Engineering, 2012, 5, 8-10
doi:10.4236/eng.2012.410B003 Published Online October 2012 (http://www.SciRP.org/journal/eng)
Red Blood Cell Mechanical Stability
Oguz K . Baskurt
Koc University School of Medicine, Rumelifeneri Kampusu, Sariyer, Istanbul, Turkey
Received 2012
ABSTRACT
It has been well documented that shear forces effective above a certain magnitude under flow conditions causes damage in blood
cells. This damage ranges from slight morphological alteration to the destruction of red blood cells (RBC). The hemolytic trauma to
RBC can easily be detected by measuring free hemoglobin level in plasma, however there are no standardized protocols to quantitate
subh emolytic trau ma. Ektacyto metr y has been used to study the alt eration s in RBC mechan ical pr operties i nduced by the appl ication
of shear stress at various levels. Additionally, a protocol for measuring the hemolytic threshold as an indicator of subhemolytic dam-
age to RBC has been developed based on ektacytometry. These standardized protocols may find applications in the pre-clinical and
clini cal evaluati on of artifici al organs and biomedical devices contacting with blood.
Keywords: Artificial Organs; Blood D a mage; Su bhemolytic Damage; Ektacytometry
1. Introduction
Red blo od cells (RBC) are exp osed to mechanical st resses dur-
ing their “everyday life”, at various levels of the circulatory
s ys t em. They respond to this effect by changing their shape and
orientation to the flow conditions; but they are not overtly
damaged by these forces, under physiological conditions.
However, if they are exposed to non-physiological conditions,
like in case of circulating through artificial organs or devices,
they might be affected resulting in deteriorations at various
degrees, ranging from slight morphological alterations to the
rupture of their membrane resulting in hemolysis [9].
The most effective factors under such conditions include the
extremely high shear forces to which RBC are exposed to dur-
ing their passage through the artificial flow environments [5, 9].
Additionally, turbulence, cavitation, contact of blood cells with
artificial su rfaces, and collisi on between cells or b etweens cells
and various parts of the device may also damage the cellular
components of blood [5, 9]. Shear forces in the human arterial
system are in the range of 1-10 Pascal (Pa) [7], while in artifi-
cial circulatory environments (e.g., ventricular assist devices,
total artificial heart, extracorporeal circulation systems, heart
valves, stents, hemodialysis systems) these forces may ap-
proach to several hundred Pa [5].
2. Mechanical Trauma to Blood
It is well known for many decades that shear forces exceeding a
critical level may cause hemolysis [1 2]. It has been demon-
strated that both magnitude and exposure time are important
determinants of the hemolytic effect of shear stress [6]. Hemo-
lytic damage to RBC can easily be quantitated by measuring
free hemoglobin in the suspending medium of blood (plasma),
which is usually expressed as the ratio to the total hemoglobin
content of whole blood. This hemolysis ratio or its normalized
forms are widely used in expressing the extent of mechanical
trauma to blood and used for the evaluation of artificial circu-
latory environments [9].
However, it should be noted that hemolysis is the end-stage
damage to RBC, corresponding to the destruction of the cell. It
has been documented that the mechani cal impact to RBC under
the hemolytic threshold also induce alterations in the cellular
properties, at varying degrees determined by the magnitude of
the effectiv e forces [9,15]. It has been argued that even physio-
logical shear forces u nder normal circul atory conditions induce
slight changes in RBC properties, which may accumulate dur-
ing the life-span of RBC (~120 days for human RBC). This
“non-lethal” trauma to RBC might be enhanced if they are ex-
posed to the conditions in artificial circulatory environment [9].
Subhemolytic trauma might be manifest by shortened life
span [3], increased trapping i n spleen [14], morphological alte-
rations [13], or deterioration in mechanical properties of the
cells [2] . Subhemolytic trauma is also determined by both the
magnitude and duration of the effective shear stresses [2,9 ].
RBC membrane st abili ty is affected by subh emolytic trau ma,
resulting in increased mechanical fragility indicated by higher
rate of hemolysis during the subsequent exposure to extreme
shear forces. Various tests to determine mechanical fragil ity has
been developed and proposed to be used in testing the artificial
circulatory environments [4 ,8 ]. Mechanical fragility is ex-
pressed as percent free hemoglobin following a standardized
mechanical stress to RBC suspensions [9]. However, the mag-
nitude of the mechanical impact to which RBC are exposed
cannot be standardized or even accurately estimated with the
propos e d pr ot oc ol s [4,8 ].
3. Detection of Subhemolytic Damage to Red
Blood Cells
Accurate quantification of subhemolytic damage to RBC is
important in studies aiming to evaluate the blood damage in-
duce by artificial organs and biomedical devices contacting
with blood. Additionally, test protocols and models based on
subhemolytic levels of mechanical trauma to RBC may help

O. K. BASKURT
9
designers to improve the safety limits of the artificial circulato-
ry systems, with respect to blood trauma.
It has been demonstrated that a Couette-type viscometer is a
useful apparatus to apply shear stresses with well-defined and
constant geometry in a wide range of magnitude. Instruments
equipped with a laser-diffraction analysis capability in addition
to a shearing apparatus have been used to analyze the altera-
tions in the deformability of RBC exposed to subhemolytic
levels of shear stress [2,10,11]. Application of 120 Pa of shear
stress for periods of 15 to 120 seconds to RBC suspensions
resulted in a deterioration in deformability, the degree of im-
pairment in elongation indexes being a function of the duration
of the mechanical stress [2]. Figure 1 demonstrates the shear
stress-elongation index curves of RBC suspensions before and
after the application of 100 Pa shear stress for 300 s. The im-
pairment of deformability under a standardized shearing proto-
col has been observed to reflect the “health” of RBC prior to
the application of the shear stress; a subhemolytic trauma to
RBC (e.g., due to circulating in a artificial organ) relates to a
higher level of impairment following the shearing. RBC with
altered membran e stabili ty (e.g., du e to exposure to lanthanides
of variou s ionic r adius) were charact erized b y different degree s
of impairment in deformability following a standard application
of shear stress (i.e., 290 Pa for 300 s) [1]. These observations
indicate that ektacytometry can be used as a standardized, sub-
hemolytic mechan ical damage test.
Figure 1. Shear stress-elongation index curves for red blood cell
suspensions before and after being exposed to 100 Pascal shear
stress for 300 seconds.
Figure 2. Number of red blood cells (RBC) in suspensions exposed
to gradually increasing shear stress before and after the application
of 100 Pa scal s hear s tress for 300 s eco nds. T he number of RB C was
estimated by the area of ellipti cal d iffraction pattern.
4. Hemolytic Threshold Determined by
Ektacytom etry
Normal RBC can withstand to shear stresses up to 300 Pa,
without being hemolysed. However, alterations in membrane
stability may significantly reduce this hemolytic threshold. It is
worthwhile to measure this hemolytic threshold as an indicator
of the accumulated subhemolytic damage to RBC during pre-
vious life-span (e.g., i n an artificial organ syste m) .
Ektacytometry might be used as an easily applicable test
protocol to measure the hemolytic threshold, as an indicator of
subhemolytic trauma to RBC. The coaxial-cylindrical shearing
apparat us of an ektacytometer ha s been used to appl y gradu ally
increasing shear stresses (in the range of 0 to 400 Pa) to RBC
suspensions and the laser diffraction images have been moni-
tored. It has been recognized that elongation indexes remained
relatively constant while the area of the elliptical diffraction
pattern has changed more significantly during the course of
shear stress application. Preliminary data indicates that the
ellipse area is an accurate indicator of the number of RBC in
the suspension under test. Figure 2 demonstrat es th e alt erati on s
in the number of RBC as a function of gradually increasing
shear stress. The curve labeled as “Before” reflects the change
in the number of RBC in suspensions before being exposed to
shear stress, which indicates a sharp decrease in RBC number
as shear stress approaches to 300 Pa. This level of shear stress
is accepted as the hemolytic threshold. The curve labeled as
“After” reflects the same test applied to RBC suspensions fol-
lowing a prior exposure to 100 Pa shear stress for 300 s, indi-
cating a very significantly reduced hemolytic threshold.
5. Conclusion
Ektacyto metry can b e develop ed into a useful app roach in stud-
ying the subhemolytic mechanical trauma to blood, to be used
in the pre-clinical and clinical evaluation of artificial organs
and biomedical devices contacting with blood.
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