, 4.5 cm or 5.4 cm diffraction pattern sizes. Deformability data were obtained in triplicate at pre-selected shear stress between

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0.5 and 50 Pa as defined by Rabai and colleagues 21. Average values of maximum deformability (EI Max) or shear stress ½ (SS ½) were used to obtain the distortion in deformability and difference in shear stress, respectively, between diffraction pattern sizes. Osmotic gradient ektacytometry Osmostic gradient ektacytometry was performed as per manufacturer’s recommendation. Specifically, 250 L of whole blood was added to 5 mL iso-osmolar polyvinylpyrrolidone 8

ACCEPTED MANUSCRIPT solution, and the solution was subject to a continuous suspending medium osmolality gradient ranging from ~50 mOsm/kg to 500 mOsm/kg at a constant shear stress of 30 Pascal (Pa). Individual parameters of the resulting curve were obtained by automated analysis.

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Cellular density analyses

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RBC densities were determined by phthalate density distribution as previously described 26.

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Briefly, n-butyl phthalate and dimethyl phthalate were mixed to achieve defined densities ranging from 1.060 to 1.136 g/mL at 20 degrees Celsius. A 50% RBC/isotonic saline suspension

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was added to each phthalate mixture in a capillary hematocrit tube, tubes were sealed and then

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centrifuged for 10 minutes at 10,000 x g. The D50 is defined as the density at which 50% of the packed RBC volume is above the phthalate mixture and 50% is below. R60 is the defined as the

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cells and the 20% least dense cells 27.

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middle density range containing 60 % of cells, i.e., the density range minus the 20% most dense

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Statistical analyses

Pearson product moment correlations and corresponding p-values were calculated using

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SigmaPlot 12.5. Where appropriate, correlations were corrected for multiple comparisons by the

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Benjamini-Hochberg procedure 28. For diffraction distortion analyses, correlations were considered significant when the p-value was < the corrected Benjamini-Hochberg significance level of 0.0171. For the osmoscan analyses, correlations were considered significant using the standard threshold p-value < 0.05.

Results

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ACCEPTED MANUSCRIPT Diffraction distortion correlates with all hemoglobin variants Patient characteristics and hematological parameters are described in Table 1. Our initial investigations focused on the association between hemoglobin variants and diffraction pattern distortions. Supplementary Figure S1 shows an example of diffraction pattern distortion arising

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from altering the camera gain to produce larger diffraction images (A-C) along with

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corresponding decreases in maximal deformability and increases in the apparent shear stress required to achieve half-maximal deformability (D-F). As previously reported, HbS is positively

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correlated with the degree of distortion when examining changes in maximal deformability (Fig 1A) 21,23. HbA2 has a similar positive and significant correlation with this measure (Fig 1C).

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HbA is negatively correlated with the distortion in deformability and fully corrects the distortion

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as its percentage increases (Fig 1D). Interestingly, HbF has a unique significant and negative correlation with diffraction pattern distortion when differences in the apparent shear stress

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required to obtain half-maximal deformation are examined (Fig 1B).

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HbF has a negative relationship with diffraction distortion regardless of transfusion status

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To further define the relationship between hemoglobin composition and diffraction pattern distortion, we stratified patients by transfusion. There is not a significant relationship between

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HbS and the difference in shear stress in transfused blood (Fig 2A). In untransfused patients, HbS may have a trending relationship with the difference in shear stress, such that the distortion is corrected as HbS approaches 100 % (Fig 2B). Correction of the distortion likely reflects an increasingly homogenous population of red cells, whether deformable or rigid. HbF, on the other hand, is inversely associated with the difference in shear stress regardless of transfusion status, although significance is not retained in the untransfused population (Figs 2C and

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ACCEPTED MANUSCRIPT 2D).Importantly, HbF does not correct the distortion even when it comprises ~29% of the blood. The relationship between HbF and difference in shear stress as a function of diffraction pattern size may be useful as an estimate of HbF in patient blood. Osmotic gradient ektacytometry is influenced by hemoglobin composition in a transfusion

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dependent manner

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To investigate the influence of hemoglobin variants on red cell characteristics, we performed osmotic gradient ektacytometry on a subset of patients with SCA. Supplementary figure S2A

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shows a representative osmotic gradient ektacytometry curve, with important parameters

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indicated. As previously reported, average osmoscans from patients with SCA exhibit increased variability, are left-shifted, and show marked decreases in deformability relative to average

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osmoscans from healthy individuals (supplementary Fig S2B) 24. Transfusion appears to

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normalize the curves by improving deformability and shifting the curves rightward to higher osmolalities relative to the curves generated with untransfused blood (Fig 3A and 3B).

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Surprisingly, HbF has a perfectly inverse relationship with curve normalization in transfused

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patients (Fig 3A) with less obvious effects on curve normalization in untransfused patients (Fig 3B). Although imperfect, HbS also shows the expected inverse relationship with curve

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normalization in transfused patients (Fig 3C). The relationship of HbS to curve normalization in untransfused patients is unclear (Fig 3D). The untransfused population includes a single patient with confirmed -thalassemia trait. Despite the fact that this patient has among the highest concentrations of HbS (92.9%; Fig 3D) and HbA2 (5.4%), as well as the lowest concentration of HbF (1.7%; Fig 3B), the osmoscan shows improvement in deformability across the entire range of osmolalities. The improvement is most pronounced in the hypotonic region. These data suggest that individual hemoglobin variants have specific contributions to alterations in osmotic 11

ACCEPTED MANUSCRIPT gradient deformability that differ depending on transfusion status. Moreover, they confirm previous reports indicating that -thalassemia improves cellular deformability in SCA29. HbA2 is inversely associated with osmotic fragility in the hypotonic arm of the osmotic

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gradient ektacytometery curve.

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In order to investigate the relationship of hemoglobin variants to surface-to-volume ratio and

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osmotic fragility, we investigated associations between HbF, HbA2 and HbS and the minimum elongation of the cell (EI Min), which occurs in the hypotonic arm of the osmoscan, as well as

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the osmolality associated with minimum elongation (O Min). Minimum elongation occurs when

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cells have obtained their maximum volume prior to lysis and is a measure of the surface-tovolume ratio of the cell. In untransfused patients, HbF is positively correlated with EI Min (Fig

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4A). A negative correlation is evident between minimum elongation and HbS in the entire

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patient cohort and in untransfused patients (Fig 4B and 4C, respectively). The osmolality associated with minimum elongation, O Min, corresponds to the 50% hemolysis point in the

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classical osmotic fragility test and provides additional information on the surface-to-volume

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ratio, intracellular osmolyte concentration and hydration state of the cell 24,25. HbF is positively correlated with osmolality at minimum elongation in the entire patient cohort and in untransfused

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patients (Fig 5A and 5B). Both HbA2 and HbS are negatively correlated with the osmolality at minimum elongation in untransfused patients (Fig 5C and 5D). These data indicate that HbF has a positive influence on the surface-to-volume ratio and osmotic fragility of sickle cells, whereas HbS is associated with decreased surface-to-volume ratio and decreased osmotic fragility. In general, these effects are most pronounced in untransfused patients, suggesting that normalized surface-to-volume ratios and osmotic fragility arising from the presence of healthy cells are sufficient to mask both positive and negative effects of alternative hemoglobins on these 12

ACCEPTED MANUSCRIPT parameters. Surprisingly, HbA2 is also associated with decreased osmotic fragility in untransfused patients.

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HbS and HbA2 are associated with the osmolality at maximal deformability in the entire

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patient population.

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The osmolality at which red cells are maximally deformable, O (EI Max), provides a second

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measure of membrane flexibility and cellular hydration 24,30. HbA2 is inversely associated with the osmolality at maximum deformability in the entire patient cohort (Fig 6B). HbS is also

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negatively correlated with the osmolality at maximum deformability when the entire cohort is examined (Fig 6C). In transfused patients, HbF is inversely correlated with the osmolality at

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maximum deformability (Fig 6A). These data confirm an inverse relationship between HbS and

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cellular hydration and flexibility. They also indicate that increasing concentrations of HbA2 are

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correlated with similar alterations in cellular hydration and/or reduced membrane flexibility. In contrast and somewhat suprisingly, HbF shows an inverse correlation only in the transfused

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population. These data suggest that high concentrations of HbF are likely a surrogate marker for

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the presence of sickled cells in the context of healthy transfused cells. HbS is inversely associated with the osmolality at half maximal deformability in the hypertonic arm of the osmoscan The hypertonic elongation index, EI hyper, is the point of half-maximal deformability in the hypertonic arm of the osmotic gradient. EI hyper and the corresponding osmolality at which it occurs, O hyper, provide information about the hydration state and intracellular viscosity of the erythrocyte 20,24,30. Our analyses indicate that HbS is inversely correlated with O hyper when the 13

ACCEPTED MANUSCRIPT entire patient population is analyzed (Supplementary Fig S3A). No significant differences are evident when the population is stratified by transfusion (Supplementary Fig S3B and S3C). These results corroborate previous observations that increasing concentrations of HbS correspond to increasing percentages of dense, dehydrated cells.

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Erythrocyte density range is correlated with several osmotic gradient ektacytometry

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parameters.

The relationship between cellular density and osmotic gradient ektacytometry has been

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comprehensively described in an earlier study using blood from normal donors 24. In that study,

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as buoyant density and MCHC increase, corresponding decreases are observed in maximum deformability and the area of the osmoscan curve. Similarly, decreases in the osmolalities at

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maximum deformability and hypertonic elongation are evident as cell density increases 24. We

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sought to confirm these observations in SCA blood by examining the relationship between RBC density range, defined as the middle range containing 60% of the cells, and osmotic gradient

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ektacytometry parameters. Results indicate a significant inverse correlation between the density

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range and maximum elongation (Fig 7A), the osmolality at maximum elongation (Fig 7B), hypertonic elongation (Fig 7C) and the area of the osmoscan curve (Fig 7D). These data are

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compatible with those previously reported and confirm that increased heterogeneity in cellular densities in SCA is associated with decreased deformability, membrane flexibility and cellular hydration. Discussion This is the first study to report the relationship of hemoglobins other than HbS with diffraction pattern distortions and osmotic gradient ektacytometry parameters in blood from 14

ACCEPTED MANUSCRIPT patients with SCA. Important findings include significant and unique correlations of diffraction pattern distortions with HbF, as well as the remaining hemoglobins investigated. Further, our data suggest that increasing HbF concentrations positively influence the surface-to-volume ratio and osmotic fragility of SCA blood from untransfused patients, as evidenced by correlations with

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minimum elongation (Fig 4A) and the osmolality at which it occurs (Figs 5A and 5B).

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Following these parameters by osmotic gradient ektacytometry may then provide a functional

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assay of therapeutic induction of HbF. Findings of significant associations of HbA2 with osmoscan parameters indicating decreased osmotic fragility (Fig 5C) and decreased membrane

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flexibility and deformability (Fig 6B) are also important as HbA2 is being investigated

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preclinically as a therapeutic target for sickle cell anemia and other hemoglobinopathies 15,31.

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It is noteworthy that the highest HbA values in our cohort, 71% and 67.9%, are sufficient to correct the diffraction pattern distortion (Fig 1D). Correction arises because transfusion supplies

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a variable number of red cells with normal deformability. Thus, increased HbA concentrations

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progressively decrease the percentage of rigid, non-deformable cells available to distort the normally elliptical diffraction pattern. The HbA concentrations required to eliminate the

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diffraction pattern distortion are closely aligned with recommended target HbA level of > 70%

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for acute indications 32 and are in agreement with current exchange transfusion guidelines suggesting targeted HbS values of less than 30% 33. In transfused patients, several lines of evidence suggest that HbF is a proxy for HbS and/or rigid cells. These include the observations that HbF is directly associated with abnormal curves from individual patients (Fig 3A) and inversely correlated with the osmolality at maximum elongation (Fig 6A). In general HbF either positively influences these parameters or has no

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ACCEPTED MANUSCRIPT relationship in untransfused patients. Thus, all future analyses will need to account for transfusion history. The effect of subpopulations of transfused cells, relative to the natural heterogeneity found in sickle blood, on the vascular complications associated with SCA is of relevance. Alterations in

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the profiles of osmotic gradient curves generated from transfused (Fig 3A) and untransfused

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patients (Fig 3B) suggest that transfusion has two major effects on the population of blood cells. The first of these is an apparent improvement in deformability across the gradient and the second

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is a right-shift of the entire curve to more normal osmolalities. We speculate that the improvement in cellular hydration represented by this right shift in the curve combined with

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improved cellular deformability decreases the likelihood of thrombus formation and intimal

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hyperplasia.

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Surprisingly, both HbS and HbA2 are inversely correlated on measures such as osmolality at minimum deformability (Figs 5D and 5C) and osmolality at maximum deformability (Figs 6C

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and 6B). Resistance to osmotic lysis is characteristic of dehydrated cells, as more water is

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required to obtain hemolytic volume 34. Importantly, it has been reported that upon dehydration sickle RBCs decrease deformability leading to an increase in whole blood viscosity 35. Increased

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viscosity, in turn, is associated with vaso-occlusive type complications 36. Whether or not these associations are indicative of a direct effect of HbA2 on erythrocyte deformability is not clear. HbA2 carries a positive charge and has been shown, along with HbS, to preferentially bind to the red cell membrane 15,37. The hemichrome of HbA2 is also more stable than that of HbA 38. One preliminary report suggested red cell abnormalities in transgenic mice expressing the human delta chain 39. A more recent preclinical study investigating the effects of transgenic activation of the human -globin gene in a thalassemic mouse model did not report red cell abnormalities, 16

ACCEPTED MANUSCRIPT however detection may have been hampered by residual thalassemia-mediated red cell abnormalities which improved but did not fully resolve in response to activation with human globin 31. In light of the data presented here, the effect of overexpressing HbA2 on cellular dehydration and deformability should be carefully investigated.

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As previously described, -thalassemia trait improves deformability across the entire range of

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osmolalities (Fig 3B and 3D). The improvement is most notable in the hypotonic arm of the curve, where deformability improves without a normalization of osmolalities. These findings

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have been specifically attributed to increased surface-to-volume ratio without an improvement in cellular dehydration. -globin gene number is primarily a determinant of RBC density

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distribution, as deformability doesn’t change as a function of gene number when density and

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MCHC are equal 40. The relationship between deformability and density is confirmed in our density range analyses, as lower density ranges are correlated with improved deformability (Fig

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7A and 7C). -thalassemia decreases hemolysis 29, resulting in decreased risk of pulmonary

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hypertension, priapism and leg ulcers 41. It is also protective against complications associated with large vessel obstructions, such as stroke and cardiomyopathy 42. However, the increased

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hematocrit is associated with an increase in whole blood viscosity 43 Moreover, deformable

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sickle cells have been shown to be more adherent to the vascular endothelium than irreversibly sickled cells and more deformable cells may play a key role in the initiation of microvascular occlusion 44. The fact that -globin status is associated with increased risk of pain, peripheral retinal vessel closure and osteonecrosis lends support to these observations 40,45-47. Although globin status is not confirmed by genetic testing in the remainder of our patients, we did examine MCV values, which are directly correlated with -globin deletion 48. We found no other MCV

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ACCEPTED MANUSCRIPT values below 85 fL at baseline, suggesting that none of the remaining patients have thalassemia trait. Importantly, interpretation of results must account for the fact that ektacytometry assays the cellular deformability of the population of cells in the blood sample rather than that of individual

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cells. As such, it is unable to account for the contributions of pancellular or heterocellular

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distribution of HbA2 and HbF, respectively, or the absence of HbA in sickle cells in transfused patients, to measured deformability changes. Comparison of population-based deformability

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assays with deformability measurements in individual cells is needed to yield important

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information regarding the effects of hemoglobin variants on red cell characteristics. In summary, the data presented herein suggest that diffraction pattern distortion and shifts in

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osmolality and elongation in hypotonic medium may be convenient and useful for monitoring the

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response to therapeutic interventions that alter hemoglobin composition. They also provide new insights into the association of HbA2 with cellular dehydration of blood from patients with sickle

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cell anemia. Future studies could examine the relationship between hemoglobin composition and

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osmotic gradient parameters from the perspective of genotype, with a particular focus on KLF1 variants associated with concomitant increases in HbF and HbA249. An investigation of the

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relationship between red cell deformability and hemoglobin composition in HbSC and thalassemic patients is also warranted.

Acknowledgements We thank Drs. Connie Noguchi (NIDDK), Hans Ackerman (NHLBI) and Christopher Saunders (South Dakota State University) for helpful discussions regarding sickle cell anemia and RBC 18

ACCEPTED MANUSCRIPT density measurements, alpha globin status, and statistical analyses, respectively. We sincerely appreciate the patients and volunteers who participated in this study. This work was supported by the Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases and the National Heart, Lung and Blood Institute, National Institutes of Health

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(NIH). The opinions expressed herein are the sole responsibility of the authors and do not

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necessarily represent the official views of the NIH.

Authorship: J.F.T., N.M., C.F. and N.L.P. designed the study. N.L.P., H.T., C.F., C.P., J.N. and

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P.V. performed experiments. N.L.P., P.V., N.M., R.E.F and M.L. wrote the manuscript.

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Conflicts of interest: The authors declare no competing interests.

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13. Fitzhugh CD, Hsieh MM, Allen D, et al. Hydroxyurea-Increased Fetal Hemoglobin Is Associated with Less Organ Damage and Longer Survival in Adults with Sickle Cell Anemia. PLoS One. 2015;10(11):e0141706. 14. Steinberg MH, Chui DH, Dover GJ, Sebastiani P, Alsultan A. Fetal hemoglobin in sickle cell anemia: a glass half full? Blood. 2014;123(4):481-485. 15. Steinberg MH, Rodgers GP. HbA2 : biology, clinical relevance and a possible target for ameliorating sickle cell disease. Br J Haematol. 2015;170(6):781-787. 16. Nagel RL, Bookchin RM, Johnson J, et al. Structural bases of the inhibitory effects of hemoglobin F and hemoglobin A2 on the polymerization of hemoglobin S. Proc Natl Acad Sci U S A. 1979;76(2):670672. 17. Maier-Redelsperger M, Noguchi CT, de Montalembert M, et al. Variation in fetal hemoglobin parameters and predicted hemoglobin S polymerization in sickle cell children in the first two years of life: Parisian Prospective Study on Sickle Cell Disease. Blood. 1994;84(9):3182-3188. 18. Bessis M, Mohandas N, Feo C. Automated ektacytometry: a new method of measuring red cell deformability and red cell indices. Blood Cells. 1980;6(3):315-327. 19. Groner W, Mohandas N, Bessis M. New optical technique for measuring erythrocyte deformability with the ektacytometer. Clin Chem. 1980;26(10):1435-1442. 20. Clark MR, Mohandas N, Shohet SB. Deformability of oxygenated irreversibly sickled cells. J Clin Invest. 1980;65(1):189-196. 21. Rabai M, Detterich JA, Wenby RB, et al. Deformability analysis of sickle blood using ektacytometry. Biorheology. 2014;51(2-3):159-170. 22. Streekstra GJ, Dobbe JG, Hoekstra AG. Quantification of the fraction poorly deformable red blood cells using ektacytometry. Opt Express. 2010;18(13):14173-14182. 23. Renoux C, Parrow N, Faes C, et al. Importance of methodological standardization for the ektacytometric measures of red blood cell deformability in sickle cell anemia. Clin Hemorheol Microcirc. 2015;62(2):173-179. 24. Clark MR, Mohandas N, Shohet SB. Osmotic gradient ektacytometry: comprehensive characterization of red cell volume and surface maintenance. Blood. 1983;61(5):899-910. 25. Da Costa L, Suner L, Galimand J, et al. Diagnostic tool for red blood cell membrane disorders: Assessment of a new generation ektacytometer. Blood Cells Mol Dis. 2016;56(1):9-22. 26. 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):3136-3141. 27. Noguchi CT, Dover GJ, Rodgers GP, et al. Alpha thalassemia changes erythrocyte heterogeneity in sickle cell disease. J Clin Invest. 1985;75(5):1632-1637. 28. Benjamini Y, Hochberg, Y. Controlling the false discovery rate: a practical and powerful approach for multiple testing. Journal of the Royal Statistical Society 1995;57(1):289-300. 29. Embury SH, Dozy AM, Miller J, et al. Concurrent sickle-cell anemia and alpha-thalassemia: effect on severity of anemia. N Engl J Med. 1982;306(5):270-274. 30. Mohandas N, Clark MR, Jacobs MS, Shohet SB. Analysis of factors regulating erythrocyte deformability. J Clin Invest. 1980;66(3):563-573. 31. Manchinu MF, Marongiu MF, Poddie D, et al. In vivo activation of the human delta-globin gene: the therapeutic potential in beta-thalassemic mice. Haematologica. 2014;99(1):76-84. 32. T. W, K. H. Best practices for transfusion for patients with sickle cell disease. Hematology Reviews. 2009;1(e22). 33. Swerdlow PS. Red cell exchange in sickle cell disease. Hematology Am Soc Hematol Educ Program. 2006:48-53. 34. Lew VL, Tiffert T, Etzion Z, et al. Distribution of dehydration rates generated by maximal Gardoschannel activation in normal and sickle red blood cells. Blood. 2005;105(1):361-367. 20

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35. Ballas SK, Mohandas N. Sickle red cell microrheology and sickle blood rheology. Microcirculation. 2004;11(2):209-225. 36. Kato GJ, Gladwin MT, Steinberg MH. Deconstructing sickle cell disease: reappraisal of the role of hemolysis in the development of clinical subphenotypes. Blood Rev. 2007;21(1):37-47. 37. Hollan SR, Szelenyi JG, Hasitz M, Szasz I, Gardos G. Haemoglobin and the red cell membrane. Physiol Bohemoslov. 1977;26(3):219-224. 38. Ranney HM, Lam R, Rosenberg G. Some properties of hemoglobin A2. Am J Hematol. 1993;42(1):107-111. 39. Nagel RL, Sharma A, Kumar R, Fabry ME. Severe red cell abnormalities in transgenic mice expressing hihg levels of normal human delta chains. Blood 1995;86:251a. 40. Embury SH, Clark MR, Monroy G, Mohandas N. Concurrent sickle cell anemia and alphathalassemia. Effect on pathological properties of sickle erythrocytes. J Clin Invest. 1984;73(1):116-123. 41. Kato GJ, McGowan V, Machado RF, et al. Lactate dehydrogenase as a biomarker of hemolysisassociated nitric oxide resistance, priapism, leg ulceration, pulmonary hypertension, and death in patients with sickle cell disease. Blood. 2006;107(6):2279-2285. 42. Francis RB. Large-vessel occlusion in sickle cell disease: pathogenesis, clinical consequences, and therapeutic implications. Med Hypotheses. 1991;35(2):88-95. 43. Serjeant BE, Mason KP, Kenny MW, et al. Effect of alpha thalassaemia on the rheology of homozygous sickle cell disease. Br J Haematol. 1983;55(3):479-486. 44. Mohandas N, Evans E. Sickle erythrocyte adherence to vascular endothelium. Morphologic correlates and the requirement for divalent cations and collagen-binding plasma proteins. J Clin Invest. 1985;76(4):1605-1612. 45. Saraf SL, Molokie RE, Nouraie M, et al. Differences in the clinical and genotypic presentation of sickle cell disease around the world. Paediatr Respir Rev. 2014;15(1):4-12. 46. Fox PD, Higgs DR, Serjeant GR. Influence of alpha thalassaemia on the retinopathy of homozygous sickle cell disease. Br J Ophthalmol. 1993;77(2):89-90. 47. Ballas SK, Talacki CA, Rao VM, Steiner RM. The prevalence of avascular necrosis in sickle cell anemia: correlation with alpha-thalassemia. Hemoglobin. 1989;13(7-8):649-655. 48. Akhavan-Niaki H, Youssefi Kamangari R, Banihashemi A, et al. Hematologic features of alpha thalassemia carriers. Int J Mol Cell Med. 2012;1(3):162-167. 49. Yu LH, Liu D, Cai R, et al. Changes in hematological parameters in alpha-thalassemia individuals co-inherited with erythroid Kruppel-like factor mutations. Clin Genet. 2015;88(1):56-61. Table 1. Patient characteristics

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Age (yrs) % Male % Transfused % on hydroxyurea HbF (%) HbS (%) HbA2 (%) HbA (%) RBC (M/L) Hgb (g/dL)

All 41.9 + 13.3 27.6 27.6 75.9 10.8 + 7.9 74.9 + 18.4

Osmoscan 42.1 + 13.3 19.1 23.8 66.7 10.1 + 8.4 76.8 + 18.7

Range 21-64 n/a n/a n/a 1.2-28.7 24.3-94.1

3.9 + 0.6

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37.6 + 23.4 2.6 + 0.7 8.4 + 1.5

38.5 + 28.7 2.6 + 0.7 8.4 + 1.3

10.7-71 1.4-4.3 4.6-11.7

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24.1 + 4.0 95.4 + 14.6 33.4 + 5.9 34.9 + 1.6 19.1 + 4.1 10.6 + 5.2 266.7 + 127.4 32.5 + 4.4 4.2 + 1.9 29.4 + 11.8 354.8 + 110.3 328.6 + 756.7

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24.1 + 4.3 95.9 + 14.4 33.8 + 5.9 35.1 + 1.6 20.0 + 4.6 10.4 + 5.1 259.3 + 126.5 33.4 + 4.4 3.8 + 1.8 28.0 + 11.6 358.1 + 113.8 305.7 + 669

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Hct (%) MCV (fL) MCH (pg) MCHC (g/dL) RDW (%) Reticulocyte (%) Reticulocyte absolute (K/L) Reticulocyte Hgb (%) Immature platelet fraction (%) Immature reticulocyte fraction (%) LDH (U/L) Pro-BNP (pg/mL)

HbS (%) 43.9 62.1 27.9 69 75.4 24.3 76 45.6

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Table 2. Hemoglobin composition of blood from transfused patients. Asterisk (*) indicates patient samples used for osmotic gradient ektacytometry.

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Figure 1. Correlations between hemoglobin composition and degree of diffraction pattern distortion. Percent distortion in deformability is calculated using differences in maximum deformability (EI Max) obtained with 3.8 cm diffraction pattern sizes and 5.4 cm diffraction pattern sizes. Differences in shear stress are calculated using the shear stress required for half-maximal deformability (SS ½) from 3.8 cm diffraction pattern sizes and 5.4 cm diffraction pattern sizes. Linear regression analyses including Pearson product moment correlation coefficient, r, and corresponding p-value of the relationship between A) EI Max-based distortions and percentage of HbS, B) SS1/2-based distortions and percentage of HbF,

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Figure 2. Correlations between percentage of sickle or fetal hemoglobin and degree of diffraction pattern distortion following stratification by transfusion. Differences in shear stress are calculated using the shear stress required for half-maximal deformability (SS ½) from 3.8 cm diffraction pattern sizes and 5.4 cm diffraction pattern sizes. Linear regression analyses of degree of diffraction pattern distortion based on differences in shear stress and A) percentage HbS in transfused patients, B) percentage HbS in untransfused patients, C) percentage of HbF in transfused patients, and D) percentage of HbF in untransfused patients. Pearson product moment correlation coefficient, r, and its associated pvalue are indicated.

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Figure 3. Individual osmostic gradient ektacytometry curves stratified by transfusion with fetal and sickle hemoglobin percentages indicated. Osmotic gradient ektacytometry curves showing deformability, as measured by the elongation index (a.u.), as a function of suspending medium osmolality (mOsm/kg). Graphs were generated using blood from A) transfused patients with HbF percentages indicated, B) representative untransfused patients with HbF percentages indicated, C) the same transfused patients with HbS percentages indicated and D) the same representative untransfused patients with HbS percentages indicated. For reference, osmotic gradient ektacytometry curves from a healthy volunteer, indicated by an inverted arrowhead, are included (----- in the graphs from transfused patients and ----- in the graphs from untransfused patients). The curve generated with blood from the patient with confirmed -thalassemia trait is indicated by an asterisk (*).

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Figure 4. Linear regression analyses of minimum deformation in the hypotonic arm of the osmoscan as a function of hemoglobin composition. Correlations between the elongation index minimum ( EI Min) and A) percentage HbF in untransfused patients, B) percentage HbS in the entire patient population and C) percentage HbS in untransfused patients Pearson product moment correlation coefficient, r, and its associated p-value are indicated. Figure 5. Linear regression analysis of osmolality at minimum deformability in the hypotonic arm of the osmoscan and the percentage of HbF, HbA2 and HbS in blood. The correlation between the

osmolality at minimum deformability (O min in mOsm/kg) and A) the percentage of HbF in the entire patient cohort, B) the percentage of HbF in untransfused patients, C) the percentage of HbA2 in untransfused patients and D) the percentage of HbS in untranfused patients. Pearson product moment correlation coefficient, r, and its associated p-value are indicated.

Figure 6. Linear regression analyses of osmolality at maximum deformability and hemoglobin composition. Correlations between osmolality at maximum elongation [O (EI Max) in mOsm/kg] and A) 23

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Figure 7. Regression analyses of select osmotic gradient parameters and cellular density range. R60 is defined as the density range minus the 20% lightest cells and the 20% densest cells and therefore represents the middle density range in which 60% of the cells can be found 27. Correlations between R60 and A) EI Max, B) O (EI Max), C) EI hyper, and D) area of the osmotic gradient curve in blood from 6 SCA patients. Where repeated measures are indicated, blood was obtained from patients in 4 month intervals. Pearson product moment correlation coefficient, r, and its associated p-value are indicated.

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