ArticlePDF Available

In vitro hemocompatibility testing: The importance of fresh blood

DOI:10.1116/1.4941850
Authors:
Sjoerd Leendert Johannes Blok at ICON Plc
Sjoerd Leendert Johannes Blok
Gerwin E Engels at HaemoScan BV
Gerwin E Engels
  • HaemoScan BV
Wim van Oeveren
Wim van Oeveren
Download full-text PDFDownload full-text PDF
Read full-text
Download citation

Abstract and Figures

The use of unactivated blood for hemocompatibility testing is essential to obtain reliable results. Here, the authors study the influence of heparinized whole blood storage time and temperature on blood activation and evaluate the importance of initiating hemocompatibility tests within 4 h of blood collection. Blood from healthy volunteers was collected and analyzed with minimal delay, after 30 min and after 60 min of storage at room temperature, 30 or 37 °C. In addition, blood was analyzed after 1, 2, or 4 h of storage at room temperature. Platelet count, mean platelet volume, platelet binding capacity to collagen and thromboxane B2 were measured to assess platelet function, complement complex C5b-9 and elastase were measured to assess activation of the inflammatory response system, and thrombin-antithrombin III was measured to assess activation of the coagulation system. Furthermore, free hemoglobin was measured in platelet poor plasma as an indicator for red blood cell damage. The authors found that storage at 30 °C significantly increased platelet and coagulation activity after 60 min and storage at 37 °C significantly increased platelet, coagulation, and white blood cell activity after 60 min. Storage at room temperature significantly decreased platelet binding to collagen after 4 h and increased platelet activity after 1 h onward and white blood cell activity after 4 h. Their results show that short-term storage of heparinized whole blood significantly influences biomarkers over time, especially at 30 and 37 °C compared to room temperature. However, blood stored at room temperature for 4 h is also affected. In particular, platelet function and white blood cell activity are significantly influenced after 4 h of stationary storage at room temperature; therefore, the authors propose that hemocompatibility tests should be initiated well within 4 h of blood collection, preferably within 2 h.
Figures - uploaded by Gerwin E Engels
Author content
All figure content in this area was uploaded by Gerwin E Engels
Content may be subject to copyright.
In vitro hemocompatibility testing - The importance of fresh blo
od.pdf
Content uploaded by Sjoerd Leendert Johannes Blok
Author content
All content in this area was uploaded by Sjoerd Leendert Johannes Blok on Jun 20, 2016
Content may be subject to copyright.
In vitro hemocompatibility testing - The importance of fresh blo
od.pdf
Content uploaded by Gerwin E Engels
Author content
All content in this area was uploaded by Gerwin E Engels on Feb 14, 2016
Content may be subject to copyright.
In vitro hemocompatibility testing: The importance of fresh blood
Sjoerd Leendert Johannes Blok, Gerwin Erik Engels, and Willem van Oeveren
Citation: Biointerphases 11, 029802 (2016); doi: 10.1116/1.4941850
View online: http://dx.doi.org/10.1116/1.4941850
View Table of Contents: http://scitation.aip.org/content/avs/journal/bip/11/2?ver=pdfcov
Published by the AVS: Science & Technology of Materials, Interfaces, and Processing
Articles you may be interested in
Thrombogenicity and hemocompatibility of biomaterials
Biointerphases 11, 029601 (2016); 10.1116/1.4938557
Early detection of cell activation events by means of attenuated total reflection Fourier transform infrared
spectroscopy
Appl. Phys. Lett. 104, 243705 (2014); 10.1063/1.4885081
Instrument and technique for the in vitro screening of platelet activation from whole blood samples
Rev. Sci. Instrum. 78, 054302 (2007); 10.1063/1.2740481
Designer gene networks: Towards fundamental cellular control
Chaos 11, 207 (2001); 10.1063/1.1345702
Dry film preparation from whole blood, plasma and serum for quantitative infrared diffuse reflectance
spectroscopy
AIP Conf. Proc. 430, 278 (1998); 10.1063/1.55780
In vitro hemocompatibility testing: The importance of fresh blood
Sjoerd Leendert Johannes Blok and Gerwin Erik Engels
HaemoScan BV, Stavangerweg 23-23, 9723 JC Groningen, The Netherlands
Willem van Oeveren
a)
HaemoScan BV, Stavangerweg 23-23, 9723 JC Groningen, The Netherlands
and Department of Cardiothoracic Surgery, University of Groningen,
University Medical Center Groningen, 9713 GZ Groningen, The Netherlands
(Received 23 December 2015; accepted 2 February 2016; published 12 February 2016)
The use of unactivated blood for hemocompatibility testing is essential to obtain reliable results.
Here, the authors study the influence of heparinized whole blood storage time and temperature on
blood activation and evaluate the importance of initiating hemocompatibility tests within 4 h of
blood collection. Blood from healthy volunteers was collected and analyzed with minimal delay,
after 30 min and after 60 min of storage at room temperature, 30 or 37 C. In addition, blood was
analyzed after 1, 2, or 4 h of storage at room temperature. Platelet count, mean platelet volume,
platelet binding capacity to collagen and thromboxane B2 were measured to assess platelet
function, complement complex C5b-9 and elastase were measured to assess activation of the
inflammatory response system, and thrombin-antithrombin III was measured to assess activation of
the coagulation system. Furthermore, free hemoglobin was measured in platelet poor plasma as an
indicator for red blood cell damage. The authors found that storage at 30 C significantly increased
platelet and coagulation activity after 60 min and storage at 37 C significantly increased platelet,
coagulation, and white blood cell activity after 60 min. Storage at room temperature significantly
decreased platelet binding to collagen after 4 h and increased platelet activity after 1 h onward and
white blood cell activity after 4 h. Their results show that short-term storage of heparinized whole
blood significantly influences biomarkers over time, especially at 30 and 37 C compared to room
temperature. However, blood stored at room temperature for 4 h is also affected. In particular, pla-
telet function and white blood cell activity are significantly influenced after 4 h of stationary stor-
age at room temperature; therefore, the authors propose that hemocompatibility tests should be
initiated well within 4 h of blood collection, preferably within 2 h. V
C2016 Author(s). All article
content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY)
license (http://creativecommons.org/licenses/by/4.0/).[http://dx.doi.org/10.1116/1.4941850]
I. INTRODUCTION
The use of medical devices in the cardiovascular system
has increased the demand for evaluation of their effects on
blood. Direct contact of biomaterials with blood results in the
activation of platelets (PLTs), the coagulation system, com-
plement cascades and white blood cells.
1
These cascades are
also activated during blood collection and storage before
hemocompatibility testing or before processing in the clinical
laboratory. Even with the use of inert storage containers,
blood activation can occur due to the absence of endothelium-
derived inhibitors. Due to irreversible activation of these cas-
cades, a loss of function can ultimately result in false negative
or positive data during analysis. Therefore, the stability and
functionality of blood samples are continuously discussed in
the clinical community, especially the lesion of stored
platelets.
2,3
In order to obtain unbiased hemocompatibility results, it
is important to use unactivated blood which is as similar to
the clinical setting as possible.
1
By using a clinical dose of
heparin (1.5 IU/ml), a dose often used during cardiovascular
procedures, blood coagulation can be prevented without
inactivating the cascades,
1
which are needed for hemocom-
patibility testing.
Braune et al. stated that the total test duration should not
exceed 4 h to ensure an appropriate function of blood cells
and blood plasma proteins,
4
since it is observed that blood
activation starts shortly after blood collection. Thus, blood
storage time and temperature between collection and initia-
tion of hemocompatibility tests could significantly influence
results of hemocompatibility testing. The ISO 10993-Part 4
standard advises that hemocompatibility tests should be per-
formed with minimal delay, usually within 4 h of blood col-
lection,
5
without reference to valid data. Following this
standard, several hemocompatibility studies described initia-
tion of hemocompatibility tests within 4 h of blood collec-
tion.
6,7
However, there are also numerous studies regarding
hemocompatibility testing without any note on the analysis
time. There are few studies describing the effects of short-
term storage on biomarkers. A previous study described the
influences of citrated whole blood- and platelet rich plasma
(PRP) storage times on platelet- morphology and function
and stated that platelet morphology and function, particularly
platelet reactivity to adherent or soluble agonists changed
rapidly outside the vascular system.
8
Another study reported
the stability of complement biomarkers in whole blood
a)
Electronic mail: w.vanoeveren@haemoscan.com
029802-1 Biointerphases 11(2), June 2016 1934-8630/2016/11(2)/029802/6 V
CAuthor(s) 2016. 029802-1
containing citrate or ethylenediaminetetraacetic acid
(EDTA) and described that C4d, C3a, factor Bb, C5a and
C5b-9 were stable at room temperature for up to 4 h and that
thawing at 37 C resulted in increased levels of complement
markers in serum and citrated plasma but not in EDTA
plasma.
9
However, to our knowledge, there are no reports on
short-term stability of the key determinants of hemocompati-
bility in heparinized whole blood.
The purpose of this study was to obtain further insight into
the effects of storage time and temperature prior to hemocom-
patibility testing. Therefore, we analyzed platelet function,
inflammatory response, coagulation and hemolysis of whole
blood over time. Blood from healthy volunteers was collected
and analyzed with minimal delay and after 30- and 60 min of
storage at room temperature, 30 or 37 C. From these data, it
was concluded that storage at room temperature was most
preferable. Subsequently, the experiment was repeated with
storage times up to 4 h at room temperature.
II. MATERIALS AND METHODS
A. Experimental setup
During the first part of this study, blood was collected and
stored at room temperature (21–25 C), 30 or 37 Cforupto
60 min. Key determinants of hemocompatibility were analyzed
and the effects of storage time and temperature were exam-
ined. These data clearly indicated that storage at room temper-
ature was preferable. Therefore, in the second part of the
study, whole blood was stored at room temperature for up to
4 h and the key determinants of hemocompatibility were ana-
lyzed and the effects of storage time were further examined. In
addition, concentrations of the generated activation markers:
Thromboxane B2 (TXB2), complement complex C5b-9, elas-
tase, thrombin-antithrombin III (TAT III) complex and free
hemoglobin (Hb) after stationary storage at room temperature
for 4 h were compared with the concentrations generated in
whole blood which had been in vitro circulated as previously
described.
10
The in vitro circulations (Hemobile, HaemoScan
BV, Groningen, The Netherlands) were performed in polyvi-
nyl chloride (PVC) circuits at 37 Cfor1h.
B. Blood collection
For the analysis of storage up to 60 min at different tem-
peratures, fresh human blood was collected by venipunc-
ture with a 19 Gauge butterfly needle from six healthy
adult volunteers (age 23–26) with a female to male ratio of
1:1 and anticoagulated with 1.5 IU heparin/ml (Leo
Pharmaceutical Products BV, Weesp, The Netherlands). In
addition, fresh human blood was collected and anticoagu-
lated in the same manner from nine healthy adult volun-
teers (age 19–28) with a female to male ratio of 5:4 for
storageupto4hatroomtemperature.Thein vitro circula-
tions were performed with fresh human whole blood from
six healthy volunteers which was collected and anticoagu-
lated as described above.
C. Storage
Heparinized whole blood was transferred to test tubes
(Cellstar
V
R
Tubes, Greiner Bio-One GmbH, Frickenhausen,
Germany) with minimal delay, and in the first part of the
study, they were stored stationary at room temperature
(21–25 C), 30 or 37 C for 30 or 60 min. Additionally, in the
second part of the study, heparinized whole blood was stored
in the same manner at room temperature for 1, 2, or 4 h.
D. Platelet function
Whole blood was centrifuged at 79gfor 5 min, and the
supernatant was used as PRP of which PLTs were counted
(cell counter Medonic CA 530, Medonic, Sweden). Platelet
count and mean platelet volume (MPV) were analyzed
(Medonic CA 530) on whole blood containing 5 mM EDTA
(preventing any further blood activation) and PLT count
was corrected for the EDTA dilution. The remaining EDTA
containing whole blood was centrifuged at 13 400gfor
1 min, and the supernatant was used as platelet poor plasma
(PPP).
Collagen is well known for its PLT binding capability;
thus, binding of PLTs to collagen was used to assess PLT
function. The capacity of PLTs to bind to collagen was ana-
lyzed in collagen-coated microtiter plates immediately after
PRP was obtained. Collagen-coated microtiter plates were
TABLE I. Activation markers of whole blood stored at room temperature, 30 or 37 C.
Room temperature 30 C37
C
Analyses 0 min 30 min 60 min p
a
30 min 60 min p
a
30 min 60 min p
a
PLT (10E9/L) 251 664 235 679 233 656 0.583 239 656 227 657 0.400 231 649 227 642 0.307
MPV (fl) 9.28 60.479 9.23 60.709 9.20 60.666 0.517 9.30 60.583 9.07 60.843 0.273 9.47 60.662 9.18 60.126 0.101
PLT-Col (%) 7.55 63.77 6.86 64.51 12.30 66.44 0.173 7.66 64.46 10.1 65.83 0.379 7.80 65.45 12.12 68.64 0.292
TXB2 (ng/ml) 0.966 61.33 2.4861.86 1.20 60.410 0.721 3.12 62.28 7.28 63.93 0.025 1.71 62.08 5.06 62.71 0.044
C5b-9 (ng/ml) 197 6401 242 6522 192 6417 0.611 296 6640 214 6464 0.562 294 6636 217 6390 0.227
Elastase (lg/ml) 1.95 61.16 1.85 60.819 2.35 61.41 0.668 2.54 61.02 3.09 61.64 0.187 2.10 60.803 4.12 61.85 0.001
TAT III (lg/ml) 1.35 60.840 2.07 62.54 2.19 61.50 0.362 1.97 62.71 5.29 63.16 0.031 1.40 61.26 4.88 61.95 0.022
Free Hb (%) 0.085 60.026 0.093 60.074 0.055 60.021 0.156 0.04360.026 0.055 60.035 0.156 0.118 60.054 0.100 60.063 0.653
a
Paired samples t-test between baseline (0 min) and 60min of storage. PLT, platelet count; MPV, mean platelet volume; PLT-Col, platelet binding to collagen;
TXB2, thromboxane B2; C5b-9, complement complex C5b-9; TAT III, thrombin-antithrombin III complex; and Free Hb, free hemoglobin.
029802-2 Blok, Engels, and van Oeveren: In vitro hemocompatibility testing 029802-2
Biointerphases, Vol. 11, No. 2, June 2016
obtained by incubation of 96 wells flat-bottom microtiter
plates (NUNC MaxiSorp
V
R
, Thermo Scientific, Inc., Roskilde,
Denmark) with 100 lg/ml bovine type I collagen (Collagen
solution from bovine skin, Sigma-Aldrich
V
R
Co. LLC., St.
Louis, MO, USA) in 50 mM sodium carbonate, pH9.6at
2–8 C overnight. After washing with phosphate-buffered sa-
line, pH 7.4 (PBS), PLT binding was achieved by incubating
PRP for 1 h at 37 C. After washing with PBS, bound PLTs
were analyzed based on the presence of acid phosphatase
11,12
by incubating with 5 mM 4-nitrophenylphosphatase in citrate
buffer þ1% (v/v) Triton X-100, pH 5.4, on a shaker for 1h.
After addition of 1 M sodium hydroxide, conversion of the
substrate was measured at 405 nm (PowerWave 200, Bio-Tek
Instruments, Inc., Winooski, VT, USA). Optical density was
related to PLT concentration using counted PLTs (Medonic
CA 530) as a standard curve and the percentage of bound
PLTs was determined.
Platelet activation leads to the activation of the arachi-
donic acid synthesis pathway to produce thromboxane A2.
1
Thromboxane A2 is highly unstable and rapidly converted to
TXB2,
13
i.e., TXB2 can be used as an indicator for PLT acti-
vation. Thromboxane B2 in PPP was analyzed by means of
an enzyme immunoassay (Cayman Chemical Company,
Michigan, USA), based on the competition between acetyl-
cholinesterase labeled TXB2 and sample TXB2. Conversion
of the substrate (Ellman’s reagent) was measured at 415 nm
(PowerWave 200).
E. Inflammatory response
Complement complex C5b-9 was analyzed as an indica-
tor for complement activation. Complement complex C5b-
9 in PPP was analyzed by means of an enzyme-linked im-
munosorbent assay (ELISA) based on a mouse anti human
C5b-9 capture antibody (DAKO, Glostrup, Denmark) and a
goat anti C5 detection antibody (Quidel, San Diego, CA,
USA).
Elastase was analyzed as an indicator for white blood cell
activation. Elastase in PPP was analyzed by means of
ELISA based on a capture antibody against human elastase
and a labeled antibody against alpha 1 antitrypsin (Affinity
Biologicals, Inc., Ancaster, Canada).
F. Coagulation
Thrombin-antithrombin III complex was analyzed to
determine thrombin formation, as an indicator for coagula-
tion activity. Thrombin-antithrombin III complex in PPP
was analyzed by means of ELISA, using antibodies of
Cedarlane Laboratories, Ltd. (Hornby, Canada).
G. Hemolysis
Free Hb was used as an indicator for hemolysis, i.e., red
blood cell damage, and was measured as described by
Harboe
14
(PowerWave 200). Percentage of hemolysis was
determined by comparison with a 100% hemolysis sample.
H. Statistical analysis
For the statistical analysis of storage up to 4 h at room
temperature, interdonor- and day-to-day variations were
eliminated by normalizing all data to a percentage of the
baseline (% baseline). Paired samples t-test was performed
for all blood parameters to assess any significant difference
(p<0.05) between the different storage times and the
FIG. 1. Influences of whole blood storage at room temperature, 30 or 37 C
on thromboxane B2, elastase and thrombin-antithrombin III release in
plasma during whole blood storage. (a) Thromboxane B2, (b) elastase and
(c) thrombin-antithrombin III. *p<0.05 compared with the baseline
(0 min).
029802-3 Blok, Engels, and van Oeveren: In vitro hemocompatibility testing 029802-3
Biointerphases, Vol. 11, No. 2, June 2016
baseline. Normally distributed variables were reported as
mean þone standard deviation.
III. RESULTS
A. Platelet function
Storage of whole blood at room temperature for up to
60 min did not change PLT count, MPV, PLT-collagen bind-
ing, or TXB2 concentrations over time (Table I). Storage of
whole blood at 30 or 37 C for up to 60 min did increase
TXB2 concentrations over time (Table I, Fig. 1). Storage of
whole blood at room temperature for up to 4 h did not affect
PLT count or MPV over time (Table II). However, PLT-
collagen binding decreased after 4 h (Table II,Fig.2) and
TXB2 concentrations increased after 1 h (p¼0.005), 2 h
(p0.000) and 4 h (p0.000) (Table II, Fig. 2). Generated
TXB2 of whole blood stored at room temperature for 4 h cor-
responded to 9% of the generated TXB2 in PVC circuits that
circulated on the Hemobile (Table III).
B. Inflammatory response
Complement complex C5b-9 and elastase of whole
blood stored at room temperature, 30 or 37 C, for up to
60 min did not change over time (Table I). Storage of whole
blood at 37 C for up to 60 min increased elastase over time
(Table I,Fig.1). Generated complement complex C5b-9 of
whole blood stored at room temperature for 4 h corre-
sponded to 0.1% of the generated complement complex
C5b-9 in PVC circuits that circulated on the Hemobile
(Table III). However, complement C5b-9 did not change
over time (Table II). Generated elastase of whole blood
stored at room temperature for up to 4 h increased over
time (Table II,Fig.2) and after 4 h corresponded to 87% of
the generated elastase in PVC circuits that circulated on the
Hemobile (Table III).
C. Coagulation
Thrombin-antithrombin III complex of whole blood
stored at room temperature for up to 60 min did not change
over time (Table I). Thrombin-antithrombin III complex of
whole blood stored at 30 or 37 C did increase over time
(Table I, Fig. 1). Generated TAT III complex of whole blood
stored at room temperature for 4 h corresponded to 14% of
the generated TAT III complex in PVC circuits that circu-
lated on the Hemobile (Table III). However, TAT III com-
plex of whole blood stored at room temperature for up to 4 h
did not change over time (Table II).
D. Hemolysis
Free Hb of whole blood stored at room temperature, 30 or
37 C, for up to 60 min did not change over time (Table I).
TABLE II. Activation markers of whole blood stored at room temperature for
1, 2, or 4-h.
Storage time (h)
Analyses (% baseline) 1 2 4 p
a
Platelet count 97.1 68.27 100 68.9 99.2 69.71 0.816
Mean platelet volume 101 67.37 102 66.5 99.8 65.91 0.913
Platelet-collagen binding 96.3 619.6 98.0 621.5 70.8 614.6 0.005
Thromboxane B2 149 638.5 236 668.8 335 6116 0.000
Complement complex C5b-9 95.8 69.15 98.1 68.08 102 67.64 0.409
Elastase 108 642.3 119 638.2 152 631.7 0.005
Thrombin-antithrombin III 86.3 644.0 101655.4 220 6170 0.086
Free hemoglobin 80.0 629.8 92.4 636.1 94.8 625.7 0.582
a
Paired samples t-test between baseline and 4 h of storage.
FIG. 2. Influences of whole blood storage at room temperature on platelet-
collagen binding, thromboxane B2, and elastase. (a) Platelet-collagen bind-
ing, (b) thromboxane B2, and (c) elastase. *p<0.05 compared with the
baseline (0 h).
029802-4 Blok, Engels, and van Oeveren: In vitro hemocompatibility testing 029802-4
Biointerphases, Vol. 11, No. 2, June 2016
Generated free Hb of whole blood stored at room tempera-
ture for 4 h corresponded to 38% of the generated free Hb in
PVC circuits that circulated on the Hemobile (Table III).
However, free Hb of whole blood stored at room temperature
for up to 4 h did not change over time (Table II).
IV. DISCUSSION
The aim of this study was to describe the influence of
storage time and temperature on the basic hemocompatibility
variables: platelet function, inflammatory response, coagula-
tion, and hemolysis in whole blood prior to hemocompat-
ibility testing. First, we determined the effect of storage
temperature—a low temperature is known to induce several
platelet changes;
15
therefore, we studied the temperature
effects between room temperature and 37 C. At room tem-
perature, none of the parameters changed up to 60 min of
storage time. However, storage at 30 or 37 C increased
blood activation after 60 min. These data clearly indicated
that storage at room temperature was preferable. To obtain
further insight into the influence of prolonged storage at
room temperature, the parameters described earlier were
also analyzed on whole blood stored at room temperature for
1, 2, or 4 h. Platelet count, MPV, complement complex C5b-
9, TAT III complex, and free Hb were not influenced by stor-
age at room temperature after 4 h as compared to baseline.
However, PLT-collagen binding decreased during 4 h of
storage at room temperature, TXB2 already increased after
1 h of storage at room temperature, and elastase increased
during 4 h of storage at room temperature. Generated throm-
boxane B2, complement C5b-9 and TAT III complex in
stored whole blood after 4 h were only 9%, 0.1%, and 14%
of the generated concentrations in circulated PVC circuits,
respectively. However, generated elastase and free Hb in
stored whole blood after 4 h were 87% and 38% of the gener-
ated concentrations in circulated PVC circuits, respectively.
However, as described earlier, free Hb did not change over
time. Therefore, these results indicate that storage at room
temperature does affect, in particular, platelets and white
blood cells.
Blood activation can have a great impact on the reliability
of blood tests, especially during hemocompatibility testing.
For instance, the use of already activated blood during
hemocompatibility testing could result in an underestimation
of blood activation initiated by the biomaterial of interest,
due to possible exhaustion of activation products. On the
other hand, it could intensify blood activation initiated by
the biomaterial, resulting in an overestimation of blood acti-
vation. Braune et al. already described the influences of
citrated whole blood and PRP storage times on platelet-
morphology and function and stated that platelet morphol-
ogy and function, particularly platelet reactivity to adherent
or soluble agonists, changed rapidly outside the vascular sys-
tem.
8
Yang et al. reported the stability of complement bio-
markers in whole blood containing citrate or EDTA.
9
However, in serum without anticoagulant, increased comple-
ment activation was observed during 4 h of storage, which
can be explained by the presence of complement cofactors:
Ca
2þ
and Mg
2þ
ions. Likewise, in heparinized blood, the
complement system is also prone to activation.
Our findings indicate that activation of platelets already
takes place after 1 h of storage at room temperature.
Furthermore, our findings indicate that reduction of platelet
binding to collagen and activation of white blood cells takes
place after 4 h. Therefore, our findings advocate hemocom-
patibility tests to be initiated well within 4 h of blood collec-
tion. However, several other arguments can be raised that
allow the use of 4 h old blood. First, a baseline can be col-
lected just prior to the use of stored blood. Second, during
biocompatibility testing, reference materials should always
be tested with the same blood as the material of interest to
be able to eliminate donor variability. Third, the activation
markers: TXB2, complement complex C5b-9, and TAT gen-
erated after 4 h of storage represent less than 15% of the gen-
erated concentrations after blood circulation in a PVC
circuit, indicating that blood after 4 h of storage still has the
capacity to respond to an activating surface. Furthermore, by
using a positive reference during in vitro circulations, higher
levels of activation markers can be achieved, resulting in an
even lower percentage of storage activation compared to
achievable activation during in vitro circulations.
Future studies may include further evaluation of 4 h
stored blood used for in vitro circulation and because hemo-
compatibility tests are also frequently performed on citrate-
anticoagulated blood, future studies may also include the
TABLE III. Generated activation markers of stored whole blood and in vitro circulations. PVC circuits: activation markers in whole blood before (baseline) and
after (circulated) in vitro circulation of hemocompatible PVC circuits at 37 C for 1 h; Storage: activation markers in whole blood before (baseline) and after
(stored) storage at room temperature for 4 h; TXB2, thromboxane B2; C5b-9, complement complex C5b-9; TAT III, thrombin-antithrombin III complex; and
Free Hb, free hemoglobin.
PVC circuits Storage
Analyses Baseline Circulated Increase (%) Baseline Stored Increase (%) DStorage/DPVC (%)
TXB2 (ng/ml) 966 61439 26408 612996 2632 475 698.8 1581 6709 233 9
C5b-9 (ng/ml) 13.7 65.65 53.18 647.9 289 22.9 622.1 23.01 620.44 0.36 0.1
Elastase (ng/ml) 1949 61160 294761319 51.1 1949 6363 2812 6968 44.2 87
TAT III (ng/ml) 13496839 8993 68634 566 659 61223 117962058 78.9 14
Free Hb (%) 0.10 60.03 0.25 60.14 159 0.03 60.03 0.04 60.03 60.4 38
029802-5 Blok, Engels, and van Oeveren: In vitro hemocompatibility testing 029802-5
Biointerphases, Vol. 11, No. 2, June 2016
effects of storage time and temperature before initiation of
hemocompatibility tests using citrate-anticoagulated blood.
V. CONCLUSION
While it has previously been stated that hemocompat-
ibility of blood contacting medical devices should be thor-
oughly tested before certification,
1
requirements regarding
the quality of blood used during hemocompatibility tests are
still in their infancy. As part of the quality of blood used dur-
ing hemocompatibility tests, information in relation to the
effects of time and temperature between blood collection
and hemocompatibility testing is still lacking. We observed
that storage at room temperature for up to 4 h does affect in
particular platelets and white blood cells. Although platelet
activation and platelet function were moderately affected,
white blood cell activation was similar to the activation
measured in blood which had been circulated in PVC cir-
cuits. Stationary storage of whole blood at room temperature
up to 4 h before initiation of hemocompatibility tests seems
unfavorable. Therefore, we propose hemocompatibility tests
to be initiated well within 4 h of blood collection, preferably
within 2 h of blood collection.
1
W. van Oeveren, Scientifica 2013, 392584 (2013).
2
M. Shrivastava, Transfus. Apheresis Sci. 41, 105 (2009).
3
D. V. Devine and K. Serrano, Clin. Lab. Med. 30, 475 (2010).
4
S. Braune, M. Grunze, A. Straub, and F. Jung, Biointerphases 8,33
(2013).
5
ISO 10993, Biological Evaluation of Medical Devices—Part 4: Selection
of Tests for Interactions with Blood (International Organization for
Standardization, Gene` ve, 2002).
6
T. C. Major, H. Handa, G. M. Annich, and R. H. Bartlett, J. Biomater.
Appl. 29, 479 (2014).
7
P. Jansen, W. van Oeveren, A. Capel, and A. Carpentier, Eur. J.
Cardiothorac. Surg. 41, e166 (2012).
8
S. Braune, M. Walter, F. Schulze, A. Lendlein, and F. Jung, Clin.
Hemorheol. Microcirc. 58, 159 (2014).
9
S. Yang, M. McGookey, Y. Wang, S. R. Cataland, and H. M. Wu, Am. J.
Clin. Pathol. 143, 558 (2015).
10
W. van Oeveren, I. F. Tielliu, and J. de Hart, Int. J. Biomater. 2012,
673163 (2012).
11
P. Bellavite, G. Andrioli, P. Guzzo, P. Arigliano, S. Chirumbolo, F.
Manzato, and C. Santonastaso, Anal. Biochem. 216, 444 (1994).
12
A. C. Eriksson and P. A. Whiss, J. Pharmacol. Toxicol. Methods 52, 356
(2005).
13
S. Saito, K. Yamasaki, S. Yamada, A. Matsumoto, T. Akatsu, N.
Takahashi, Y. Shibasaki, T. Suda, and T. Fukuhara, Bone Miner. 12,15
(1991).
14
M. Harboe, Scand. J. Clin. Lab. Invest. 11, 66 (1959).
15
M. G. Egidi, A. D’Alessandro, G. Mandarello, and L. Zolla, Blood
Transfus. 8, 73 (2010).
029802-6 Blok, Engels, and van Oeveren: In vitro hemocompatibility testing 029802-6
Biointerphases, Vol. 11, No. 2, June 2016
... Then, blood was recirculated for 4 hours, and timing samples were collected every 30 minutes; in addition, "still" control samples were collected at 0 and 4 hour time points. 11,13 Timing blood samples were processed immediately as described below. A 5 ml blood sample was centrifugated at 2,000g for 10 minutes at room temperature to obtain platelet-poor plasma. ...
... 41,42 Thus, Block et al. showed that alterations of platelet function in freshly collected heparin-anticoagulated human blood occurred even within four hours of storage, which reiterates the importance of utilization of freshly collected blood for hemocompatibility testing. 13 Shear-mediated alterations of platelet morphology result in platelet fragmentation and ejecting of MPs. 43 Microparticles are submicron membrane vesicles generated by platelets and other blood cells, recently been declared to play a major role in promoting coagulation, inflammation, and immune response. ...
Impella 5.5 Versus Centrimag: A Head-to-Head Comparison of Device Hemocompatibility
Article
Full-text available
  • Nov 2020
Despite growing use of mechanical circulatory support, limitations remain related to hemocompatibility. Here, we performed a head-to-head comparison of the hemocompatibility of a centrifugal cardiac assist system-the Centrimag, with that of the latest generation of an intravascular microaxial system-the Impella 5.5. Specifically, hemolysis, platelet activation, microparticle (MP) generation, and von Willebrand factor (vWF) degradation were evaluated for both devices. Freshly obtained porcine blood was recirculated within device propelled mock loops for 4 hours, and alteration of the hemocompatibility parameters was monitored over time. We found that the Impella 5.5 and Centrimag exhibited low levels of hemolysis, as indicated by minor increase in plasma free hemoglobin. Both devices did not induce platelet degranulation, as no alteration of β-thromboglobulin and P-selectin in plasma occurred, rather minor downregulation of platelet surface P-selectin was detected. Furthermore, blood exposure to shear stress via both Centrimag and Impella 5.5 resulted in a minor decrease of platelet count with associated ejection of procoagulant MPs, and a decrease of vWF functional activity (but not plasma level of vWF-antigen). Greater MP generation was observed with the Centrimag relative to the Impella 5.5. Thus, the Impella 5.5 despite having a lower profile and higher impeller rotational speed demonstrated good and equivalent hemocompatibility, in comparison with the predicate Centrimag, with the advantage of lower generation of MPs.
... The antibiofilm activity and hemocompatibility studies of SPIONs require the assessment of these nanoparticles with biological materials, which are bacterial biofilms and blood cells (ex.: red blood cells and platelets), respectively [4,5,[9][10][11][12][13][14]. These analyses of SPIONs have allowed the development of their applications in materials science and biomedical engineering [15][16][17][18][19][20]. ...
Analysis of Nanoparticles Characteristics with TOPSIS for Their Manufacture Optimization
Article
Full-text available
  • Jan 2022
The present study focuses on the comparative analysis of superparamagnetic iron oxide nanoparticles (SPIONs) characteristics with the TOPSIS method. The prediction of the characteristics of SPIONs is required for better manufacturing of these nanoparticles. Although the characteristics of these nanoparticles have been investigated, no research has been done on their comparison in order to determine which one of their surface functionalities would be more appropriate for their diverse applications. The objective of this study was to analyze the characteristics of SPIONs without or with surface charge with a prediction model and TOPSIS in order to determine the best nanoparticles. Moreover, the effect of inappropriate consideration of their cost criterion on their ranks was explored with the modified TOPSIS. This analysis showed that the characteristics of SPIONs such as antibiofilm activity, hemocompatibility, activity with hydrogen peroxide, rheological properties, and the labour of their chemical synthesis could affect their ranking. Neutral SPIONs, negatively charged SPIONs, and positively charged SPIONs were ranked as the first, second, and third candidates, respectively. However, the improvement of the activity of positively charged SPIONs with hydrogen peroxide showed an increase to 0.3 instead of 0.2, which resulted in a better rank of these nanoparticles in comparison with that of the same nanoparticles in the first analysis series. One of the advantages of this study was to determine the impact of the characteristics of SPIONs on their ranking for their manufacturing. The other advantage was getting the information for further comparative study of these nanoparticles with the others. The results of this work can be used in manufacturing engineering and materials science.
... For the experiment, several boundary conditions for success were defined: (i) experiments must conclude within a 4-h time frame from the moment of blood drawing from the animal, because of a decrease in blood vitality in vitro. 3,4 (ii) Red blood cell count must be relatively stable for the duration of the experiment, because a decline in red blood cell count alters the Xray attenuation coefficient of the free-flowing blood in the CT scans. This would complicate the comparisons of consecutive CT scans, because no additional contrast agent besides hemoglobin was used. ...
In-Vitro Visualization of Thrombus Growth in Artificial Lungs Using Real-Time X-Ray Imaging: A Feasibility Study
Article
Full-text available
  • Sep 2021
Purpose Extracorporeal membrane oxygenation has gained increasing attention in the treatment of patients with acute and chronic cardiopulmonary and respiratory failure. However, clotting within the oxygenators or other components of the extracorporeal circuit remains a major complication that necessitates at least a device exchange and bears risks of adverse events for the patients. In order to better predict thrombus growth within oxygenators, we present an approach for in-vitro visualization of thrombus growth using real-time X-ray imaging. Methods An in-vitro test setup was developed using low-dose anticoagulated ovine blood and allowing for thrombus growth within 4 h. The setup was installed in a custom-made X-ray setup that uses phase-contrast for imaging, thus providing enhanced soft-tissue contrast, which improves the differentiation between blood and potential thrombus growth. During experimentation, blood samples were drawn for the analysis of blood count, activated partial thromboplastin time and activated clotting time. Additionally, pressure and flow data was monitored and a full 360° X-ray scan was performed every 15 min. Results Thrombus formation indicated by a pressure drop and changing blood parameters was monitored in all three test devices. Red and white thrombi (higher/lower attenuation, respectively) were successfully segmented in one set of X-ray images. Conclusion We showed the feasibility of a new in-vitro method for real-time thrombus growth visualization by means of phase contrast X-ray imaging. In addition, with more blood parameters that are clinically relevant, this approach might contribute to improved oxygenator exchange protocols in the clinical routine.
... To evaluate the potential of mAbs in eliciting a pro-thrombotic environment, TAT analysis was performed upon 4 h of stimulation. The incubation time was set at 4 h based on the lifespan of the assay and with the aim of capturing late inflammatory responses [38]. Indeed, LPS as the positive control exhibited a much stronger coagulation activation after 4 h, as opposed to only 2 h incubation. ...
The blood endothelial cell chamber – An innovative system to study immune responses in drug development
Article
Full-text available
The risk for adverse immune-mediated reactions, associated with the administration of certain immunotherapeutic agents, should be mitigated early. Infusion reactions to monoclonal antibodies and other biopharmaceuticals, known as cytokine release syndrome, can arise from the release of cytokines via the drug target cell, as well as the recruitment of immune effector cells. While several in vitro cytokine release assays have been proposed up to date, many of them lack important blood components, required for this response to occur. The blood endothelial cell chamber model is an in vitro assay, composed of freshly drawn human whole blood and cultured human primary endothelial cells. Herein, its potential to study the compatibility of immunotherapeutics with the human immune system was studied by evaluating three commercially available monoclonal antibodies and bacterial endotoxin lipopolysaccharide. We demonstrate that the anti-CD28 antibody TGN1412 displayed an adaptive cytokine release profile and a distinct IL-2 response, accompanied with increased CD3⁺ cell recruitment. Alemtuzumab exhibited a clear cytokine response with a mixed adaptive/innate source (IFNγ, TNFα and IL-6). Its immunosuppressive nature is observed in depleted CD3⁺ cells. Cetuximab, associated with low infusion reactions, showed a very low or absent stimulatory effect on proinflammatory cytokines. In contrast, bacterial endotoxin demonstrated a clear innate cytokine response, defined by TNFα, IL-6 and IL-1β release, accompanied with a strong recruitment of CD14⁺CD16⁺ cells. Therefore, the blood endothelial cell chamber model is presented as a valuable in vitro tool to investigate therapeutic monoclonal antibodies with respect to cytokine release and vascular immune cell recruitment.
... The importance of using fresh blood within 4 hr of blood collection (and preferably within 2 hr) has been reported. 29 Interestingly, examination of our platelet count data expressed relative to the No Material control according to Reference 6 showed the HDPE reference biomaterial to be consistently outside (60-80%) the assay validation condition of 80-120% (see Appendix). This low level of HDPE platelet reactivity has been reported by others using alternative blood preparation/exposure methods. ...
In vitro methodology for medical device material thrombogenicity assessments: A use condition and bioanalytical proof-of-concept approach
Article
Full-text available
Device manufacturers and regulatory agencies currently utilize expensive and often inconclusive in vivo vascular implant models to assess implant material thrombogenicity. We report an in vitro thrombogenicity assessment methodology where test materials (polyethylene, Elasthane™ 80A polyurethane, Pebax®), alongside positive (borosilicate glass) and negative (no material) controls, were exposed to fresh human blood, with attention to common blood-contact use conditions and the variables: material (M), material surface modification (SM) with heparin, model (Mo), time (T), blood donor (D), exposure ratio (ER; cm2 material/ml blood), heparin anticoagulation (H), and blood draw/fill technique (DT). Two models were used: (1) a gentle-agitation test tube model and (2) a pulsatile flow closed-loop model. Thrombogenicity measurements included thrombin generation (thrombin-antithrombin complex [TAT] and human prothrombin fragment F1.2), platelet activation (β-thromboglobulin), and platelet counts. We report that: (a) thrombogenicity was strongly dependent (p < .0001) on M, H, and T, and variably dependent (p < .0001 - > .05) on Mo, SM, and D (b) differences between positive control, test, and negative control materials became less pronounced as H increased from 0.6 to 2.0 U/ml, and (c) in vitro-to-in vivo case comparisons showed consistency in thrombogenicity rankings on materials classified to be of low, moderate, and high concern. In vitro methods using fresh human blood are therefore scientifically sound and cost effective compared to in vivo methods for screening intravascular materials and devices for thrombogenicity.
... Blood is a complex biological fluid containing approximately 45% cellular components suspended in plasma (5 billion cells per milliliter of blood) and it represents an active indicator of various pathological disorders [1,2]. Many medical diagnostic procedures require a reliable separation of plasma from blood as it includes proteins, metabolites, circulating nucleic acids (CNAs), and other biomarker organisms. ...
Influence of Hydrodynamics and Hematocrit on Ultrasound-Induced Blood Plasmapheresis
Article
Full-text available
  • Jul 2020
Acoustophoretic blood plasma separation is based on cell enrichment processes driven by acoustic radiation forces. The combined influence of hematocrit and hydrodynamics has not yet been quantified in the literature for these processes acoustically induced on blood. In this paper, we present an experimental study of blood samples exposed to ultrasonic standing waves at different hematocrit percentages and hydrodynamic conditions, in order to enlighten their individual influence on the acoustic response of the samples. The experiments were performed in a glass capillary (700 µm-square cross section) actuated by a piezoelectric ceramic at a frequency of 1.153 MHz, hosting 2D orthogonal half-wavelength resonances transverse to the channel length, with a single-pressure-node along its central axis. Different hematocrit percentages Hct = 2.25%, 4.50%, 9.00%, and 22.50%, were tested at eight flow rate conditions of Q = 0:80 µL/min. Cells were collected along the central axis driven by the acoustic radiation force, releasing plasma progressively free of cells. The study shows an optimal performance in a flow rate interval between 20 and 80 µL/min for low hematocrit percentages Hct ≤ 9.0%, which required very short times close to 10 s to achieve cell-free plasma in percentages over 90%. This study opens new lines for low-cost personalized blood diagnosis
... Stents are used in percutaneous coronary intervention to restore blood flow in patients with coronary artery disease (CAD) which is characterized by the occlusion of a coronary artery due to plaque formation (Garg and Serruys, 2010;Li et al., 2010). Researchers are dedicated to the development of new biocompatible materials for use in stent devices, that on the one hand provide enough strength for vessel support during constrictive re-modeling while also allowing for good cytoand hemocompatibility (Van Oeveren et al., 1999;Williams, 2008;Huang et al., 2014;Zhang et al., 2014;Blok et al., 2016). Complications still arise from stent placement, the main issues being in-stent restenosis and stent thrombosis resulting from inadequate mechanical properties or poor biocompatibility (Zhang et al., 2014). ...
Cytocompatibility Evaluation of a Novel Series of PEG-Functionalized Lactide-Caprolactone Copolymer Biomaterials for Cardiovascular Applications
Article
Full-text available
  • Aug 2020
Although the use of bioresorbable materials in stent production is thought to improve long-term safety compared to their durable counterparts, a recent FDA report on the 2-year follow-up of the first FDA-approved bioresorbable vascular stent showed an increased occurrence of major adverse cardiac events and thrombosis in comparison to the metallic control. In order to overcome the issues of first generation bioresorbable polymers, a series of polyethylene glycol-functionalized poly-L-lactide-co-ε-caprolactone copolymers with varying lactide-to-caprolactone content is developed using a novel one-step PEG-functionalization and copolymerization strategy. This approach represents a new facile way toward surface enhancement for cellular interaction, which is shown by screening these materials regarding their cyto-and hemocompatibility in terms of cytotoxicity, hemolysis, platelet adhesion, leucocyte activation and endothelial cell adhesion. By varying the lactide-to-caprolactone polymer composition, it is possible to gradually affect endothelial and platelet adhesion which allows fine-tuning of the biological response based on polymer chemistry. All polymers developed were non-cytotoxic, had acceptable leucocyte activation levels and presented non-hemolytic (<2% hemolysis rate) behavior except for PLCL-PEG 55:45 which presented hemolysis rate of 2.5% ± 0.5. Water contact angles were reduced in the polymers containing PEG functionalization (PLLA-PEG: 69.8 • ± 2.3, PCL-PEG: 61.2 • ± 7.5) versus those without (PLLA: 79.5 • ± 3.2, PCL: 76.4 • ± 10.2) while the materials PCL-PEG550, PLCL-PEG550 90:10 and PLCL-PEG550 70:30 demonstrated Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 1 August 2020 | Volume 8 | Article 991 Pacharra et al. Cytocompatible PLCL-PEG Copolymers for Cardiovascular Applications best endothelial cell adhesion. PLLA-PEG550 and PLCL-PEG550 70:30 presented as best candidates for cardiovascular implant use from a cytocompatibility perspective across the spectrum of testing completed. Altogether, these polymers are excellent innovative materials suited for an application in stent manufacture due to the ease in translation of this one-step synthesis strategy to device production and their excellent in vitro cyto-and hemocompatibility.
Enhanced localization of cefazoline sodium in the ocular tissue using thermosensitive-mucoadhesive hydrogels: Formulation development, hemocompatibility and in vivo irritation studies
Article
Bacterial keratitis is an eye infectious disease which became a global concern due to its impact to visual impairment in worldwide. Cefazolin (CFZ) is the first-line drug for the treatment of bacterial keratitis and only available in the drop dosage form. However, the administration of eye drops results in lack of bioavailability, which is below 5%. Therefore, an innovation is required in an attempt to overcome these problems. In this study, the development of a mucoadhesive-thermosensitive gel in situ preparation was carried out. A combination of Pluronic F127 and F68 was used as a thermosensitive agent. To increase the contact time, a mucoadhesive agent, namely hyaluronic acid was added to the formulation. Several steps of evaluation were performed to ensure that the developed formula possessed desired characteristics, including determination of gelation temperature, pH test, viscosity test, rheology test, mucoadhesive test, drug content test, in vitro drug release test, in vivo irritation test on experimental animals, ex vivo permeation test, and hemolysis test. The results that the formulations developed exhibited desired characteristics, with gelation temperatures of around 37 °C. The formulation could also control the release and improve the localization of CFZ in the ocular tissue compared to control solution. Furthermore, the incorporation of CFZ into this approach did not change the antimicrobial activity of CZ against Pseudomonas aeruginosa. Importantly, no toxicity and irritation were observed after the application of this approach. However, further research is needed to evaluate the pharmacokinetic and pharmacodynamic in the appropriate animal models.
Product evaluation: blood compatibility studies
Chapter
  • Jan 2022
Blood compatibility is defined as the ability of a material to stay in contact with blood for a clinically relevant amount of time without altering the blood or blood components. This definition implies that all materials comes into contact with blood directly or indirectly. Here, we elaborate on the various factors that affect blood-material interactions and response of the blood or blood components to the materials. Blood-material interaction depends on various factors varies from surface properties of the materials to the end application. Performance of any biomaterials largely depends on the way materials interact with the blood. The vast research in this area focus on how we analyze blood compatibility, how a materials reacts with blood and what factor decides blood compatibility of a biomaterial. As new materials/medical devices are emerging, concept of blood compatibility is establishing new era. Hemocompatibility is one of the most important criteria for in vivo applicability of any blood contacting biomaterial. A biomaterial cannot be defined as blood compatible or non compatible solely based on the reaction, its largely depends on the end use of the biomaterial/medical devices. Thus an extensive in vitro analysis based on ISO 10993:4, “Biological evaluation of medical devices-part4: selection of tests for interaction with blood,” is crucial before the clinical evaluation of biomaterials or devices. In vitro analysis also paves the way for a controlled way to understand the effect of material on blood and the improvisation of developing biomaterials and medical devices for successful clinical applications.
Impact of Polyethylene Glycol Functionalization of Graphene Oxide on Anticoagulation and Haemolytic Properties of Human Blood
Article
Full-text available
  • Aug 2021
Graphene oxide (GO) is one of the most explored nanomaterials in recent years. It has numerous biomedical applications as a nanomaterial including drug and gene delivery, contrast imaging, cancer treatment, etc. Since most of these applications need intravenous administration of graphene oxide and derivatives, the evaluation of their haemocompatibility is an essential preliminary step for any of the developed GO applications. Plentiful data show that functionalization of graphene oxide nanoparticles with polyethylene glycol (PEG) increases biocompatibility, thus allowing PEGylated GO to elicit less dramatic blood cell responses than their pristine counterparts. Therefore, in this work, we PEGylated graphene oxide nanoparticles and evaluated the effects of their PEGylation on the structure and function of human blood components, especially on the morphology and the haemolytic potential of red blood cells (RBCs). Further, we studied the effect of PEGylation on some blood coagulation factors, including plasma fibrinogen as well as on the activated partial thromboplastin (aPTT), prothrombin time (PT) and platelet aggregation. Our findings provide important information on the mechanisms through which PEGylation increases GO compatibility with human blood cells. These data are crucial for the molecular design and biomedical applications of PEGylated graphene oxide nanomaterials in the future.
Changes in platelet morphology and function during 24 hours of storage
Article
Full-text available
For in vitro studies assessing the interaction of platelets with implant materials, common and standardized protocols for the preparation of platelet rich plasma (PRP) are lacking, which may lead to non-matching results due to the diversity of applied protocols. Particularly, the aging of platelets during prolonged preparation and storage times is discussed to lead to an underestimation of the material thrombogenicity. Here we study the influence of whole blood- and PRP-storage times on changes in platelet morphology and function. Blood from apparently healthy subjects was collected according to a standardized protocol and examined immediately after blood collection, four hours and twenty four hours later. The capability of platelets to adhere and form stable aggregates (PFA100, closure time) was examined in sodium citrate anticoagulated WB using the agonists equine type I collagen and epinephrine bitartrate (collagen/epinephrine) as well as equine type I collagen and adenosine-5'-diphosphate (collagen/ADP). Circulating platelets were quantified at each time point. Morphology of platelets and platelet aggregates were visualized microscopically and measured using an electric field multi-channel counting system (CASY). The percentage of activated platelets was assessed by means of P-selectin (CD62P) expression of circulating platelets. Furthermore, platelet factor 4 (PF4) release was measured in platelet poor plasma (PPP) at each time point. Whole blood PFA100 closure times increased after stimulation with collagen/ADP and collagen/epinephrine. Twenty four hours after blood collection, both parameters were prolonged pathologically above the upper limit of the reference range. Numbers of circulating platelets, measured in PRP, decreased after four hours, but no longer after twenty four hours. Mean platelet volumes (MPV) and platelet large cell ratios (P-LCR, 12 fL - 40 fL) decreased over time. Immediately after blood collection, no debris or platelet aggregates could be visualized microscopically. After four hours, first debris and very small aggregates occurred. After 24 hours, platelet aggregates and also debris progressively increased. In accordance to this, the CASY system revealed an increase of platelet aggregates (up to 90 μm diameter) with increasing storage time. The percentage of CD62P positive platelets and PF4 increased significantly with storage time in resting PRP. When soluble ADP was added to stored PRP samples, the number of activatable platelets decreased significantly over storage time. The present study reveals the importance of a consequent standardization in the preparation of WB and PRP. Platelet morphology and function, particularly platelet reactivity to adherent or soluble agonists in their surrounding milieu, changed rapidly outside the vascular system. This knowledge is of crucial interest, particularly in the field of biomaterial development for cardiovascular applications, and may help to define common standards in the in vitro hemocompatibility testing of biomaterials.
Development and hemocompatibility testing of nitric oxide releasing polymers using a rabbit model of thrombogenicity
Article
Full-text available
  • Jun 2014
Hemocompatibility is the goal for any biomaterial contained in extracorporeal life supporting medical devices. The hallmarks for hemocompatibility include nonthrombogenicity, platelet preservation, and maintained platelet function. Both in vitro and in vivo assays testing for compatibility of the blood/biomaterial interface have been used over the last several decades to ascertain if the biomaterial used in medical tubing and devices will require systemic anticoagulation for viability. Over the last 50 years systemic anticoagulation with heparin has been the gold standard in maintaining effective extracorporeal life supporting. However, the biomaterial that maintains effective ECLS without the use of any systemic anticoagulant has remained elusive. In this review, the in vivo 4-h rabbit thrombogenicity model genesis will be described with emphasis on biomaterials that may require no systemic anticoagulation for extracorporeal life supporting longevity. These novel biomaterials may improve extracorporeal circulation hemocompatibility by preserving near resting physiology of the major blood components, the platelets and monocytes. The rabbit extracorporeal circulation model provides a complete assessment of biomaterial interactions with the intrinsic coagulation players, the circulating platelet and monocytes. This total picture of blood/biomaterial interaction suggests that this rabbit thrombogenicity model could provide a standardization for biomaterial hemocompatibility testing.
Are there sufficient standards for the in vitro hemocompatibility testing of biomaterials?
Article
Full-text available
Scitation is the online home of leading journals and conference proceedings from AIP Publishing and AIP Member Societies
Obstacles in Haemocompatibility Testing
Article
Full-text available
  • Jan 2013
ISO 10993-4 is an international standard describing the methods of testing of medical devices for interactions with blood for regulatory purpose. The complexity of blood responses to biomaterial surfaces and the variability of blood functions in different individuals and species pose difficulties in standardisation. Moreover, in vivo or in vitro testing, as well as the clinical relevance of certain findings, is still matter of debate. This review deals with the major remaining problems, including a brief explanation of surface interactions with blood, the current ISO 10993 requirements for testing, and the role of in vitro test models. The literature is reviewed on anticoagulation, shear rate, blood-air interfaces, incubation time, and the importance of evaluation of the surface area after blood contact. Two test categories deserve further attention: complement and platelet function, including the effects on platelets from adhesion proteins, venipuncture, and animal derived- blood. The material properties, hydrophilicity, and roughness, as well as reference materials, are discussed. Finally this review calls for completing the acceptance criteria in the ISO standard based on a panel of test results.
In vitro haemocompatibility of a novel bioprosthetic total artificial heart
Article
Full-text available
The CARMAT total artificial heart (TAH) is an implantable, electro-hydraulically driven, pulsatile flow device with four bioprosthetic valves. Its blood-pumping surfaces consist of processed bioprosthetic pericardial tissue and expanded polytetrafluorethylene (ePTFE), potentially allowing for the reduction of anti-coagulation. This pre-clinical study assessed the in vitro haemocompatibility of these surfaces. Coupons of pericardial tissue and ePTFE were placed in closed tubular circuits filled with 12.5 ml of fresh human blood exposed to the pulsatile flow at 120 ml/min for 4 h (37°C). Silicone- and heparin-coated polyvinyl chloride (PVC) tubes served as positive and negative controls, respectively. Fresh blood from six donors was used to fill four sets of 12 circuits. Blood samples were taken at baseline and from each circuit after 4 h. Coupons of materials were examined with scanning electron microscopy. The platelet count was 202 ± 45 10(9) l(-1) at baseline. Four hours after circulation, the platelet counts were 161 ± 30 10(9) l(-1) (compared with baseline, P = 0.0207) for pericardial tissue, 162 ± 35 10(9) l(-1) (P = 0.0305) for ePTFE and 136 ± 42 10(9) l(-1) for positive controls (P = 0.0021). Baseline plasma fibrinogen was 2.9 ± 0.5 mg/dl compared with 3.0 ± 0.5 mg/dl for pericardial tissue and 3.1 ± 0.7 mg/dl for ePTFE, indicating no marked fibrinogen consumption. Thromboxane B2 levels for positive controls were 33.3 ± 8.7 ng/ml compared with 16.2 ± 11.5 ng/ml for pericardial tissue (P = 0.0015) and 15.2 ± 4.7 ng/ml for ePTFE (P < 0.0001). Platelet adhesion was 2.87 ± 1.01 10(9) cm(-2) for positive controls compared with 1.06 ± 0.73 10(9) cm(-2) for pericardial tissue (P < 0.0001) and 0.79 ± 0.75 10(9) cm(-2) for ePTFE (P < 0.0001). Thrombin-antithrombin III complex levels were 3.8 ± 0.5 μg/ml for positive controls compared with 1.9 ± 0.9 for pericardial tissue (P < 0.0001) and 2.1 ± 1.0 for ePTFE (P < 0.0001). With an electro-microscopic examination at ×600, only small depositions of platelets, erythrocytes and fibrin were noticed on the pericardial tissue samples and ePTFE samples. Silicone surfaces showed marked areas of thrombi, and PVC tubings a thin protein layer. Haemocompatibility of the TAH blood-contacting surfaces was confirmed by in vitro studies showing a limited consumption of fibrin, limited thromboxane B2 release and platelet adhesion, and minor blood cell depositions on the surfaces. These results will be validated in clinical studies, with the aim of reducing anti-coagulation when using the CARMAT TAH.
Effect of Blood Sampling, Processing, and Storage on the Measurement of Complement Activation Biomarkers
Article
Recent studies have shown that complement hyperactivation contributes to development of thrombotic microangiopathy. The evaluation of complement biomarkers is known to be influenced by inappropriate specimen handling. However, there has been no study fully addressing this topic. Blood from each donor was subjected to 62 different handling conditions prior to complement assays. Complement biomarkers (C4d/C3a/factor Bb/C5a/C5b-9) are stable at room temperature (RT) for up to 4 hours in whole blood containing citrate or EDTA. However, under similar conditions, levels of C4d and C3a were significantly higher in serum than those in plasma. Thawing of the samples on ice or at RT had no significant effect on complement levels. In contrast, thawing at 37°C resulted in striking increases in levels of the complement system in serum and citrated plasma but not in EDTA plasma. Up to four freeze/thaw cycles on ice or RT did not substantially increase the levels of C3a, factor Bb, C5a, and C5b-9 but had a significant effect on C4d. Long-term storage of citrated plasma at -80°C for up to 6 years had no significant effect on levels of complement factors. The results from this study thus provide crucial guidelines for future investigations using complement biomarkers to define the role of complement system in disease. Copyright© by the American Society for Clinical Pathology.
A stable analogue of throm☐ane A2, 9,11-epithio-11,12-methanothrom☐ane A2, stimulates bone resorption in vitro and osteoclast-like cell formation in mouse marrow culture
Article
Throm☐ane A2 (TXA2) is a powerful promoter of platelet aggregation and smooth muscle contraction. However, this compound is highly unstable and is rapidly hydrated to a more stable metabolite, throm☐ane B2 (TXB2). TXA2 has been considered to be involved in bone resorption, in particular bone loss caused by inflammatory diseases and by orthodontic treatment. However precise mechanisms of bone resorption caused by TXA2 have not yet been proved because of its highly unstable nature.Recently, a chemically stable analogue of TXA2, 9,11-epithio-11,12-methanothrom☐ane A2 (STA2), was successfully synthesized. Using this synthetic compound, we examined its in vitro bone resorbing activity and induction of osteoclast-like cells in a mouse marrow culture system in comparison with related compounds with bone resorbing activity. Like prostaglandin E2 (PGE2), a well-known bone resorbing agent. STA2 time-and dose-dependently stimulated the release of 45Ca from prelabelled mouse calvariae. Both STA2 and PGE2 induced the accumulation of cAMP in mouse calvariae. The TXA2, agonist. ONO-3708, inhibited STA2-induced release of 45Ca, TXB2 induced neither bone resolor cAMP accumulation. When mouse marrow cells were cultured with STA2 for 8 days, osteoclast-like multinucleated cells appeared in parallel with the increase of the amount of STA2 added. Again TXB2 showed no effect on osteoclast-like cell formation. These results indicate a role for TXA2 in some form of bone resorption.
The Platelet Storage Lesion
Article
  • Jun 2010
The gradual loss of quality in stored platelets as measured collectively with various metabolic, functional, and morphologic in vitro assays is known as the platelet storage lesion. With the advent of pathogen reduction technologies and improved testing that can greatly reduce the risk for bacterial contamination, the platelet storage lesion is emerging as the main challenge to increasing the shelf life of platelet concentrates. This article discusses the contribution of platelet production methods to the storage lesion, long-established and newly developed methods used to determine platelet quality, and the significance for clinical transfusion outcome. Highlighted are the novel technologies applied to platelet storage including platelet additive solutions and pathogen inactivation.
The platelet storage lesion
Article
The continuous increase in the demand for platelet transfusion has necessitated the need to establish standards for determining the quality of platelets during storage. Bacterial contamination of platelet products and deleterious changes in structure and function referred to as the platelet storage lesion (PSL), have restricted the platelet shelf life to 5 days. The PSL and platelet health variables have been well studied and documented. The precise correlation between in vitro assays and in vivo platelet recovery and survival is yet to be established. This review presents an overview of the current understanding of PSL and the novel approaches being developed to negate the storage lesion.