Centrifuge-Free Isolation of Liquid Plasma from Clinical Samples of Whole Blood
Mikhail Fomovsky, Galina Fomovska, Tom Bormann, Pall Corporation, Port Washington, NY
MATERIALS AND METHODS
Conclusions – The study results demonstrate the efficient performance of the filtration device in the rapid centrifuge-free production
of plasma. Samples of 100-800 µL cell-free plasma can be collected from 0.8-3.0 mL of whole blood in 2-5 min. The quality of plasma
collected using the filter device is comparable to plasma prepared by centrifugation with respect to the degree of RBC hemolysis, total
protein, total cholesterol, and the HPLC protein profile.
Key Words – Blood filtration device, plasma separation membrane, liquid plasma.
Filtered plasma samples were analyzed for the presence of residual blood cells, free hemoglobin, concentration of total protein, total
cholesterol, and HPLC profiling of plasma proteins against those in the control plasma. Control plasma was prepared from the same
blood samples by a common centrifugation method.
Centrifugal fractionation of whole blood followed by aspiration of the cell-free fraction is currently the typical technique for plasma
sampling. It is a laborious and time-consuming procedure requiring highly skilled laboratory personnel.
Pall Life Sciences presents a new device which enables rapid non-centrifugal isolation of cell-free liquid plasma from whole blood in
a simple POC-capable procedure.
Positive results are achieved by utilizing the separation capability of Pall filter materials, including Pall’s unique Vivid™ Plasma
Separation polysulfone asymmetric membrane in a device designed for this application.
The mechanism of blood separation by Pall Vivid Plasma Separation membrane via size exclusion in a 3-dimensional filtration
structure is presented in Figure 1.
Figure 1
Blood Filtration by Vivid Plasma Separation Membrane Via Size Exclusion Filtration
Blood
Flow
Large-pore
Region
Fine-pore
Region
Captured
Red
Cells
0.14
0.12
2.50
0.04
0.02
2.00
AU
B
0.00
Collected Plasma Samples
0.00
The level of free hemoglobin in plasma collected using the filtration device is comparable to that in centrifugal control plasma.
Table 1
Recovery of Liquid Plasma and Target Analytes on Filtration Device with Vivid Plasma Separation Membrane and Different Pre-Filters
Pre-filter
Media
Loaded Blood
Volume (µL)
Plasma
Recovery (µL)
Total Protein Conc.
% to Control Plasma
Total Cholesterol Conc.
% to Control Plasma
Vivid PSM-GF
non
850
108
102
101
Volume of Plasma
Vivid PSM-GF
GF/AD
3000
700
101
101
100-800 µL of liquid, cell-free plasma can be collected in 2-5 min. from 0.8-3.0 mL of blood by filtration using an R&D prototype of a
Pall filtration device.
Vivid PSM-GF
LKB
1600
280
100
103
Vivid PSM-GX
non
850
274
83
85
Vivid PSM-GX
GF/AD
2700
704
101
98
The concentration of total protein and total cholesterol in obtained plasma samples is greater than 90% of that measured in control
centrifugal plasma (Table 1).
Vivid PSM-GX
LKB
1600
325
92
95
Figures 5 and 6 show the similarity of HPLC protein profiles of plasma collected via the plasma separation device against the profiles
of control centrifugal plasma.
Vivid PSM-GR
non
850
292
77
80
Vivid PSM-GR
GF/AD
2700
630
90
93
The process of plasma separation on the tested device does not cause significant hemolysis of RBCs and facilitates enrichment of
isolated plasma with free hemoglobin (Figures 3 and 4).
Analyte Recovery
Figure 3
Samples of Plasma Separated from Whole Blood Using Pall Filtration Device or Blood Centrifugation Procedure C1, C2, and C3 Three samples of centrifugal plasma; 1, 2, 3, 4, 5, 6, 7, 8, 9 – Samples of plasma obtained from 850 µL of whole blood using Pall’s
plasma separation device.
Separated
Cell-Free
Plasma
Filtered Plasma
Contact: Phone: 800.521.1520 (USA and Canada) • (+)800.PALL.LIFE (Outside USA and Canada) • www.pall.com/oem • E-mail: LabSupport@pall.com.
Vivid PSM-GR
LKB
1800
322
95
95
Three variants of Vivid Plasma Separation membrane (GF, GX, and GR) were tested alone or in combination with micropore pre-filters,
such as glass fiber GF/AD or polymeric LKB media.
Using Vivid Plasma Separation membrane in combination with a pre-filter layer enables a significant increase in blood volume capacity
for the same size device and enhances recovery of target analytes above 90%.
Control
Centrifuged Plasma
Fibrinogen zone
0.50
Blood Separation
Media
RESULTS
2.00
1.00
0
A – cross view of the device; B – collection of liquid plasma after blood sample processing on the device; 1 – top part; 2 - bottom
part; 3 – plasma separation membrane; 4 – blood loading port (inlet); and 5 – plasma sampling port (outlet). Blood sample is loaded
onto the upstream surface of the plasma separation membrane through the blood loading port (4). After absorption of blood by the
membrane matrix (3) liquid plasma is collected from the downstream surface of the membrane through the plasma collection port
(5) using a syringe (B).
HSA
zone
IgG
zone
3.00
0.10
0.06
INTRODUCTION
A wide variety of modern diagnostic tests involve obtaining and subsequent testing of liquid plasma.
Figure 5
HPLC Protein Profiles of Plasma Collected by Filtration Device with Single Layer Vivid Plasma Separation Membrane (GF)
and Control Plasma
0.16
0.08
Figure 2
Schematic of the Filtration Device
A
Demonstration of the similarity of HPLC protein profiles of plasma collected via the plasma separation device against the profiles of
control centrifugal plasma.
20.00
40.00
60.00
Minutes
80.00
100.00
120.00
140.00
Green – protein profile of plasma from the filtration device; Blue – protein profile of centrifugal plasma.
Figure 6
HPLC Protein Profiles of Plasma Collected by Filtration Device with Vivid Plasma Separation Membrane (GX) and GF/AD Pre-Filter
and Control Plasma
3.00
2.50
IgG
zone
HSA
zone
2.00
AU
Results – It was demonstrated that 100-800 µL of liquid, cell-free plasma can be collected in 2-5 min. from 0.8-3.0 mL of blood by a
simple procedure without the need for centrifugation. The process of plasma separation on the tested filter device does not cause
additional red blood cell (RBC) hemolysis – hemoglobin concentrations measured using the device plasma and control plasma samples
are comparable. The recovery of total protein and total cholesterol is greater than 90%. The HPLC protein profile of the collected
plasma in the minor fraction is also very similar to the control plasma.
The device was tested using human blood collected from healthy donors into blood collection tubes containing EDTA anticoagulant.
Whole blood samples of 0.8-3.0 mL were loaded onto the device and collected after separation as demonstrated in Figure 2B.
Figure 4
Free Hemoglobin Level in Plasma Collected Using Pall Filtration Device Compared to Control (OD415 Data)
Control PL
Materials and Methods – The filtration device comprises a plasma separation membrane and a housing having an inlet, a downstream
chamber, and an outlet. The plasma separation membrane is positioned in the housing across the fluid flow path such that, when a
blood sample is loaded on the upstream surface of the membrane and absorbed by its matrix, liquid plasma is collected from the
downstream surface through the outlet. The device was tested using human blood collected from healthy donors into blood collection
tubes containing EDTA anticoagulant. Whole blood samples of 0.8-3.0 mL were loaded onto the device. Plasma was collected,
measured, and analyzed for the presence of residual blood cells, free hemoglobin, concentration of total protein, total cholesterol, and
HPLC profiling of plasma proteins. Control plasma was prepared from the same blood by a common centrifugation method.
The filtration device comprises a plasma separation membrane and a housing having an inlet, a downstream chamber, and an outlet.
The plasma separation membrane is positioned in the housing across the fluid flow path such that, when a blood sample is loaded on
the upstream surface of the membrane and absorbed by its matrix, liquid plasma is collected from the downstream surface through
the outlet. A schematic of the device is presented in Figure 2A.
RESULTS (continued)
Control PL
Objectives – Demonstrate the effectiveness of a novel filtration device for the rapid isolation of liquid plasma from 0.8-3.0 mL of whole
blood without the use of a centrifuge. Illustrate the application of the device for plasma sample preparation in terms of volume of
collected plasma, hemolysis, and target analyte recovery.
RESULTS (continued)
Control PL
ABSTRACT
2.00
1.00
Fibrinogen zone
0.50
0.00
0.00
20.00
40.00
60.00
Minutes
80.00
100.00
120.00
140.00
Green – protein profile of plasma from the filtration device; Blue – protein profile of centrifugal plasma.
CONCLUSIONS
Pall’s plasma filtration device enables the rapid centrifuge-free production of plasma from 0.8-3.0 mL samples of blood in 2-5 min.
The quality of plasma collected using the filtration device is comparable to that of plasma prepared by centrifugation with respect
to the degree of RBC hemolysis, concentration of total protein, total cholesterol, and the HPLC protein profile.
Use of Vivid Plasma Separation membrane in combination with glass fiber or a polymeric pre-filter enables a significant increase
in blood volume capacity for the same size device and enhances recovery of target analytes above 90%.
© 2012, Pall Corporation. Pall,
, and Vivid are trademarks of Pall Corporation. ® indicates a trademark registered in the USA. 6/12, GN12.8070.