Effect of inositol hexaphosphate-loaded red blood cells (RBCs) on the rheology of sickle RBCs

B L O O D C O M P O N E N T S

Effect of inositol hexaphosphate–loaded red blood cells (RBCs)on the rheology of sickle RBCs_3779 627..636

Yann Lamarre, Vanessa Bourgeaux, Aurélien Pichon, Max R. Hardeman, Yannick Campion,

Marise Hardeman-Zijp, Cyril Martin, Jean-Paul Richalet, Françoise Bernaudin, Françoise Driss,

Yann Godfrin, and Philippe Connes

BACKGROUND: The recent in vitro demonstration thatinositol hexaphosphate–loaded red blood cells (IHP-RBCs) may reduce the risks of sickling of sickle RBCs(SS RBCs) exposed to hypoxia make these modifiedRBCs potentially useful in transfused sickle cell anemia(SCA) patients.STUDY DESIGN AND METHODS: Hemorheologicproperties of IHP-RBCs, normal RBCs (AA RBCs), SSRBCs, SS RBCs plus AA RBCs, and SS RBCs plusIHP-RBCs were compared under normoxia and/or afterhypoxic challenges.RESULTS: Although IHP-RBCs have reduced deform-ability compared with SS RBCs or AA RBCs, IHP-RBCsexhibited lower aggregability than AA RBCs and SSRBCs and, when mixed with SS RBCs, the aggregationlevel was below the one of SS RBCs alone or SSRBCs plus AA RBCs. Blood viscosity of SS RBC plusIHP-RBC suspension was lower than the viscosity ofSS RBCs alone and greater than viscosity of SSRBCs plus AA RBCs. The hypoxic challenge was detri-mental for deformability and viscosity of SS RBCs aloneor SS plus AA RBC suspension but not for SS plus IHP-RBC suspension.CONCLUSION: Our results support the fact that IHP-RBCs could be useful in SCA by decreasing RBCaggregation and blunting the adverse effects of hypoxiaon RBC deformability and blood viscosity.

Blood transfusions are commonly used in sicklecell anemia (SCA), notably in case of seriousevents such as severe anemia or acute chest syn-drome,1,2 or as a chronic therapy to prevent

stroke3,4 or any recurrence of sickle-related accident.5,6

Transfusion therapy with normal red blood cells (RBCs)is used to compensate for the altered oxygen-carryingcapacity related to anemia and to improve micro-vascular perfusion by decreasing the proportion of RBCscontaining abnormal hemoglobin S (HbS; sickle RBCs[SS RBCs]). However, it is recommended not to exceed aposttransfusion hematocrit (Hct) of 35% to avoid blood

ABBREVIATIONS: AA RBCs = normal red blood cells; AI =aggregation index; EI(s) = elongation index(-es); IHP-

RBCs = inositol hexaphosphate–loaded red blood cells;

LORCA = laser-assisted optical rotational cell analyzer;

PVP = polyvinylpyrrolidone; SCA = sickle cell anemia;

SS RBCs = sickle red blood cells.

From the UMR Inserm U665, Pointe-à-Pitre, Guadeloupe; the

Université des Antilles et de la Guyane, Guadeloupe; ERYtech

Pharma, Bâtiment Adénine, Lyon, France; the Université Paris

13, Sorbonne Paris Cité, Laboratoire Réponses Cellulaires et

Fonctionnelles à l’Hypoxie, EA 2363 UFR-SMBH, Bobigny,

France; the Department of Translational Physiology, Academic

Medical Center, Amsterdam, the Netherlands; EA 647 Centre de

Recherche et d’Innovation sur le Sport, Université Claude

Bernard Lyon 1, Villeurbanne, France; Pediatrics, Referral

Center for Sickle Cell Disease, Centre Hospitalier Intercommu-

nal of Créteil, Créteil, France; and the Service d’Hématologie

Biologique, Centre Hospitalier Universitaire Bicêtre, Le Kremlin-

Bicêtre, France.

Address reprint requests to: Philippe Connes, PhD, UMR

Inserm U665, Pointe-à-Pitre, F-97159 Guadeloupe; Université

des Antilles et de la Guyane, F-97157 Guadeloupe; e-mail:

[email protected].

Received for publication February 27, 2012; revision

received May 2, 2012, and accepted May 16, 2012.

doi: 10.1111/j.1537-2995.2012.03779.x

TRANSFUSION 2013;53:627-636.

Volume 53, March 2013 TRANSFUSION 627

hyperviscosity7 that may compromise tissue perfusion8

and lead to serious adverse events.9 Therefore, the rheo-logic behavior of transfused homologous RBCs is of spe-cific interest to avoid macro- and microvascular disorders.

Recently, the use of inositol hexaphosphate–loadedRBCs (IHP-RBCs) has been proposed as an alternate strat-egy in SCA transfusion therapy.10 IHP is an allosteric Hbeffector that binds tightly to Hb (1000 times more thannatural 2,3-disphosphoglycerate effector) and reducesthe oxygen affinity for Hb leading to a two- to threefoldgreater ability of transfused RBCs to release oxygen.Unlike normal cells, IHP-RBCs present a rightward-shiftedoxygen dissociation curve compared to SS patient RBCs,which may prevent HbS deoxygenation in physiologicconditions. Transfusion of IHP-RBCs to SCA patients wasthus hypothesized to be able to inhibit sickling of RBCscontaining HbS and to improve oxygen supply to tissues.11

Bourgeaux and colleagues10 compared the in vitro effect ofnormal RBCs and IHP-RBCs on the sickling rate of patientRBCs under hypoxic challenge. They demonstrated that 1)IHP-RBC suspension was seven times more effective inreducing sickling than normal RBCs (at the same dose)under hypoxic stress, 2) 10% IHP-RBCs was as effective as50% normal RBCs to offset sickling, and 3) IHP-RBCsdisplay lower iron content than normal RBCs.10 Thosefindings on sickling inhibition suggest that IHP-RBCscould be developed with the aim to decrease transfusionvolumes thus reducing problems of blood availability andalloimmunization. In addition, a recent study performedin two sickle mice models demonstrated that transfusionof IHP-RBCs may decrease inflammation to a greaterextent than normal RBCs.12

As previously described,10 incorporation of IHP intonormal RBCs is obtained after a step of poration againsta hypotonic buffer (RBCs swelling) and then a step ofRBCs resealing against a hypertonic buffer (RBCs shrink-ing). Extreme swelling and shrinking may submit RBCmembranes to extreme deformation that could inducecytoskeleton rearrangement and change the rheologicproperties of RBCs. In addition, the binding of IHPto Hb and the membrane skeleton13 might influencethe rheologic functionality of IHP-RBCs and impact theposttransfused hemorheologic profile of SCA patients.Thus, even though IHP-RBCs are intended to betransfused at low level in comparison with normal RBCs,it appeared important to study the hemorheologicbehavior of both suspensions when mixed with patientRBCs.

In a first set of experiments, we compared thehemorheologic properties of IHP-RBCs, normal (storedand washed) RBCs (AA RBCs) and SS RBCs in humans. Ina second set of experiments, we compared the effects ofadding either human IHP-RBCs or human AA RBCs on therheology of human SS RBC suspension under normoxiaand under hypoxic challenge.

MATERIALS AND METHODS

Patient blood samplesBlood samples were collected into ethylenediaminetet-raacetate tubes from 12 patients with SCA consultingfor clinical routine visits. Samples were used forroutine biologic analyses. As authorized by Article L.1245-2 of the French Public Health Code (http://www.cnrs.fr/infoslabos/reglementation/rechhomme.htm), theremaining blood in a “routine” blood sample can be usedfor scientific purposes after the patients receive theinformation of the protocol and give written informedconsent. This study applied this ethic rule and wasdone in accordance with the Declaration of Helsinki.All 12 patients were homozygous for the bS allele. Onesubject was excluded from the experiment becausehe had sickle SC disease. Hemorheologic experimentswere performed on fresh samples and within 4 hours asrecommended.14

Preparation of human IHP-RBCs and controlAA RBCsIHP was loaded into human RBCs using the method ofporation (reversible hypotonic lysis) with AN69 hollowfibers (Gambro, Lyon, France). Human leukoreducedRBCs (EFS, Lyon, France) were used as the starting bio-logic material. The RBCs were washed three timeswith 0.9% NaCl (vol/vol) and cells were concentrated to80% Hct. IHP was added at a final concentration of10.5 mmol/L leading to a suspension of 70% Hct. Theporation of RBCs was performed at 1.5 mL/min against ahypotonic buffer of 50 mOsm/kg running countercurrent(15 mL/min) into the column. RBCs were then resealedwith the addition (10% vol/vol) of a hypertonic solution(1900 mOsm/kg) composed of 0.4 g/L adenine, 15.6 g/Linosine, 6.4 g/L sodium pyruvate, 4.9 g/L monosodiumphosphate dihydrate, 10.9 g/L disodium phosphatedodecahydrate, 11.5 g/L glucose monohydrate, 50 g/LNaCl. The suspension was then incubated at 37°C for 30minutes. After three washes with 0.9% NaCl, 0.2% glucosemonohydrate, the final product was resuspended andadjusted to a Hct of 50% with the AS-3 buffer.15 Washednormal RBCs (i.e., normal Hb) were stored in the samebuffer (AS-3 buffer, Hct of 50%) and used as controls in thestudy (AA RBCs). Mean cell volume (MCV) and mean cellHb concentration (MCHC) of IHP-RBCs were 78 � 4 fLand 29.7 � 0.1 g/dL, respectively. AA RBCs exhibitedgreater MCV (90 � 4 fL) and MCHC (33.1 �

0.6 g/dL) than IHP-RBCs. The biochemical characteristicsof IHP-RBCs have been previously published.10 All prod-ucts were stored at 2 to 8°C until use. Hemorheologic char-acterization of human IHP-RBCs and AA RBCs wasperformed.

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628 TRANSFUSION Volume 53, March 2013

Treatment of SS RBCs with IHP-RBCs or AA RBCsThree suspensions of RBCs were prepared into sealedtubes: SS RBCs, SS RBCs mixed with IHP-RBCs (70:30 vol/vol), and SS RBCs mixed with AA RBCs (70:30 vol/vol).The proportions used were those used in the study ofBourgeaux and colleagues.10 The two latter suspensionswere made in the same plasma as that used for the SSRBC suspension. Hct was adjusted at 42% for all thethree suspensions to make hemorheologic comparisonspossible.

Hypoxic challenge on SS RBC suspensionsIHP-RBCs are expected to deliver O2 more easily than SSRBCs and, therefore, are expected to inhibit sickling of SSRBCs under hypoxic challenge, that is, when the PaO2 isdecreased to a value where RBCs containing HbS are atrisk for sickling. Therefore, some of the SS RBC, SSRBC plus IHP-RBC, and SS RBC plus AA RBC suspensionsprepared were also used to test the effects of hypoxicchallenge on the hemorheologic properties of these sus-pensions. The suspensions were deoxygenated for 20minutes of gas bubbling at 37°C with 99.6% nitrogen(PO2 ª 0 mmHg) and then partially reoxygenated for 20minutes of gas bubbling at 37°C with 5% O2 (HXO2) aspreviously done.10 At the end of the procedure, hemorheo-logic characteristics were determined.

Hemorheologic measurementBlood viscosity was measured at high shear rate (225/sec)with a cone plate viscometer (with ULA spindle, Brook-field LV, Middleboro, MA) at 25°C. The RBC elongationindexes (EIs) were determined at 37°C and at nine shearstresses (0.3, 0.57, 1.08, 2.04, 3.87, 7.34, 13.92, 26.38, and50 Pa) by laser diffraction analysis, using a laser-assistedoptical rotational cell analyzer (LORCA, MaxSis, RRMechatronics, Hoorn, the Netherlands). Twenty-fivemicroliters of prepared blood suspension was mixed with5 mL of polyvinylpyrrolidone (PVP; viscosity, 30 mPa/sec)and sheared into the Couette viscometer. The system cal-culates a mean RBC EI for each shear stress. The higherthis index is, the more deformable the RBCs are.14

Osmotic gradient ektacytometry was also performedon the various RBC suspensions prepared (250 mL of pre-pared blood suspension was mixed with 5 mL of PVP).RBCs were sheared at a constant shear stress into theLORCA MaxSis (10 Pa; 37°C) and submitted to an osmoticgradient ranging from 50 to 550 mOsm/kg using two PVPbottles of equal viscosity and different osmolalities (50and 550 mOsm/kg). The changes in EI with the osmoticgradient were recorded and then analyzed. The curvesobtained may be analyzed as follows:16 1) EImax isthe maximum RBC deformability obtained during the

osmotic gradient and reflects the amount of membranesurface area for RBC deformation; 2) the osmolality atwhich the hypotonic minimum occurs, designed Omin,provides a measure of the mean surface area-to-volumeratio of the cell population and has been demonstrated tocoincide with the osmolality value at which 50% of theRBCs hemolyze in an osmotic fragility test; and 3) O′ is thepoint found in the hypertonic arm of the osmotic gradientprofile reflecting the RBC water content.

Stability test was performed in the LORCA MaxSis byshearing RBC suspension (25 mL of prepared blood sus-pension into 5 mL PVP) at 70 Pa for 30 minutes. Mean EIvalue was collected every minute. RBC aggregation (aggre-gation index [AI]) was determined at 37°C via syllectom-etry (i.e., laser backscatter versus time) using the LORCA(RR Mechatronics).17

Blood viscosity and RBC deformability at variousshear stresses were determined in suspensions containingSS RBCs, IHP-RBCs, AA RBCs, SS plus IHP-RBCs, and SSplus AA RBCs under normoxic conditions and in SS RBCs,SS plus IHP-RBCs, and SS plus AA RBCs after hypoxicchallenge (HXO2). Osmotic gradient ektacytometry andRBC aggregation measurements were performed insuspensions containing SS RBCs, IHP-RBCs, AA RBCs,SS plus IHP-RBCs, and SS plus AA RBCs under normoxicconditions. The stability test was performed in SS RBCs,SS plus IHP-RBCs, and SS plus AA RBCs under normoxicconditions. The normoxic condition was obtained withRBC suspension equilibrated with air by continuousmixing on a tube roller for 30 minutes (PO2 ª 140 mmHg).18

Hemorheologic measurements were performed accordingto the recent guidelines for hemorheologic techniques.14

Statistical analysisValues are expressed as mean � standard deviation (SD).Comparisons of blood rheologic properties of 1) SS RBCs,IHP-RBCs, and AA RBCs and 2) SS RBCs, SS plus IHP-RBCs, and SS plus AA RBCs in normoxic condition wereperformed using a one-way analysis of variance and Tukeytest for pairwise comparisons. A paired t test was used totest the effect of hypoxic challenge on the rheology of SSRBCs, SS plus IHP-RBCs, and SS plus AA RBCs. The sig-nificance level was defined as p values less than 0.05(Statistica, Version 5.5, Statsoft, Tulsa, OK).

RESULTS

Hemorheologic characterization of IHP-RBCsTable 1 shows the RBC deformability (EI values) at nineshear stresses in SS RBC, IHP-RBC, and AA RBC suspen-sions. Except for the two lowest shear stresses (i.e., 0.3 and0.57 Pa), IHP-RBCs exhibited significant lower deform-ability than SS RBCs. In contrast, AA RBCs were signifi-

BLOOD TRANSFUSION AND SICKLE CELL ANEMIA


cantly more deformable than both SS RBCs and IHP-RBCs at the nine shear stresses. Figure 1 showsa typical pattern of osmotic gradient ektacytometryobtained for SS RBC, IHP-RBC, and AA RBC suspensions.Compared with AA RBCs, the curve is shifted on the left forSS RBCs and further shifted on the left for IHP-RBCs. O′and Omin are lowered in both IHP-RBCs (344 and<50 mOsm/kg, respectively) and SS RBCs (416 and60 mOsm/kg, respectively) compared with AA RBCs(504 and 147 mOsm/kg, respectively). O′ and Omin were

further decreased in IHP-RBCs compared with SS RBCs.The EImax of AA RBCs (0.531) was greater than EImax ofboth SS RBCs (0.458) and IHP-RBCs (0.442); EImax of SSRBCs was close to IHP-RBCs EImax. The RBC AI followedthe rank order SS RBCs > AA RBCs > IHP-RBCs (Table 1),whereas blood viscosity (at same Hct) followed the rankorder SS RBCs > IHP-RBCs > AA RBCs (Table 1). On thewhole, these findings demonstrate that IHP-RBCs aredehydrated and have reduced deformability and aggrega-bility compared to AA and SS RBCs.

Effect of IHP-RBCs on thehemorheologic properties of sicklecell bloodTable 2 shows the changes in RBCdeformability with shear stressesincreasing, in three suspensions: SSRBCs, SS plus IHP-RBCs, and SS plus AARBCs under normoxic conditions. At thetwo lower shear stresses (i.e., 0.3 and0.57 Pa), RBC deformability was not sig-nificantly different between the threeRBC suspensions. At 1.08, 2.04, and26.38 Pa, RBC deformability had the fol-lowing rank order: SS plus AA RBCs > SSRBCs = SS plus IHP-RBCs. At the fourremaining shear stresses (i.e., 3.87, 7.34,13.92, and 50 Pa), RBC deformabilityfollowed the rank order SS plus AARBCs > SS RBCs > SS plus IHP-RBCs.The osmotic gradient eckatcytometrycurves of the three RBC suspensionsexhibited the following differences(Fig. 2): EImax of SS plus AA RBCs(0.472) > SS RBCs (0.459) > SS plus IHP-RBCs (0.427); O′ of SS plus AA RBCs(434 mOsm/kg) > SS RBCs (404 mOsm/kg) ª SS plus IHP-RBCs (396 mOsm/kg);and Omin of SS plus AA RBCs(109 mOsm/kg) > SS RBCs (98 mOsm/kg) > SS plus IHP-RBCs (74 mOsm/kg).The stability test demonstrated a 50%decrease of deformability below base-line with time in SS RBCs whereas thedeformability reduction was only 25 and22% of baseline in SS plus AA RBCs andSS plus IHP-RBCs, respectively (Fig. 3).AI followed the rank order SS RBCs >SS plus AA RBCs > SS plus IHP-RBCs(Table 2) whereas blood viscosity fol-lowed the rank order SS RBCs >SS plusIHP-RBCs > SS plus AA RBCs (Table 2).In summary, the addition of IHP-RBCsto SS RBCs does not change the deform-

TABLE 1. RBC deformability, blood viscosity, and RBC aggregationmeasured in SS RBCs, IHP-RBCs, and AA RBCs (n = 5)

Hemorheologic parameters SS RBCs IHP-RBCs AA RBCs

EIAt 0.3 Pa 0.03 � 0.01 0.03 � 0.03 0.08 � 0.02*At 0.57 Pa 0.09 � 0.01 0.08 � 0.03 0.14 � 0.01*At 1.08 Pa 0.16 � 0.02 0.13 � 0.03† 0.23 � 0.01*At 2.04 Pa 0.24 � 0.03 0.16 � 0.03† 0.34 � 0.01*At 3.87 Pa 0.32 � 0.03 0.19 � 0.04† 0.43 � 0.01*At 7.34 Pa 0.39 � 0.03 0.22 � 0.04† 0.50 � 0.01*At 13.92 Pa 0.44 � 0.05 0.25 � 0.04† 0.56 � 0.01*At 26.38 Pa 0.47 � 0.05 0.27 � 0.04† 0.59 � 0.00*At 50 Pa 0.50 � 0.06 0.29 � 0.04† 0.62 � 0.00*

Blood viscosity (mPa/sec) 6.09 � 0.81 5.32 � 0.42† 4.32 � 0.51*AI (%) 60.10 � 8.20 43.54 � 9.20† 49.4 � 11.1*

* Significant difference between AA RBCs and both SS RBCs or IHP-RBCs (p < 0.05).† Significant difference between SS RBCs and IHP-RBCs (p < 0.05).

Fig. 1. Typical osmotic gradient ektacytometry in SS RBCs, IHP-RBCs, and AA RBCs

at 10 Pa. EI (i.e., RBC deformability) was measured for the three RBC suspensions

with RBCs being sheared at 10 Pa and submitted to an osmotic gradient ranging

from 50 to 550 mOsm/kg. The peak of EI reflects the amount of membrane surface

area for RBC deformation. The hypoosmotic point is the osmolality at which EI

reaches a minimum in the hypotonic region; it is the same as the osmolality at

which 50% of the RBCs hemolyze in a standard osmotic resistance test. This index

thus provides a measure of the average surface area-to-volume ratio of RBCs. The

hyperosmotic point is the osmolality in the hypertonic region (right leg of the curve)

at which EI reaches half of its maximum value. It provides information on the RBC

hydration status. See text for comments.

LAMARRE ET AL.


ability profile of SS RBCs but increases the whole RBCmembrane stability of the blood suspension anddecreases the extent of SS RBC aggregation.

Effect of hypoxic challenge (hypoxia-partialreoxygenation) and IHP-RBCs on thehemorheologic properties of sickle cell bloodHypoxic challenge significantly decreased RBC deform-ability under normoxic values in SS RBC and SS plus AARBC suspension whereas RBC deformability remainedunchanged in SS plus IHP-RBCs (Fig. 4A). The samepattern of evolution was found for blood viscosity with

hypoxic challenge increasing signifi-cantly blood viscosity in SS RBCs andSS plus AA RBCs whereas the changeremains nonsignificant for SS plus IHP-RBCs (Fig. 4B). Because light backscat-tering technique for RBC aggregationmeasurement is affected by the level ofHb oxygenation,18 we did not analyzethe effect of hypoxic challenge on theRBC AI. On the whole, the addition ofIHP-RBCs to SS RBCs was efficient inlimiting the adverse effects of hypoxiaon SS RBC deformability and SS bloodviscosity whereas the addition of AARBCs to SS RBCs had no positive effect.

DISCUSSION

The major findings of this study are that1) IHP-RBCs have reduced deformabil-ity compared with SS RBCs or AARBCs, and when added to SS RBCs,the deformability of the RBCs isnot improved and contrasts with theimprovement of the deformability ofSS plus AA RBCs; 2) IHP-RBCs exhibitlower aggregability than AA RBCs and SSRBCs and when mixed with SS RBCs, theaggregation level is below the aggrega-tion level of SS RBCs alone or SS RBCsplus AA RBCs; 3) blood viscosity ofIHP-RBC suspension mixed with SSRBCs is lower than viscosity of SS RBCsalone and greater than viscosity of SSRBCs plus AA RBCs; and 4) hypoxic (pluspartial reoxygenation) challenge wasdetrimental for the deformability andviscosity of SS RBCs alone or SS plus AARBC suspension but not for SS plus IHP-RBC suspension.

In the context of SCA, a furtherreduction of RBC deformability could

impair microcirculation and tissue perfusion.19,20 The AARBCs appeared to be well deformable with EI values beingclose to values obtained in fresh AA RBCs coming fromhealthy donors.21 The addition of 30% AA RBCs into SSRBC suspension improved the deformability of the wholesuspensions. This last result is of primary importancesince AA RBCs, in a context of preventive therapy, are mas-sively transfused to patients with the goal to diluteSS RBCs below 30%. In contrast, the deformability of IHP-RBCs was reduced compared with both SS RBCs and AARBCs. When mixed with SS RBCs, the whole RBC deform-ability of the suspension (SS RBCs plus IHP-RBCs)was not improved and was either slightly reduced or

TABLE 2. RBC deformability (n = 12), blood viscosity (n = 11), andRBC aggregation (n = 9) measured in SS RBCs, SS plus IHP-RBCs,

and SS plus AA RBCsHemorheologic parameters SS RBCs SS plus IHP-RBCs SS plus AA RBCs

EIAt 0.3 Pa 0.03 � 0.01 0.04 � 0.03 0.05 � 0.02At 0.57 Pa 0.08 � 0.02 0.08 � 0.03 0.10 � 0.03At 1.08 Pa 0.15 � 0.02 0.14 � 0.03 0.18 � 0.04*At 2.04 Pa 0.23 � 0.04 0.20 � 0.03 0.26 � 0.04*At 3.87 Pa 0.30 � 0.05‡ 0.26 � 0.03 0.34 � 0.05At 7.34 Pa 0.37 � 0.06‡ 0.31 � 0.04 0.42 � 0.05At 13.92 Pa 0.41 � 0.07‡ 0.35 � 0.04 0.47 � 0.05At 26.38 Pa 0.44 � 0.07 0.40 � 0.10 0.50 � 0.06*At 50 Pa 0.47 � 0.08‡ 0.40 � 0.05 0.53 � 0.06

Blood viscosity (mPa/sec) 6.59 � 0.83 6.00 � 0.69† 5.64 � 0.54*AI (%) 52.82 � 9.68 46.29 � 10.14† 50.59 � 10.36*

* Significant difference between SS plus AA RBCs and both SS RBCs or SS plus IHP-RBCs (p < 0.05).

† Significant difference between SS RBCs and SS plus IHP-RBCs (p < 0.05).‡ Significant difference between SS plus AA RBCs and SS RBCs and between SS RBCs

and SS plus IHP-RBCs (p < 0.05).

Fig. 2. Typical osmotic gradient ektacytometry in SS RBCs, SS plus IHP-RBCs, and

SS plus AA RBCs at 10 Pa. See text for comments.



unchanged compared to SS RBC deformability alone. Thereduction in deformability for IHP-RBCs might be relatedto changes in cytoskeleton membrane properties thatoccurred during the process used for IHP entrapmentinside RBCs. Those findings suggest that in contrast tonormal RBCs, IHP-RBCs that display reduced deformabil-ity might not be adequate for massive transfusions such asthe one done with AA RBCs.

Osmotic gradient ektacytometry performed on IHP-RBCs also demonstrated that IHP-RBCs are very dehy-drated compared with AA RBCs. In addition, the IHP-RBCosmotic gradient curve shifted on the left indicatedgreater dehydration than for SS RBCs. The lower MCV ofIHP-RBCs confirms this hypothesis and indicates thatIHP-RBCs are microcytotic. These smaller RBCs couldprobably flow more easily through small capillaries thanSS RBCs with greater MCV and could compensate, tosome extent, for the “bad side” of reduced deformability incomparison with AA RBCs.

The results obtained with the stability test demon-strate that SS-RBCs, in addition to having impaireddeformability, exhibit poor membrane stability. Applying ashear stress of 70 Pa resulted in a deformability loss of 50%indicating membrane fragmentation in SS RBCs.22 In con-trast, when IHP-RBCs or AA RBCs were added to the SS RBCsuspension, the losses of deformability were only 22 and25%, respectively. The effect of IHP-RBCs and AA RBCs onthe membrane stability of SS RBC suspensions was similar.RBC membrane stability and deformability are two distinctmembrane properties that are independently regulated bycytoskeletal protein associations.22 The fact that IHP-RBCdeformability is reduced despite the fact that membrane

stability is close to the membrane stabil-ity of AA RBCs when mixed with SS RBCssuggests that some membrane proteinsinteractions have been probably modi-fied during the process used for IHPencapsulation into RBCs.

Experiments conducted on theaggregation properties of IHP-RBCsdemonstrate a reduced aggregability incomparison with SS RBCs and AA RBCs.RBC aggregation has been proven toplay an important role in blood flowhemodynamics in various vessels.23,24

Increased RBC aggregation causes anincrease in blood flow resistance in largevenous blood vessels (i.e., >1 mm diam-eter) where the shear forces are not highenough to disperse RBC aggregates.23,24

There is also experimental evidencesuggesting a significant influence ofincreased RBC aggregation on microcir-culatory and arterial hemodynamicmechanisms where the shear forces are

high. For example, Kim and colleagues25 reported thatincreased RBC aggregation decreased functional capillarydensity in the microcirculation of the rat spinotrapeziusmuscle. One of the possible mechanisms is that enhancedRBC aggregation increases the difficulty to fully disperseRBC aggregates before they can be able of entering intosmall capillaries.24 Finally, a clinical study performed inpatients suffering from peripheral occlusive arterialdisease observed a negative relationship between transcu-taneous oxygen pressure and RBC aggregation level, sug-gesting that increased RBC aggregation affects oxygensupply to tissues.26 Therefore, the reduced aggregability ofIHP-RBCs observed in our study could be beneficial forSCA patients from a hemodynamic point of view.

When mixed with SS RBCs, the AI was lower than for SSRBCs alone or SS plus AA RBCs confirming the aggregationimpact of IHP-RBCs. RBC aggregation has not been exten-sively studied in SCA but a recent study demonstrated thatpatients are characterized by a 2.5- to threefold greater RBCaggregates strength than healthy subjects.21 The increasedshear forces needed to disperse RBC aggregates in SCA maydisturb blood flow, especially at the microcirculatory level,since RBCs are only able to pass through narrow capillariesas single cells rather than as aggregates.21 Thus, one couldsuggest that the lower AI obtained in SS plus IHP-RBCs incomparison with SS RBC or SS plus AA RBC suspensionsshould improve blood flow in microcirculation, as well asin macrocirculation.

Although the deformability of IHP-RBCs has beenfound to be impaired, the lower MCV and RBC aggregabil-ity of these RBCs resulted in a lower blood viscosity thanfor SS RBCs. Blood viscosity has been measured at high

Fig. 3. Typical stability test performed at 70 Pa for 30 minutes in SS RBCs (�),

SS plus IHP-RBCs (¥), and SS plus AA RBCs (�). The decrease in EI over time (%) was

greater in SS RBCs than in SS plus IHP-RBCs or SS plus AA RBCs.

LAMARRE ET AL.


shear rate (i.e., 225/sec) and it is usually thought that RBCaggregation has little impact on blood viscosity in thiscondition. However, the minimal shear rate to disperseRBC aggregates is particularly elevated in SCA with amean value of 315/sec.21 Thus, blood viscosity of the dif-ferent RBC suspensions in our study was also influencedby the RBC aggregation properties with some RBC aggre-gates still present at the shear rate used. The mixing of SSRBCs with IHP-RBCs decreased blood viscosity of SS RBCsand the decrease was more marked with the use of AARBCs. Increased blood viscosity has been associated withclinical severity in SCA with blood viscosity being posi-tively correlated with end organ failure27 or the occurrenceof frequent vasoocclusive crises.28 When SCA patients aretransfused, physicians may pay attention to the posttrans-

fusion blood viscosity since it may compromise tissue per-fusion8 and lead to serious adverse events.9 The decreaseof blood viscosity of SS RBC suspensions obtained withthe use of IHP-RBCs is therefore interesting since it maylimit blood hyperviscosity in SCA patients. When usingnormal AA RBCs for simple transfusion or exchange trans-fusion, it is recommended not to exceed a posttransfusionHct of 35% to avoid posttransfusion blood hyperviscosity.7

Since the benefits of using IHP-RBCs on the blood viscos-ity of SS RBCs (30:70 vol/vol) is a little bit less than thebenefits obtained with the use of AA RBCs in the sameproportion, one could recommend using a lower volumeof IHP-RBCs than AA RBCs in the perspective of transfu-sion therapy. This should also play down the negativeeffects of IHP-RBCs on the SS RBC deformability.

Fig. 4. (A) Effect of hypoxic challenge on RBC deformability (EI) measured at 1.08 Pa in SS RBC (n = 6), SS plus IHP-RBC (n = 7),

and SS plus AA RBC (n = 6) suspensions. *Significant difference (p < 0.05). (B) Effect of hypoxic challenge on blood viscosity deter-

mined at 225/sec Pa in SS RBC (n = 6), SS plus IHP-RBC (n = 6), and SS plus AA RBC (n = 6) suspensions. *Significant difference

(p < 0.05). NX = normoxic condition: RBC suspensions were equilibrated with air by continuous mixing on a tube roller for 30

minutes. HXO2 = hypoxic (plus partial reoxygenation) challenge: RBC suspensions were deoxygenated for 20 minutes of gas

bubbling at 37°C with 99.6% nitrogen (PO2 ª 0 mmHg) and then partially reoxygenated for 20 minutes of gas bubbling at 37°C with

5% O2.



SCA patients alternate steady-state phases withpainful crisis phases, with the frequency and gravity of thelatter ones varying from one patient to another. Duringpainful vasoocclusive crisis, RBC deformability severelydecreases below steady-state value29 and blood viscosityexhibits a large increase above baseline value.30-32

Dynamic changes in RBC deformability and blood viscos-ity reflect a change in the health condition of SCA patients.Hypoxia triggers HbS polymerization and sickling of SSRBCs in SCA.33,34 Indeed, any SS RBCs residing in an area oflow oxygen tension long enough to permit nucleation andthe explosive growth of polymer will become sickle andcapable of obstructing flow and initiating vasoocclusion.35

Since IHP-RBCs have been demonstrated to prevent sick-ling of SS RBCs under hypoxic challenge,10 we tested theeffects of IHP-RBCs on the deformability and viscositychanges of SS RBC suspensions under hypoxia. The shearstress used to test the effects of the hypoxic protocol onRBC deformability was 1.08 Pa because it was close to themean value found in microcirculation. For example, inconjunctival microvessels, the shear stress may vary froma maximum of 9.55 Pa in the smallest capillaries to 0.28 Pain the highest postcapillary venules with a mean value of1.54 Pa for all microvessels.36 The hypoxic challenge wassimilar to the one used by Bourgeaux and coworkers,10

which has been demonstrated to affect sickling in SSRBCs. Our study demonstrates the efficacy of the hypoxicchallenge on the rheologic changes of SS RBCs since, after20 minutes nitrogen and 20 minutes 5% O2, RBC deform-ability was significantly reduced below the prehypoxicchallenge values. When mixed with AA RBCs, the effect ofhypoxia on the deformability of SS RBCs was partiallyblunted but still present since deformability significantlydecreased below prehypoxic values. The diluting effect ofAA RBCs did not counteract the increase of sickle and rigidSS RBCs number caused by hypoxia. In contrast, themixed SS plus IHP-RBC suspension was not affectedby hypoxia that is probably the direct consequence ofthe sickling inhibition of SS RBCs by IHP-RBCs underhypoxia.10 During the partial reoxygenation state with 5%O2, IHP-RBCs release their oxygen to the adjacent SS RBCsmaking them better oxygenated and less able to becomesickle and rigid.10,11 The positive hemorheologic effect ofIHP-RBCs on SS RBC suspensions during hypoxia wasobserved for blood viscosity too since hypoxia signifi-cantly increased blood viscosity above prehypoxic valuesin SS RBCs alone and SS plus AA RBC suspensions but notin SS plus IHP-RBC suspension.

Although the use of IHP-RBCs to dilute SCA bloodseems to decrease RBC aggregation, blood viscosity and tobe effective in preventing SS RBC sickling and rheologicchanges under hypoxia, the low baseline RBC deformabil-ity of these RBCs could be a concern when infused in vivointo blood circulation. However, a very recent study12 hasinvestigated the in vivo effects of chronic transfusion

therapy of murine IHP-RBCs or murine AA RBCs on thesurvival rate and the inflammatory response of Berkeleysickle mice over a period of 7 weeks. As for humanIHP-RBCs, murine IHP-RBCs have lower deformability(EImax = 0.21) than murine AA-RBCs (EImax = 0.27).These studies were designed with partial RBC exchangesallowing a constant proportion of 15% to 20% murine IHP-RBCs or AA RBCs into Berkeley sickle mice circulation.12

Sickle mice receiving murine IHP-RBCs exhibited a sig-nificant decrease in reticulocyte counts and a reducedinflammatory state compared to mice treated with murineAA-RBCs. In addition, the chronic transfusion therapy ofsickle mice with murine AA RBCs or IHP-RBCs increasedtheir survival rate compared with mice that did not receiveany blood transfusion, but the survival rate at 7 weeks oldwas greater with murine IHP-RBCs (86%) than with AARBCs (75%).12 The authors also tested the effects of a singletransfusion of IHP-RBCs in transgenic HbS/S SAD miceexposed to acute hypoxic stress and demonstrated thatlung VCAM-1 mRNA was reduced sevenfold compared tomice who received AA RBCs.12

In conclusion, our in vitro findings suggest that boththe use of classical RBCs (i.e., AA RBCs) and IHP-RBCscould improve the rheology of SCA patients. The use of AARBCs provides the greater benefits on the baselinedeformability and blood viscosity of SCA patients. IHP-RBCs have been demonstrated to lower RBC aggregation,and to a lesser extent than AA RBCs, to lower blood viscos-ity of SCA blood. In addition, one of the most importantfindings of this study is the beneficial hemorheologiceffects of IHP-RBCs on SS RBCs when stressed by hypoxia,with greater benefits obtained in SS plus IHP-RBC sus-pension than in SS plus AA RBC suspension. In summary,the present results provide a strong basis to go further indepth in the assessment of the effects of IHP-RBCs intransfusion therapy in SCA.

ACKNOWLEDGMENTS

We thank Drs F. Lionnet, M. Benkerou, A. Kamdem, C. Arnaud,

and I. Hau, as well as nurses of medical departments, for supply-

ing blood samples.

CONFLICT OF INTEREST

YG is the head of Erytech Pharma. VB and YC are employees of

Erytech Pharma. The other authors have no conflict of interest.

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Effect of inositol hexaphosphate-loaded red blood cells (RBCs) on the rheology of sickle RBCs

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