1. Background
Materials and Methods
In vitro blood testing is used in numerous settings including in
laboratories and clinical offices. These diagnostic tests are used to
detect a variety of medical complications. The reliability of such
tools may be compromised by factors such as blood age, blood-
device interactions, and the blood drawing procedure. Hemolysis,
which refers to the rupture of red blood cells, releases hemoglobin
into the plasma. Therefore, we have characterized healthy and
damaged blood at different time points after it has been drawn to
evaluate the viability of blood and to determine which blood
parameters are likely to be affected.
• To study and compare blood characteristics at different blood
ages (time after blood draw)
• To compare the blood characteristics and fragility parameters of
different species (porcine, human).
The results of this research are intended to improve in vitro
blood testing methodologies.
Blood Parameters:
To study the change in blood characteristics due to blood
aging, the blood was tested on different days. When not in use, the
blood was kept refrigerated. The following conditions were used for
testing:
• Hematocrit was adjusted to 36%
• Anticoagulation was achieved with either acid citrate dextrose
solution A (ACDA) (for porcine and human blood) or 3.2%
sodium citrate (for human blood only)
• Blood Age: fresh blood (Day 1), Day 3 blood, and Day 8 blood
• Blood Species: porcine and human
Day 1 human Day 8 human
plasma plasma
Rocker Bead Fragility Test:
Four tubes were rocked, with 15 stainless steel beads in each, in
order to test blood fragility on Day 1. The following conditions were
used for this test:
• 3 mL of blood
• 24º rocking angle
• 30 minute run time
• 30 cycles per minute
Blood Characterization Tests:
The density of each sample was found by weighing 1.000 ml of
blood on a balance and making three measurements.
A Complete Blood Count (CBC) System was used to determine
the RBC count, WBC count, platelet count, and mean cell
volume (MCV) of each blood sample.
pH and partial pressure of oxygen (P O2) and carbon dioxide
(P CO2) were measured using a blood gas analyzer.
Plasma protein concentration was measured optically using a
total solids refractometer.
Blood and plasma viscosity and elasticity were measured by
loading 0.5 ml of blood into an oscillatory rheometer, which uses a
piston to generate oscillatory blood flow through a small diameter
tube at known shear stresses. Viscosity was measured over a
shear rate range of 5 to 1000 1/s.
The Impact of Blood Age and Mechanical Damage
on Measured Physical Parameters of Red Blood Cells
Naomi Dereje, Luke Herbertson, Richard Malinauskas
OSEL/Division of Applied Mechanics
Results
Blood
Age
Viscosity
(cP) @
1000 1/s
Plasma
Protein
(g/dL)
Glucose
(mg/dL)
P O2
(mmHg)
P CO2
(mmHg)
pH
RBC Count
(*1012
/L)
MCV (fL)
Platelet
Count
(*109
/L)
Plasma
Free Hb
(mg/dL)
Day 1 2.52±0.21 5.7±0.7 89±29 34±6 38±1 7.4±0 3.94±0.62 90.3±10.4 165±30 1.9±0.8
Human
3.2% Na
Cit
Day 3 2.88±0.61 6.3±0.7 60±13 50 42 7.3 4.08±0.12 84.4±2.1 114±57 28.2±8.6
Day 8 2.64±0.45 6.1±0.3 23±15 176 18 7.4 4.08±0.28 83.3±5.2 134±26 136.8±45.0
Day 1 2.47±0.04 6.1±0.6 418±40 71±6 67±15 6.8±0.1 6.78±0.81 55.1±2.7 247±80 14.0±5.7
Porcine
ACDA
Day 3 2.68±0.22 6.5±0.9 411±45 72±9 46±17 6.9±0.1 6.09±0.19 54.1±1.9 260±77 31.1±9.9
Day 8 2.64±0.18 6.6±0.6 411±34 82±7 36±3 6.9±0 6.03±0.28 55.0±2.6 278±78 53.7±10.3
The following table displays the different characteristics of
each blood pool. For sodium citrate human blood we observed
most of the parameters measured were not affected by blood
age, however platelet count and P CO2 decreased while P O2
increased. For ACDA porcine blood, the difference is seen in the
platelet count, which increased instead. Other trends are similar,
but the anticoagulant appears to improve the long term viability of
the blood.
Blood Age Effects:
The figure to the right
compares the plasma free
hemoglobin levels of human
blood and porcine blood on each
test day, showing the damage
effects of blood aging. It should
be noted that the human blood
was anticoagulated with sodium
0
20
40
60
80
100
120
140
160
Day 1 Day 3 Day 8
PlasmaFreeHemoglobin
(mg/dL)
Blood Age
Human Porcine
n=4
Blood Damage Effects:
Both the human and porcine blood hemolysis levels were
affected similarly by the rocker bead fragility test, shown by the
graph below. The initial hemolysis level (t = 0) was always higher
for the porcine blood than it was for human blood. The RBC count
decreased in porcine blood with damage, but stayed constant in
human blood.
0
50
100
150
0 30
PlasmaFree
Hemoglobin(mg/dL)
Time (minutes)
Human n=2 Porcine n=4
Conclusions
RBC Count
(*1012
/L)
MCV (fL)
Platelet
Count
(*109
/L)
Plasma Free
Hb (mg/dL)
Human
Healthy 3.64 92.2 164 2.1
Damaged 3.68 92.2 167 112.7
Porcine Healthy 6.56 57.7 227 29.2
Damaged 5.86 57.9 270 137.3
• Human and porcine blood are affected similarly by mechanical
damage.
• Human blood with sodium citrate resulted in more hemolysis
due to aging than ACDA porcine blood.
• ACDA blood is more viable than sodium citrate blood over time,
as seen from the plasma free hemoglobin levels, glucose
concentration, and platelet count.
• P O2 increases in human blood as blood age increases.
Objectives and Significance
n=3
citrate, whereas the porcine blood contained ACDA. Thus, the
anticoagulant also likely plays a role in how the blood is damaged
over time.
Anticoagulant Effects:
ACDA resulted in much higher glucose levels compared to
sodium citrate. The blood gas levels were higher, and the pH was
lower, when using ACDA as opposed to sodium citrate.