Whole blood separation method and devices using the same

The present invention relates to a method and to a device for separating plasma from whole blood. The method and device utilize a permeable non-glass fiber matrix containing a polyol which is capable of clumping red blood cells. The matrix, in the absence of such a polyol, would otherwise be porous to red blood cells. The polyol-containing matrix has a first surface and a second surface such that a whole blood sample which is applied to the first surface flows directionally toward the second surface. Plasma separated from whole blood becomes available at the second surface of the matrix and can be tested for the presence of a particular analyte, such as glucose or fructosamine, as provided by multi-layer test devices of the present invention.

FIELD OF THE INVENTION 
The present invention relates generally to the chemical analysis of 
analytes present in whole blood and more specifically, to a method and to 
a device for separating plasma or serum from whole blood, thus providing a 
convenient and accurate means for such chemical analysis. 
BACKGROUND INFORMATION 
Presently, numerous test devices are available for the analysis of body 
fluids in order to determine the presence or concentration of a particular 
analyte. For example, tests are available for detecting glucose, 
fructosamine, albumin, calcium, urea, uric acid, bilirubin, cholesterol, 
and other soluble analytes present in whole blood or the fluid part of 
blood, namely the plasma or serum, after whole blood has been separated. 
Many of these test devices utilize chromogenic or other visual responses to 
indicate the presence or absence, or the concentration, of an analyte 
being detected. Cellular components of whole blood, and in particular the 
red blood cells, have a deep red color which substantially interferes with 
chromogenic or other visual tests. Therefore, the highly-colored red blood 
cells as well as other interfering substances present in blood including 
hemoglobin and white blood cells are separated from the plasma or serum 
before a blood sample is assayed for a particular analyte. 
Conventionally, the plasma or serum is separated from the cellular material 
of whole blood by centrifugation. The cellular material collects at the 
bottom of the centrifuge tube and the supernatant plasma or serum is 
decanted and tested for a particular analyte. Centrifugation, however, is 
time consuming, involves extra manipulative steps and requires equipment 
that is generally not present outside of the clinical laboratory. Thus, 
reliance on centrifugation makes field testing, such as testing at the 
doctor's office or at the patient's home, difficult. 
Certain methods other than centrifugation have been developed to separate 
the cellular components of whole blood from plasma or serum. Some of the 
earlier methods, such as that described in U.S. Pat. No. 4,543,338 to 
Chen, involve the use of a carrier membrane impregnated with a test 
reagent and coated with a semipermeable membrane. The semipermeable 
membrane effectively acts as a means for filtering out cells or large 
molecules, such as hemoglobin, but allows the passage of smaller molecules 
and ions which then contact the testing reagents impregnated in the 
bibulous matrix. These methods, however, typically require an extra 
manipulative step, such as rinsing with water or wiping off the test 
device so as to remove cellular material retained on the semipermeable 
membrane. Such techniques can be cumbersome and laborious. Moreover, if 
the red blood cells are not completely removed or rinsed from the 
semipermeable barrier, interference with the assay remains a problem. 
Another method for separating whole blood is described in U.S. Pat. No. 
4,477,575 to Vogel et al. which describes separating plasma or serum from 
whole blood using a layer of glass fibers having a defined average 
diameter and density. As well, Baumgardner et al. in U.S. Pat. No. 
5,186,843 describe the use of glass fibers in a single separation layer. 
Blood separation devices utilizing glass fiber filters, however, tend to 
separate serum at a relatively slow speed and tend to retain significant 
quantities of serum or plasma in the interstices of the glass fiber 
matrix. 
Alternative approaches to blood separation involve incorporating 
agglutinating reagents or other separation reagents in a matrix. For 
example, Aunet et al. in U.S. Pat. No. 4,933,092, Daubney et al. in 
published Canadian Patent Application No. 2,104,976, and Limbach in 
European Patent Application 0 194 502 all describe the use of polymeric 
agglutinating agents, such as cationic polymers, which, for example, in 
Daubney and in Limbach are combined with additional agglutinating agents, 
such as lectins. As well, Barkes et al. in European Patent Application No. 
0 436 897, describe the use of lectins and thrombin incorporated into a 
suitable carrier matrix. However, such matrices incorporated with these 
types of agglutinating agents exhibit problems similar to those associated 
with glass fiber matrices. For example, the separation may occur at a 
relatively slow speed and the amount of plasma or serum separated may be 
limited to 50% of the absorption volume of the matrix, often requiring the 
use of external pressure, such as in European Patent Application 0 194 
502, in order to obtain the maximum efficiency and quantity of plasma or 
serum. 
Other separating agents, such as water-soluble salts, amino acids, 
carbohydrates and large polymers, such as polyethylene glycol, polyvinyl 
alcohol and the like, have been incorporated into single matrix test 
strips. Fetter in U.S. Pat. Nos. 3,552,925 and 3,552,928 describes a test 
device having a bibulous matrix impregnated with an inorganic salt or 
amino acid at a first region on the matrix and test reagents impregnated 
at an adjacent second region. While the salts or amino acids used in this 
process can separate the cellular components from the whole blood, they 
also introduce contaminating ions or molecules into the plasma or serum 
and precipitate a portion of the soluble plasma or serum constituents, 
thus rendering a quantitative assay for the soluble constituent 
unreliable. Rapkin et al. in U.S. Pat. No. 4,678,757 describe the use of 
carbohydrates, such as mannitol, impregnated or coated onto a carrier, 
preferably coated onto an impermeable carrier. The described device, 
whether having a permeable or impermeable carrier, however, only provides 
for capillary and longitudinal transport of the blood. Therefore, the 
blood separation matrices of Rapkin et al. are not described as being 
useful in test devices that operate primarily by gravitational force, as 
do many of the multi-layer test devices currently used by doctors or 
patients. While Kiser et al. in U.S. Pat. No. 5,306,623 describe 
separation matrices which can operate by wicking gravity flow, Kiser et 
al. use large polymeric separating reagents, such as polyethylene glycol, 
polystyrene sulfonic acid, hydroxypropyl cellulose, polyvinyl alcohol, 
polyvinylpyrrolidone, and polyacrylic acid. Upon application of a whole 
blood sample, such large polymers contained within a matrix would be 
solubilized, potentially blocking the pores of the matrix and most 
certainly rendering the sample more viscous, thereby slowing the 
separation process and decreasing the yield of plasma. 
Based on the shortcomings of these methods, there exists a need for a 
device that provides rapid and efficient methods for the separation of 
plasma or serum from whole blood. In particular, there is an increasing 
awareness of the importance of, and accordingly a need for, being able to 
carry out diagnostic assays at the doctor's office or, better yet, at 
home. Therefore, there exists a need to minimize any extra manipulative 
steps, such as the rinsing or wiping of test devices or the application of 
external pressure. Moreover, there is a need for rapid separation which is 
reliable and does not contain interfering substances. The present 
invention satisfies these needs and provides related advantages as well. 
SUMMARY OF THE INVENTION 
The present invention relates to a method and to a device for separating 
plasma from whole blood. The method and device utilize a permeable 
non-glass fiber matrix containing a polyol which is capable of clumping 
red blood cells. The matrix, in the absence of such a polyol, would 
otherwise be porous to red blood cells. The polyol-containing matrix has a 
first surface and a second surface such that a whole blood sample which is 
applied to the first surface flows directionally toward the second 
surface. Plasma separated from whole blood becomes available at the second 
surface of the matrix and can be tested for the presence of a particular 
analyte, such as glucose or fructosamine, as provided by multi-layer test 
devices of the present invention.

DETAILED DESCRIPTION OF THE INVENTION 
There is an increasing awareness of the importance of being able to carry 
out diagnostic assays at the doctor's office or, better yet, at home. For 
example, a diabetic's blood glucose level fluctuates significantly 
throughout a given day, being influenced by diet, activity, and treatment. 
Depending on the nature and severity of the individual case, some diabetic 
patients measure their blood glucose levels up to seven times a day. 
Clearly, the results of these tests should be available to the patient 
immediately. 
Because of the frequent fluctuation of glucose levels in a given day, tests 
which are independent of a patient's diet, activity, and/or treatment and 
which provide longer term indications of blood glucose levels have been 
developed. These tests measure the concentration of glycated proteins or 
"frucosamines." Proteins, such as those present in whole blood, plasma, 
serum and other biological fluids react with glucose, under non-enzymatic 
conditions, to produce glycated proteins. The extent of the reaction is 
directly dependent upon the glucose concentration of the blood. 
Measurement of serum or plasma fructosamine levels is useful for 
monitoring diabetic control because fructosamine concentrations in serum 
or plasma reflect an average of blood glucose level over approximately a 
half month period. 
Scandinavian investigators recently showed that doctors and patients who 
were made aware of their glycated protein test results had better glycemic 
control than those who were unaware of such results. Moreover, it is now 
believed that glycated proteins can be the causative agents of 
complications associated with diabetes, which include retinopathy, 
nephropathy, neuropathy and cardiovascular disease. Therefore, any delay 
in information transfer, such as a doctor's delay in reporting clinical 
test results to a patient, decreases the value of the test result. Again, 
this emphasizes the importance in being able to perform diagnostic assays 
at the doctor's office or at home. 
For an assay to be useful in the doctor's office or home, the test should 
be relatively free of sensitivity to small changes in the conditions under 
which the assay is carried out and the measurements should be accurate and 
reliable. Equally as important, if not more so, the assay must have a 
simple and convenient protocol which does not involve extra manipulative 
steps. To enhance the simplicity and convenience of such tests, the 
preferable body fluid for testing such analytes as glucose and 
fructosamine is whole blood which can simply be taken from a finger or 
earlobe puncture. Such simple and rapid determinations of an analyte in 
blood is especially desirable in the case of an emergency. 
As described above, the simplicity and accuracy of such tests depends to a 
large extent on the whole blood separation layer contained within the test 
device and the ability of the blood separation matrix to provide 
uncontaminated plasma or serum. The present invention provides a method 
and device, namely a blood separation matrix, which is capable of such 
simple and accurate blood separation. Of particular importance, the matrix 
contains a polyol which causes red blood cells contained in whole blood to 
clump. The clumped red blood cells either can be retained in the matrix or 
can be filtered by a filter material. The method and matrix can be used in 
various test devices which analyze whole blood for a particular analyte, 
such as, the fructosamine and glucose test devices provided by the present 
invention. 
As used herein, the term "plasma" means the substantially colorless fluid 
obtained from a whole blood sample after red blood cells have been removed 
by the separation process and device of the present invention. Because 
plasma is serum plus the clotting protein fibrinogen, the term "plasma" is 
used broadly herein to include both plasma and serum. 
In order to obtain plasma from whole blood, the present invention provides 
a permeable non-glass fiber matrix containing a polyol which is capable of 
clumping red blood cells. The matrix is porous to red blood cells in the 
absence of such a polyol. The matrix has a first surface for sample 
application and a second surface where plasma is received or becomes 
available. If desired, the matrix additionally can contain a polycationic 
polymer. Also useful, though not required, is a permeable filter material 
or membrane supporting the separation matrix which serves as a final 
filter to red blood cells and/or provides a reagent layer for effecting an 
assay. Each of these components of the invention, as well as test devices 
which use the blood separation method and matrix of the present invention 
are disclosed in detail. 
MATRIX 
The separation matrix of the present invention is a permeable matrix which 
does not contain glass fibers and, therefore, is termed "a permeable 
non-glass fiber matrix." The term "permeable" means liquid-permeable, such 
as permeable to plasma, as well as permeable or porous to red blood cells 
when the matrix is provided in the absence of a polyol. As used herein, 
the phrase "matrix being porous to red blood cells in the absence of a 
polyol" means that without the polyol contained in or on the matrix the 
red blood cells would simply pass through the matrix, virtually 
immediately. In the absence of the polyol, red blood cells are not 
retained, by filtration or otherwise, in the matrix. 
The polyol contained within or on the matrix chemically reacts with the 
whole blood sample so as to clump the red blood cells. As used herein, 
"clump" or "clumping" means the collection into a mass or group, red blood 
cells distributed in a whole blood sample. While not wishing to be bound 
by any theory or mechanism, the clumping can be the result of 
agglutination, coagulation, or the like, or some other chemical 
interaction between the polyol and the red blood cells. Thus, the present 
invention is not strictly a filtration process, for example, based on the 
pore size of the matrix, as described, for example, in U.S. Pat. No. 
4,543,338 to Chen, or as used in a glass fiber prefilter, such as that 
described in U.S. Pat. No. 4,477,575 to Vogel et al. Rather, it is the 
presence of a polyol which provides the matrix with its ability to 
separate plasma from whole blood by clumping the red blood cells. 
Surprisingly, the clumping of the red blood cells by the polyol does not 
substantially block the flow of the whole blood sample or plasma through 
the matrix. Thus, sufficient amounts of plasma become available at the 
second surface of the matrix. Sufficient quantities of plasma can rapidly 
be obtained for the specific applications exemplified herein, namely 
analyzing the concentration of frucotosamine or glucose in drops of whole 
blood. The present invention can be used with other applications and 
diagnostic assays as well, including ones which use a larger volume of 
blood or which require more plasma. With as small as a 3/16" diameter 
circle of a polyol-containing matrix of the present invention, as much as 
10 .mu.l of plasma can be obtained from a drop of blood. Therefore, a 
matrix of the present invention can be used for separating larger volumes 
of blood than a drop of blood. Accordingly, the matrix can be used in 
diagnostic applications which analyze larger quantities of blood, such as 
for example, some known cholesterol tests. 
A useful permeable matrix can be a woven or non-woven material and can be 
an absorbent or a non-absorbent material which may or may not be 
hydrophilic. Especially suitable materials for the matrix include, for 
example, woven or non-woven, absorbent or non-absorbent, nylon, rayon, 
cotton, and polyester. In one embodiment of the invention, the matrix is a 
non-woven, non-absorbent polyester. The polyester is preferably a 
poly(paraphenylene terephthalate), such as that used in a preferred 
polyester sold as Sontara.RTM. (DuPont, Inc., Wilmington, Del.). Another 
preferred matrix is the woven, absorbent nylon Tetex.RTM. 3-3710 (Tetko, 
Inc., Lancaster, N.Y.). 
Depending upon the porosity or other properties of the matrix, the clumped 
red blood cells either are retained in the matrix or are filtered out by 
the filter material as described below. Some of the above-described matrix 
materials, such as the non-woven, non-absorbent polyesters, do not have 
"pores" in the traditional sense, i.e., that can be measured, for example, 
by pore size (microns). In the absence of a polyol of the present 
invention such materials essentially have no limit as the porosity and are 
porous to red blood cells, which have an average size of 5 .mu.m. With 
such macroporous materials, if the polyol is not present the red blood 
cells pass through the matrix almost immediately. For those matrix 
materials which can be characterized based on pore size, the matrices used 
in the present invention can have a pore size generally of from about 2 
.mu.m to about 10 .mu.m. Such pores sizes can be useful for retaining the 
clumped red blood cells. Depending upon the porosity, thickness, which is 
generally 200 to 1100 .mu.m, and other properties of the matrix, such as 
absorbency, the clumped red blood cells are either retained in the matrix 
or captured in a final filter material as described below. 
The polyol-containing matrix has a first surface for sample application and 
a second surface where plasma is received or becomes available for testing 
or additional separation. Generally, the first and second surfaces are 
presented as opposite sides of the matrix. The whole blood sample flows in 
a direction from the first surface toward the second surface, under 
conditions which provide such directional flow, such as, gravitation, 
vacuum, or external pressure. To enhance the simplicity of the method, if 
desired, separation can be performed by gravity alone. Preferably, the 
separation matrix provides for flow in a vertical direction, preferably by 
gravitation. 
Rapkin et al. in U.S. Pat. No. 4,678,757, describe the use of 
carbohydrates, such as mannitol, impregnated or coated onto a carrier, 
preferably coated onto an impermeable carrier. However, the described 
device of Rapkin et al., whether having a permeable or impermeable 
carrier, only provides for capillary and lateral transport of the blood. 
There can be divergent contact times provided by vertical flow, a 
relatively short period of contact, versus lateral flow, a slow process 
which can involve continuous interaction between a whole blood sample and 
a matrix. Because of these variable contact times, it is not predictable 
that what works by lateral flow would similarly work under vertical flow. 
Unexpectedly, with the present invention, even with matrices which are 
porous to red blood cells in the absence of a polyol, a matrix containing 
a polyol can effectively separate plasma from whole blood even when the 
blood sample flows vertically through the matrix. 
POLYOL 
The separation method and device include a permeable non-glass fiber matrix 
containing a polyol. As used herein, the terms "matrix containing a 
polyol" and "polyol-containing matrix" mean that the polyol is separately 
added to the matrix and is not a component originally found in the 
composition or make up of the matrix, such as cellulose filter paper. 
Further, "matrix containing a polyol" means a polyol can be impregnated 
into the matrix or coated into or onto the matrix or covalently or 
non-covalently bound to the matrix. In a preferred embodiment, the polyol 
is impregnated into the matrix. 
As used herein, the term "polyol" means a polyhydroxy alcohol which is an 
alkyl or aromatic containing more than one hydroxyl group. The term "poly" 
as used in "polyol" does not infer that the alkyl or aromatic compound is 
a large polymer made up of repeating monomeric units, but, instead, means 
that more than one hydroxyl group is present in the compound. As discussed 
more fully below, with the exception of polysaccharides, the polyols used 
in the present invention are simple sugars or sugar alcohols, 
oligosaccharides, or other naturally or non-naturally occurring 
non-polymeric alkyl or aromatic compounds. Therefore, the term "polyol" 
encompasses sugars, alcohol derivatives of sugars, herein termed "sugar 
alcohols," and other naturally or non-naturally occurring non-polymeric 
polyols. 
As used herein, "sugar" includes monosaccharides, oligosaccharides, and 
polysaccharides. A monosaccharide is a simple sugar which is as a linear, 
branched, or cyclic polyhydroxy alcohol containing either an aldehyde or a 
ketone group. Exemplary monosaccharides include, but are not limited to, 
mannose, glucose, talose, galactose, xylose, arabinose, lyxose, ribose and 
fructose. An oligosaccharide is a linear or branched carbohydrate that 
consists from two to ten monosaccharide units joined by means of 
glycosidic bonds. Oligosaccharides which can be used in the present 
invention include, but are not limited to disaccharides such as sucrose, 
trehalose, lactose and maltose. Examples of larger oligosaccharides which 
can be used in the invention include the cyclodextrins, such as 
alpha-cyclohexylamylose, beta-cycloheptaamylose, and 
gamma-cyclooctoamylose, as well as other oligosaccharides well known in 
the art. A polysaccharide is any linear or branched polymer having more 
than ten monosaccharides linked together by glycosidic bonds. Exemplary 
polysaccharides include, but are not limited to, ficoll, polysucrose, and 
hydroxyethyl starch. 
Encompassed within "sugar" are those sugars which are naturally occurring 
as well as those which are known but which have not yet been identified as 
occurring naturally in plants or animals. For example, there are five 
known naturally occurring aldohexoses, including D-glucose, D-mannose, 
D-talose, D-galactose, and L-galactose. However, the aldohexose structure 
has four chiral carbons and thus, sixteen possible stereoisomers, all of 
which are known, although only the five listed above have been identified 
as occurring naturally in plants or animals. Thus, "sugar" encompasses 
enantiomers in either the D or L forms of a sugar as well as racemic 
mixtures thereof. 
A polyol of the present invention also can be a "sugar alcohol." A "sugar 
alcohol" is an alcohol derivative of a mono- or an oligosaccharide which 
is generally formed by reduction of the aldehyde or ketone moiety on the 
mono- or oligosaccharide. Exemplary sugar alcohols include, but are not 
limited to, mannitol, sorbitol, arabitol, inositol, galactitol, 
erythritol, and threitol. Also included within the definition of "sugar 
alcohol" are the alcohol derivatives of those mono- and oligosaccharides 
described above. 
Where chiral carbons are present in the sugar alcohol, the sugar alcohol 
may be in the D or L form, such as D-threitol or L-threitol, or in a 
racemic mixture of both the D and L forms. The sugar alcohol can, but does 
not have to, be naturally occurring. That is, the sugar alcohol can be a 
derivative of a known, naturally occurring sugar, or, alternatively, it 
can have a D or L configuration known to exist but not necessarily 
identified as occurring in nature. The sugar alcohol also can be a sugar 
which is found naturally in its reduced alcohol form or it can be an 
alcohol derivative of a sugar which derivative is not known to exist in 
nature. 
In addition to sugar or sugar alcohols, the polyol can be a non-polymeric 
naturally occurring or non-naturally occurring polyol, which includes 
linear, branched, or cyclic alkyl or aromatic compounds containing more 
than one hydroxyl group. As used herein the term "non-polymeric" means the 
alkyl or aromatic compounds are not polymers. Polymers are defined as high 
molecular weight compounds consisting of long chains that may be open, 
closed, linear, branched, or crosslinked, which chains are composed of 
repeating units, called monomers, which may be either identical or 
different. As used herein, those polyols which are "naturally occurring" 
are ones which occur in nature and those which are "non-naturally 
occurring" are not found in nature. Generally, these naturally occurring 
or non-naturally occurring alkyl or aromatic compounds range in size from 
three to twenty carbons (C.sub.3 to C.sub.20), and more preferably, from 
three to ten carbons (C.sub.3 to C.sub.10). Examples of such naturally 
occurring, non-polymeric polyols are glycerol, a three-carbon trihydroxy 
alcohol that occurs in many lipids, and quinic acid, a 
1,3,4,5-Tetrahydroxycyclohexanecarboxylic acid, which acid can be in the 
salt form. Examples of non-naturally occurring, non-polymeric polyols 
include pentaerythritol and dipentaerythritol. 
Kiser et al. in U.S. Pat. No. 5,306,623 describe the use of large polymeric 
separating reagents, such as polyethylene glycol, polystyrene sulfonic 
acid, hydroxypropyl cellulose, polyvinyl alcohol, polyvinylpyrrolidone, 
and polyacrylic acid. Upon application of a whole blood sample, such large 
polymers contained within a matrix would be solubilized, potentially 
blocking the pores of the matrix and most certainly rendering the sample 
more viscous, thereby slowing the separation process and decreasing the 
yield of plasma. Therefore, with the exception of the above-described 
polysaccharides, the invention does not involve the use of large alkyl 
polymers as the primary separating agent. The matrix described in U.S. 
Pat. No. 5,306,623 is different from the instant invention in a number of 
other aspects as well. For instance, the Examples given in U.S. Pat. No. 
5,306,623 involve the use of matrices which have a very small pore size, 
less than 1 .mu.m, which would act as a filter to red blood cells in the 
absence of the disclosed polymers. As discussed above, the present 
invention is not strictly a filtration process, rather it involves the use 
of polyols which clump red blood cells and in the absence of such polyols 
the matrix would be porous to red blood cells. Moreover, the matrices 
taught by Kiser et al., in addition to a polymer, also contain the test 
reagents, which reagents may substantially influence the separation and 
test results. The separation matrix of the present invention does not 
contain the test reagents. The test reagents, as disclosed in greater 
detail below, are either on the filter material or additional test reagent 
layers and the like. 
In one embodiment, to apply the polyol to the matrix, the polyol can simply 
be dissolved in an aqueous solution generally, at a concentration of about 
20% when used alone, and at about 10% concentration when combined with a 
polycationic polymer, which is generally present in a concentration of 
about 0.5% to 5% as discussed more fully below. If desired, multiple 
layers of matrices containing polyol at lower concentrations, such as four 
layers of matrix containing 5% polyol, also can be used. The polyol and, 
if present, the polycationic polymer can alternatively be dissolved in 
physiological saline (0.85% NaCl), phosphate buffered saline (PBS), an 
organic solvent, or the like. 
POLYCATIONIC POLYMER 
In addition to the polyol, a polycationic polymer can, but does not have 
to, be added to the matrix. Similar to the addition of a polyol to the 
matrix, the polycationic polymer can also be physically impregnated, 
coated into or onto, or covalently or non-covalently bound to the matrix. 
The polycationic polymer is also useful for clumping, as well as 
stabilizing clumped, red blood cells, the latter of which is described for 
example, in the published Canadian Patent Application No. 2,104,976 to 
Daubney et al., which is incorporated herein by reference. 
The polycationic polymer component can be any polymer having more than one 
cationic site and are generally based on monomers which contain an amine 
group. Suitable polycationic polymers include, for example, hexadimethrine 
bromide, trimethylenehexamethylenediammoniumbromide, polylysine, 
polyallylamine, polyarginine, poly(N,N-dimethylaminoethylmethacrylate, 
copolymers of N,N-dimethylaminoethylmethacrylate and methylmethacrylate, 
polyethyleneimine, poly(diallyldimethylammonium chloride), 
poly(1,1-dimethyl-3,5-dimethylenepiperidinium chloride), and mixtures 
thereof. The polymerized positively charged amino acids, such as 
polylysine, can have the amino acids in either the D or L forms, such as 
poly-L-lysine or poly-D-lysine, or a racemic mixture thereof, such as 
poly-D,L-lysine. 
As described above, in one embodiment, to apply the cationic polymer to the 
matrix, the polymer can be dissolved in an a solution such as water, 
physiological saline, PBS, an organic solvent, or the like, and the matrix 
then dipped into the polymer containing solution. Generally, the polymer 
is in a concentration of about 0.5% to 5%. Where both polyol and polymer 
are contained in the matrix, the order of adding polyol and polymer to the 
matrix is irrelevant. For example, polyol and polymer can be 
simultaneously or sequentially dissolved in such aqueous solutions or 
solvents as those described above and both polyol and polymer 
simultaneously applied to the matrix, as described in the Examples below. 
Alternatively, polyol and polymer can be applied to the matrix 
sequentially in any order. 
Non-hemolytic detergents, such as Pluronic (Pragmatics, Inc., Elkhart, 
Ind.), can be added to the aqueous solutions or solvents described above, 
generally at a concentration of 0.01% to 0.1%. Such detergents help 
maximize impregnation of a polyol into the matrix, thereby improving the 
flow rate of the whole blood sample and the plasma. Other optional agents 
which can further enhance the flow rate, include, for example, 
polyvinylpyrrolidone or similar polymers and other fillers which give the 
matrix and the below described filter material stiffness. 
FILTER 
Though not required, a filter material can be used in combination with the 
matrix of the present invention. Suitable filter materials include, for 
example, nylon, cellulose acetate, polysulfone, synthetic fibers, and 
polycarbonate. The filter can, but does not have to, be a membrane. 
Illustrative filters and membranes include, for example, BTS polysulfone 
membrane (Memtek, Inc., San Diego, Calif.), Ahlstrom synthetic fiber 
sheets, such as 94-30 A (Ahlstrom Filtration, Inc., Mt. Holly Spring, 
Pa.), Biodyne A.RTM. nylon membrane (Pall Corp., East Hills, N.Y.), 
Ultrabind 450 (Gelman, Ann Arbor, Mich.), and Nucleopore.RTM. 
polycarbonate (Costar, Corp., Cambridge, Mass.). 
The need for any additional filter material depends to a large extent on 
the porosity, thickness, absorbency or other properties of the matrix. For 
example, the clumped red blood cells, depending upon the above properties 
of the matrix, can be retained in the matrix. Alternatively, or in 
addition thereto, a final filter material can be used to capture or retain 
any additional clumps of red blood cells. Where present, the filter 
material can generally have a porosity of up to about 12 .mu.m and 
preferably will have a pore size of less than 10 .mu.m, and more 
preferably 5 .mu.m or less. 
A filter material can be placed underneath the polyol-containing separation 
matrix, thereby supporting the matrix. Because the filter or membrane is 
at the second surface of the matrix where the plasma becomes available, 
the filter material can also serve as a reagent layer. The filter material 
can contain at least one chemical reagent for determining the presence of 
an analyte in the plasma. Determining the presence of an analyte can be a 
qualitative or quantitative determination. 
In preferred embodiments of the invention, the blood separation method and 
device comprise a non-woven, non-absorbent polyester matrix impregnated 
with mannitol and either a nylon or polysulfone membrane below the matrix. 
Preferably, the matrix additionally contains hexadimethrine bromide. The 
membrane can additionally contain at least one chemical reagent for 
analyzing the concentration of an analyte such as glucose present in a 
whole blood sample. 
In one embodiment of the invention, provided in FIG. 2 and Example 2, the 
filter membrane contains test reagents for determining glucose 
concentration in whole blood. Referring to FIG. 2, the glucose test device 
of the present invention has a polyol-containing blood separation matrix 
15 which can be held in position by a mask 14 and a plastic support member 
16, below the latter of which is a membrane 17 containing glucose test 
reagents, held in position by a plastic support member 18. 
Chemical reagents for determining the presence or absence or the 
concentration of various analytes, such as glucose or fructosamine, are 
well known in the art. For example, such test reagents which produce a 
signal in response to glucose typically involve a glucose oxidase enzyme 
reaction. Glucose and glucose oxidase enzyme react to produce hydrogen 
peroxide. A peroxidase, such as horse radish peroxidase, and a redox 
indicator, such as o-tolidine, o-dianisidine, 3,3,5,5-tetramethylbenzidine 
(TMB), 4-aminoantipyrine, and others well known in the art, can be 
oxidized in the presence of hydrogen peroxide to produce a colored 
product. Such reagents for determining glucose presence and concentration 
are disclosed, for example, in European Patent Application 0388782 to 
Chen, and U.S. Pat. No. 5,304,468 to Phillips et al., both of which are 
incorporated herein by reference. 
As described above, though not required, the filter material can serve as a 
final filter in the blood separation process. In another embodiment, the 
filter is provided without the presence of chemical test reagents. For 
example, referring to an alternative embodiment of the invention, as shown 
in FIG. 1, a multi-layer fructosamine test device containing the blood 
separation matrix of the present invention in combination with a membrane 
can be used to determine the concentration of fructosamine present in 
whole blood. Referring now to FIG. 1, a fructosamine multi-layer test 
device 1 has a blood separation matrix 4 and membrane 5, below which is 
the reagent layers, including a buffer layer 6 and an indicator layer 7 as 
well as clear plastic window 8 for reading the test results on the 
indicator layer 7. A mesh layer 3, used to press the multi-layers 
together, and the separation matrix 4 are contained in a guard piece 11 
and sealed with the membrane 5. Layers 6, 7, and 8 are contained in 
opening 12 of well 13 which is on plastic support member 9. Pieces 11 and 
13 containing the respective layers are ultrasonically welded together 2. 
Test reagents for determining the presence or concentration of fructosamine 
such as the appropriate buffers and indicator reagents, including 
chromogenic dyes, or fluorescent reagents, are known in the art, for 
example, as described in U.S. patent application Ser. No. 08/269,351, 
which is incorporated herein by reference. 
The buffer layer 6 of the fructosamine test generally contains a buffer 
having a pH value of at least 9. Various known buffers can be contained in 
the buffer layer so long as the buffer provides sufficiently high pH such 
that the fructosamines are converted to their eneaminol form. The 
eneaminol form of fructosamine is a chemically active reducing substance 
that reacts with a suitable indicator capable of being reduced by 
fructosamine. To achieve this, the pH of the buffer should be at a pH 
value between about 9 and about 13 and, for optimum results, the pH is at 
a pH value of between 10 and 12. Examples of such buffers include 
potassium hydrogen phosphate, sodium hydrogen phosphate, sodium hydroxide, 
guanidinium salts, certain amino acids, and other suitable buffers as are 
well known in the art, or combinations thereof. 
The indicator layer 7 of the fructosamine test device contains any 
indicator capable of being reduced by fructosamine such as certain dyes, 
including chromogenic dyes, or fluorescent reagents. Examples of suitable 
chromogenic dyes which change color based on the amount of fructosamine 
present in a liquid sample include tetrazolium dyes such as Neotetrazolium 
chloride (NT), Tetranitroblue tetrazolium chloride (TNBT), Blue 
tetrazolium chloride (BT), Iodonitrotetrazoilum chloride, Nitroblue 
tetrazolium chloride (NBT), Nitro blue monotetrazolium chloride, Thiazolyl 
blue tetrazolium bromide (MTT), Tetrazolium violet, 
2,3,5-Triphenyl-2-H-tetrazolium chloride, Thiocarbamyl nitro blue 
tetrazolium chloride (TCNBT), Tetrazolium XTT (XTT), 
2-2'-Benzothiazolyl-5-styryl-3-(4'-phthalhydrazidyl) tetrazolium chloride 
(BSPT), Distyryl nitroblue tetrazolium chloride (DSNBT). An example of a 
suitable fluorescent reagents is 5-Cyano-2,3-ditolyl tetrazolium chloride 
(CTC). 
The following examples are intended to illustrate but not limit the 
invention. 
EXAMPLE 
Fructosamine Test 
This Example provides the preparation and testing of a multi-layer 
fructosamine test device using a whole blood separation matrix of the 
present invention. Because the presence of red blood cells normally 
interferes substantially with the analysis of fructosamine in whole blood, 
this Example compares testing for fructosamine in a whole blood sample 
using the present invention versus testing for fructosamine in a serum 
sample. 
A. Blood Separation Layer 
Mesh: A Tetko mesh #7-280/44 (Tetko, Inc. Rueschlikon, Switzerland) was 
placed in a detergent solution of 1% Pluronic (Pragmatics, Inc.) for 1 
minute. Excess detergent was removed and the mesh was dried by heating at 
60.degree. C. for 10 minutes. Mesh was stored in desiccated plastic bags 
until ready for use at which time a 3/16" circle of the mesh was placed in 
the fructosamine multi-layer test device. 
Blood Separation Matrix: A solution of 10% mannitol and 1.25% 
hexadimethrine bromide in physiological saline (0.85% NaCl) was 
impregnated onto Sontara.RTM. #8007 (DuPont, Inc.) on an automated 
impregnation/drying unit (AFM Engineering, Santa Anna, Calif.). The drying 
temperature was 100.degree. C. for approximately 10 minutes. 
Membrane: An untreated BTS polysulfone membrane of 0.85 p/n pore size 
(Memtek, Inc.) was cut into a 3/16" for use as an additional filter below 
the blood separation matrix. 
B. Reagent Layers: 
Buffer Layer: A 1M solution of aqueous sodium phosphate (aqueous NaH.sub.2 
PO.sub.4) containing 1M guanidinium carbonate buffer was titrated with 
sodium hydroxide (NaOH) to yield a 100 ml solution at pH 11. After 
addition of 0.5% Surfactant 10G detergent (Pragmatics, Inc.) the mix was 
impregnated onto Whatman 540 paper and dried for 10 minutes at 100.degree. 
C. 
Dye Layer: A 10 mM methanolic solution of nitroblue tetrazolium chloride 
(NBT) containing 200 .mu.M N-ethylmethoxyphenazine ethylsulfate and 1% 
Gantrez.RTM. AN 119 was impregnated onto Whatman 54 paper and dried for 15 
minutes at 60.degree. C. 
The layers (3/16" circles) were assembled as follows: 
Tetko Mesh (top) 
Mannitol Containing Matrix 
Polysulfone Membrane 
Buffer Layer 
Dye Layer (bottom) 
The layers were held in place by an injection molded plastic part. Whole 
blood and serum (15 .mu.l) from the same donor were applied at the top and 
reaction rates were measured at the bottom as follows: 
______________________________________ 
.DELTA.K/S 
Sample Starting K/S 
(1 to 2 min.) 
______________________________________ 
whole blood 0.214 0.184 
serum 0.201 0.184 
______________________________________ 
The results demonstrate that the reaction rate with the whole blood sample 
is the same as that with the serum sample. These results indicate that 
there was no interference from red blood cells and, therefore, that the 
whole blood separation of the present invention successfully removed the 
red blood cells present in the whole blood sample. 
EXAMPLE II 
Glucose Test 
This Example demonstrates the preparation and testing of a rapid glucose 
test containing a blood separation matrix of the present invention. The 
Example tests for glucose in spiked whole blood samples. 
A. Blood Separation Matrix: 
A #8007 Sontara.RTM. impregnated with 10% mannitol and 1.25% hexadimethrine 
bromide was prepared as described above, except that the sugar alcohol and 
polymer were dissolved in water. 
B. Filter Membrane and Chemical Reagent Layer: A sheet of 0.45 .mu.m 
Biodyne A.RTM. nylon (Pall Corp.) was dipped into an aqueous solution 
containing the following reagents: 
______________________________________ 
0.30 M Citrate Buffer 
1.25% Gelatin 150 Bloom 
1106 U/ml Glucose Oxidase 
479 U/ml Horseradish Peroxidase 
0.43% 4-Aminoantipyrine (AAP) 
1.52% N-ethyl-N-(2-hydroxyl-3-sulfopropyl)-m-toluidine 
Sodium Salt (TOOS) 
0.25% Pluronic L64 (Poly(oxyethylene-co-oxypropylene) 
block polymer 
1% Gantrez .RTM. L139 
______________________________________ 
After dipping, the sheet was dried for 20 minutes at 50.degree. C. The 
blood separation matrix was mounted on top of the nylon membrane 
containing the test reagents and the two held together by adhesive. A drop 
each of four glucose spiked blood samples were applied to the first 
surface of the mannitol containing Sontara.RTM. matrix. The results are as 
follows: 
______________________________________ 
Glucose Level K/S 
(mg/dl) (at 45 seconds) 
______________________________________ 
98 0.732 
180 0.986 
297 1.312 
450 1.747 
______________________________________ 
These results demonstrate that the color produced was proportional to the 
glucose concentration in the blood sample and that a linear dose/response 
curve was accurately obtained, indicating good performance. 
Although the invention has been described with reference to the disclosed 
embodiments, those skilled in the art will readily appreciate that the 
specific examples detailed are only illustrative of the invention. It 
should be understood that various modifications can be made without 
departing from the spirit of the invention. Accordingly, the invention is 
limited only by the following claims.