Use of Vivid™ Plasma Separation Membrane
for Sensitive Detection of Troponin in Whole Blood Samples
Andrew Dubitsky and Galina Fomovska; Pall Life Sciences, Port Washington, NY, USA
When cardiac TnI is present in blood, it is primarily complexed to Troponin T (TnT) and Troponin C (TnC). Troponin I is most commonly used
as the target molecule for ELISA detection, but is poorly soluble in its isolated form. For this reason, we used radiolabeled troponin ITC
complex and recombinant troponin IC complex to model cardiac troponin behavior in whole blood and plasma.
Levels of influent troponin complex were varied between 2 ng/mL and 20 ng/mL. Volumes of influent blood between 30-50 µL were applied
to discs of Vivid GF Plasma Separation membrane. Results show that up to 20% of troponin complex spiked into whole blood may associate
with the cellular fraction and is then unavailable for ELISA detection. Pre-treating Vivid GF membrane with a combination of casein and
surfactant enhances the plasma separation process and allows transmission of 95-100% of the available troponin complex.
Vivid Plasma Separation membrane has been designed to provide reproducible, high efficiency yields of high quality plasma from small
volumes of whole blood. Blood volumes of up to 50 µL/cm2 of membrane can be accommodated. This study shows that Vivid GF Plasma
Separation membrane, after pre-treatment, is useful for small volume TnI assays. It is likely that this pre-treatment will also improve detection
of other cardiac markers, as well as other low-abundant or relatively insoluble plasma components.
INTRODUCTION
Fresh whole blood collected in heparinized tubes was used for all experiments. Centrifuged plasma was prepared by spinning at 3000 x g
for 5 minutes.
Preparation of Casein Solutions
Hammerstein grade casein (BDH) is poorly soluble at neutral pH. Casein was dissolved by adding powder to buffer and then heating to
approximately 90 °C (194 °F), then cooling slowly to room temperature.
Analysis of Separated Plasma
40–45 µL whole blood was added to 13 mm discs of Vivid GF membrane laid on top of 13 mm discs of Supor® 5000 PES membrane. Five minutes after
addition of blood, the membranes were separated and the plasma was spun out of the Supor membrane by centrifugation at 10,000 g for 15 minutes.
The effect of blocking filtration of troponin ITC complex in PBS was confirmed
by a partial repeat of the experiment, using native (unlabeled) troponin ITC
28
complex at 20 ng/mL diluted in centrifuged plasma.
24
Troponin I concentrations were measured in plasma generated from untreated
Vivid Plasma Separation membrane, as well as those blocked with casein and
surfactant. 2% sucrose was included in the pre-treatment step, as sucrose
can increase wettability of treated membranes after drying (Figure 2).
20
Relative effectiveness of different blocking treatments was examined
by measuring troponin complex concentrations after plasma generation
with treated and untreated membrane. Results were normalized to troponin
recovery from centrifuged whole blood (Figure 5).
12
In this test, the TnI concentration of unfiltered plasma was close to the target
level. Filtration of spiked plasma through untreated Vivid GF membranes
resulted in approximately 60% loss of TnI, while membranes blocked with
casein and surfactant yielded concentrations similar to the target influent.
Presence of sucrose did not affect recovery, but plasma penetrated the
blocked membrane more quickly when sucrose was present.
8
Influent
Plasma
Untreated
F
l
o
w
Captured
Red Cells
Separated
Cell-Free Plasma
Troponin I as Model Analyte
Troponin I, a commonly used marker for myocardial infarction, was chosen as a model protein. Normal circulating levels of TnI are less than
1 ng/mL. Elevated levels indicate breakdown of cardiac muscle cells. The range of clinical interest is between 2-50 ng/mL.
Centrifuged plasma samples were spiked with 15 or 25 ng/mL of unlabeled native Troponin ITC complex. 300 µL of spiked plasma was added
to 47 mm discs of Vivid GF membrane. The plasma was then collected from the membrane by centrifugation. Concentration of TnI in plasma
recovered from the membranes was determined using an ELISA kit from Calbiotech.
Results were very consistent, with Vivid GF membrane discs binding 40% of available troponin ITC complex at both the 15 ng/mL and
25 ng/mL influent levels compared to measurements of the influent plasma.
Binding of Troponin ITC Complex to Untreated and Treated Vivid GF Membrane:
Radiochemical Method
After finding a decrease in troponin concentration with filtration of plasma, other proteins were eliminated from the challenge solution for
examination of binding of the troponin complex to Vivid GF membrane. This experiment was performed to evaluate effectiveness of casein
and surfactants for reducing binding to the membrane.
Membrane treatments included 0.05% Tween 20 and 0.8% PVP surfactants, singly or in combination, along with 0.5% Hammerstein grade
casein. This grade of casein has been shown to lower protein binding levels on membranes more effectively than BSA, and is used at lower
concentrations than non-fat dry milk.
Troponin I is normally present in whole blood complexed to TnC and TnT, and is poorly soluble by itself. Native troponin ITC complex and a
recombinant single chain troponin IC molecule are both recognized as calibration standards for TnI clinical tests. Both complexes were used
in this study.
PBS was spiked with 20 ng/mL Iodine troponin ITC. 30 µL challenge solution was applied to the top of Vivid GF membrane discs. Liquid
plasma was recovered by centrifugation. Aliquots of influent and effluent were used to calculate concentrations, based on specific activity.
Vivid Membrane and Microfluidic Rapid Assays
Table 1
Blocking with Casein Greatly Reduced Non-Specific Binding of Troponin Complex
Goal of Study
Initial tests for plasma generated by Vivid membrane showed TnI levels below expected values based on the amount of single chain troponin
IC or native troponin ITC complex spiked into the blood. Radio-labeled troponin IC and troponin ITC complexes were then used to track the
distribution of molecules in plasma, cells and membrane. Simple pre-treatment steps were developed that eliminated effects of the membrane
on concentration measurements. These treatments also increased the plasma yield from the membrane.
Condition
1
2
3
4
5
6
7
8
Treatment
Casein
+
+
+
+
Tween 20
+
+
+
+
PVP
+
+
+
+
% Binding to Disc
Disc 1
Disc 2
26.3
18.7
28.5
22.5
22.3
25.7
23.3
26.9
3.3
6.2
4.6
5.5
5.2
5.4
4.4
6.8
22
Vivid GF membrane discs were placed on top of pre-weighed Supor 5000
membrane discs. 45 µL whole blood was added to the top of the Vivid discs.
After 5 minutes, the membranes were separated and the wetted Supor
membrane was re-weighed. The plasma volume was calculated from the net
weight of the Supor collection membrane.
18
Partitioning of Troponin Complex in
Centrifuged Blood from Multiple Donors
In order to examine the effect of Vivid GF membrane on plasma
separated from whole blood, the distribution of troponin complex
in whole blood had to be determined. Is all the troponin found in
the plasma, or does some amount of troponin complex associate
with cells? This is critical because troponin concentration in plasma
generated via membrane filtration is compared to centrifuged
plasma. Additionally, TnI concentration is always measured in serum
or plasma, and not in whole blood.
To answer this question, an experiment was designed using
centrifugation to prepare plasma. Whole blood from multiple donors
was spiked with 125Iodine troponin ITC or 125Iodine troponin IC
complex at 10 ng/mL. After centrifugation, aliquots of plasma
and packed cell fractions were counted. Concentrations were
determined from the specific activity of the labeled troponin.
Results were very similar for both the native troponin ITC and
recombinant single chain troponin IC complexes. An average of
80% of the troponin was recovered in the plasma; 20% remained
associated with the packed cells. Partitioning of the troponin
complex in centrifuged blood provided a baseline for comparison
when looking at plasma generated by the Vivid GF membrane.
Note: Casein treatment alone renders the membrane difficult to re-wet. Plasma or whole blood penetrates quickly
into untreated Vivid GF membrane, but very slowly or not at all after casein treatment. If Tween 20 or PVP surfactant
was added to the casein during the blocking step, blood or plasma was again rapidly absorbed into the filter matrix.
A combination of casein and surfactant (Tween 20 chosen) was used in all following experiments.
Contact: 1.800.521.1520 (USA and Canada) • (+)800.PALL.LIFE (Outside USA and Canada) • www.pall.com/oem • E-mail: [email protected]
• Treatment with sucrose alone resulted in an improvement, but there was
still an average loss of 15%.
• Treatment with casein and surfactant (+/) sucrose resulted in less than
5% loss of complex, even at casein levels of 0.05%.
Volume Plasma Recovered - Six Donors
Figure 5
Improved Recovery of Troponin IC in Plasma Generated From
Treated Vivid GF Membranes
Average, All Donors
110
100
90
80
70
60
50
Untreated Sucrose
0.5%
Casein,
Tw20
Casein,
Sucrose,
Tw20
0.05%
Casein,
Tw20
0.05%
Casein,
Sucrose,
Tw20
DISCUSSION
14
12
10
Vivid GF plasma separation membranes are used for a variety of POC diagnostic applications. The Vivid GF membrane generates high quality
plasma from small whole blood volumes. The resultant plasma is transferred to another layer of membrane or capillary system for analysis.
8
6
4
Untreated Sucrose
Untreated Vivid membrane binds significant amounts of troponin complex, as demonstrated by spiking into PBS, plasma and whole blood,
and measured by ELISA and radiometric label. This binding is effectively eliminated using standard membrane blocking methods. Effective
pre-treatment formulations include 0.05–0.5% casein plus surfactant. Tween 20 was chosen because of compatibility with whole blood assays.
The addition of sucrose resulted in faster re-wetting of the membrane, but did not effect either transmission of analyte or plasma volume.
0.5%
Casein, 0.05%
0.05%
Casein, Sucrose, Casein, Casein,
Tw20
Tw20
Tw20 Sucrose,
Tw20
Other common protein blocking agents, such as BSA, may work but were not used in this study as casein is known to have more effective
blocking properties for membrane-based assays. BSA may be evaluated for membrane treatment if casein interferes with downstream analysis.
As in any case where blocking is required for a successful assay result, the formulation mix should be optimized for best test results.
Figure 4
Partitioning of Tropinin Complex Between Plasma and Packed Cells
After Centrifugation
Whole Blood Spiked with 125I Tropinin ITC Complex
Partitioning of Tn ITC in Whole Blood
W
Tn ITC in Cell Fraction
Measured Concentration After Centrifugation
Tn ITC in Plasma
Percentage Found in Plasma and Cells
120
10
The partitioning of troponin complex between blood and plasma fractions shows that some troponin complex associated with the cellular
fraction is unavailable for detection. Use of a radiochemical method allowed careful examination of the distribution of analyte both in centrifuged
plasma and in membrane-generated plasma. Experiments with 125Iodine-labeled troponin complex showed that all of the analyte was not present
in the free plasma fraction, either after centrifugation or membrane separation. There was very good correlation between the concentration of
troponin complex in plasma generated by centrifugation and plasma generated by separation with casein treated membranes.
100
8
6
4
2
0
1
2
3
Donor
4
80
CONCLUSIONS
60
40
20
0
5
Vivid Plasma Separation membrane has been optimized for the generation of plasma from small volumes of whole blood. Utilization of the
membrane eliminates the need for centrifugation minimizing the turn around time for whole blood diagnostic assays. The rapid generation of
high quality plasma enables the detection of rare or poorly soluble analytes such as TnI. Pre-treatment of the membrane with a combination
of casein, orDonor
other protein blocking agents, andDonor
surfactants has been shown to improve the recovery of target analytes. Pre-treatment of the
membrane effectively decreases loss of analyte due to non-specific binding to the membrane, and increases plasma volume recovery relative
to untreated membrane.
1
2
3
Donor
4
5
Whole Blood Spiked with 125I Single Chain Rec Troponin IC Complex
Partitioning of Tn IC in Whole Blood
Tn IC in Cell Fraction
Measured Concentration After Centrifugation
10
120
Tn ITC in Plasma
Percentage Found in Plasma and Cells
The robust performance and utility of Vivid Plasma Separation membrane enables whole blood assays to be performed at the point-of-care,
thus minimizing the dependency on a centralized lab and improving the determination of key diagnostic indicators.
100
8
6
4
2
0
D
Blocking the membrane allows full recovery of troponin:
16
Pre-treatments increased the amount of plasma delivered to a drainage layer.
Without blocking, GF membranes produced an average of 9 µL plasma. After
blocking with casein, GF membranes produced an average of 15 µL plasma.
Treatment with sucrose alone also increased the plasma recovery.
n
Microfluidic-based assays typically require 5-15 µL plasma for detection. As patients come into the hospital with chest pain, it is important
that a rapid, accurate diagnosis is made. The use of Vivid membrane and a microfluidic test requires only a finger stick volume of whole blood,
which is applied directly to the top of the Vivid membrane. A capillary system contacts the bottom of the Vivid membrane, drawing plasma
into the detection system in minutes. This eliminates the need to draw and send the sample to a STAT lab for analysis, thus shortening the
time to results.
125
Effect of Blocking on Plasma Recovery
% in Plasma and Cells
Fine Pore
Region
Filtration of Spiked Plasma Through Untreated Vivid GF Membrane: Reduction in TnI Concentration
Measured by ELISA
B
l
o
o
d
Casein, Tw 20,
Sucrose
Figure 3
Plasma Yield Increased with Membrane Pre-Treatment
µL Plasma On Disc
Large Pore
Region
RESULTS
Casein, Tw 20
• Untreated membrane showed an average of 27% loss of troponin.
Details of experiments are shown with the results.
Figure 1
Vivid GF Plasma Separation Membrane: Mode of Operation
Whole blood from six donors was spiked with 10 ng/mL 125Iodine troponin
IC complex. After blood was applied to membrane discs, separated plasma
was analyzed for troponin IC recovery and compared to same donor whole
blood spiked and centrifuged. 40 µL of whole blood was applied to 13 mm
membrane discs. Average values shown, error bars = 1 SD.
16
% in Plasma and Cells
Plasma generated on the bottom of the membrane can be collected from the
bottom surface by contact with another porous material, or with a capillary
bed. While this system has been demonstrated to work well for quantitative
determination of abundant proteins and hormones, it is harder to obtain
accurate reproducible results for poorly soluble or low-abundant proteins.
Whole Blood and Plasma
Plasma From Spiked Whole Blood Generated By Untreated and Treated Vivid GF Membrane:
Transmission of Troponin Complex
Figure 2
Improved Recovery of TnI After Filtration Through
Treated Vivid GF Membrane
ng/ml recovered
Vivid Plasma Separation membrane uses a graded pore structure to separate
plasma from whole blood. The membrane is formed from polysulfone and is
inherently wettable with blood. Pores at the top surface are approximately
50x larger than pores at the bottom surface (from 100 µm to 2 µm). As seen
in the SEM at right, when blood is added to the larger pores on top of the
membrane, the cells penetrate partway through the cross section without
plugging the pores. Plasma can then flow through to the bottom of the
membrane.
Troponin ITC complex (Hytest) and recombinant single chain troponin IC (Instrumentation Laboratories) were radio-labeled with 125Iodine at
PerkinElmer NEN. The level of 125Iodine incorporation was such that we were able to use labeled complex only to achieve 10-20 ng/mL levels in
plasma or whole blood without addition of unlabeled troponin complex.
Filtration of Spiked Plasma Through Vivid GF
Membrane: ELISA Results
ng/ml recovered
Vivid Plasma Separation Membrane
Radio-Labeling
RESULTS (continued)
% Centrifuged Plasma Values
Vivid Plasma Separation membrane extracts plasma from small volumes of whole blood. The membrane has an asymmetric pore structure
which traps cells inside the membrane, allowing plasma to flow through to a receiving material or capillary. A pre-treatment process is described
that enables the use of Vivid membrane for microfluidic POC rapid detection of cardiac troponin I (TnI).
RESULTS (continued)
%
METHODS
ng/mL
ABSTRACT
1
2
3
4
Donor
5
6
80
60
40
20
0
1
2
3
4
Donor
5
6
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