HDL determination in whole blood

High density lipoprotein-cholesterol determination is made employing a device which allows for removal of red blood cells, measurement of sample volume, removal of LDL and VLDL, and quantitation of HDL-cholesterol on a quantitation strip.

INTRODUCTION 
1. Technical Field 
The field of this invention is noninstrumented quantitative determination 
of high-density lipoprotein-cholesterol in whole blood. 
2. Background 
High-density lipoprotein (HDL) consists of a number of heterogeneous 
particles that vary with respect to size, content of lipid, and 
apolipoprotein. The major apolipoproteins found in HDL are A(I) and A(II) 
and these proteins constitute about 90% of total HDL protein. The roles of 
HDL in lipid transport are (1) to act as a reservoir of C apoprotein 
required for triglyceride transport; (2) to act as a "scavenger" of 
surplus cholesterol and phospholipids liberated from lipolysed 
triglyceride-rich lipoproteins; and (3) to transport surplus cholesterol 
from peripheral tissues to the liver for excretion and catabolism, both 
directly and indirectly, via other lipoproteins and the lipid transfer 
proteins. 
HDL concentration has been found to correlate inversely with coronary heart 
disease. Epidemiologic studies have emphasized the importance of HDL as a 
negative risk factor. There is, therefore, substantial demand for an easy 
method for quantitation of this lipoprotein. Currently, the determination 
of HDL-cholesterol is extremely difficult and impractical to quantitate 
directly. The primary methods depend on the measurement of the plasma 
content of HDL-cholesterol after selective separation. Several methods are 
available for separation, such as ultra-centrifugation, electrophoresis, 
precipitation as in soluble complexes between lipoproteins, polyanions and 
divalent cations, gel or membrane filtration; and precipitation with 
antibodies to the apolipoproteins. 
Ultracentrifugation, followed by precipitation with heparin and manganese 
chloride is the most commonly used reference method. Ultracentrifugation 
separates VLDL on the basis of differential density, while 
heparin-manganese chloride removes LDL by precipitation. HDL is estimated 
as cholesterol in the plasma fraction of a density greater than 1.063 
g/ml. Ultra-centrifugation requires expensive instrumentation and 
significant technical skill and has therefore found limited application. 
Electrophoretic techniques lack precision and accuracy in the range of 
20-40 mg/dL, where the greatest clinical interest lies. Gelpermeation 
chromatography is too complex and time consuming for routine analysis. 
There is, therefore, a clear interest in the development of techniques 
having relatively simple protocols and equipment for the quantitative 
determination of cholesterol in high-density lipoprotein. 
RELEVANT LITERATURE 
See also Allen, M. P., Delizza, A., Ramel, U., Jeong, H., and Singh, P. A 
Non-instrumented Quantitative Test System and its Application for 
Determining Cholesterol Concentration in Whole Blood, Clin. Chem. 1990; 
36(9) 1591-1597; and Ramel, U., Allen, M. P., and Singh, P. Sample Pad 
Assay Initiation Device and Method of Making U.S. Pat. No. 4,959,324, 
issued Sep. 25, 1990. 
SUMMARY OF THE INVENTION 
High-density lipoprotein-cholesterol is quantitated from blood by removal 
of apoB containing lipoproteins with a membrane, followed by enzymatic 
reaction of cholesterol and cholesterol esters to produce hydrogen 
peroxide. The hydrogen peroxide is then quantitated on a quantitation 
strip to which a coupling dye is bound, which reacts with the product of 
the peroxidase to produce a detectable colored product.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS 
Method and devices are provided for the determination of high-density 
lipoprotein-cholesterol (HDL-cholesterol) from blood. The method and 
device provide for initial removal of a substantial proportion of the red 
blood cells present in the blood, removal of substantially all of the very 
low density lipoprotein (VLDL) and low density lipoprotein (LDL), which 
lipoproteins contain apo B lipoprotein. The resulting HDL-cholesterol 
concentrated plasma fraction is then treated with enzymes which react with 
cholesterol and cholesterol esters to produce hydrogen peroxide. 
Preferably, the hydrogen peroxide is then measured by transport of the 
hydrogen peroxide on a quantitation strip in conjunction with a 
peroxidase, where a compound is conjugated to the strip. The reaction of 
the peroxidase with a coupling compound results in the coupling compound 
reacting with another compound bound to the strip with production of a 
highly colored product. The distance of the colored front from the sample 
site or other designated position is quantitatively related to the amount 
of HDL-cholesterol in the blood sample. 
A series of membranes are provided which allow for a number of functions in 
producing the desired sample, which is free of red blood cells and free of 
cholesterol associated with lipoproteins other than HDL. The first stage 
is the application of the blood sample to one or more membranes, desirably 
having diminishing pore size. The membranes are selected to efficiently 
remove red blood cells without lysis. Significant lysis of red blood cells 
results in the discoloration of the sample, which discoloration interferes 
with the measurement for the determination of HDL-cholesterol and more 
importantly hemoglobin may chemically interfere with the assay system. 
Generally, the pore size will be in the range of 0.5 to 50 .mu.m, whereas 
with two filters, the first filter will have a pore size in the range of 
about 5 to 50 .mu.m, while the second filter will have a pore size in the 
range of about 0.5 to 10 .mu.m. The filters are selected to minimize the 
sample holdup in the filter and should substantially reduce the red blood 
cells (RBC) which are transferred to the next membrane layer. 
The next membrane layer normally serves to remove VLDL and LDL, which 
include apolipoproteins B, C and E and various techniques may be employed 
for the removal of the cholesterol associated with VLDL and LDL. 
Alternatively one may use two treated glass fibre membranes for removal of 
LDL and VLDL at the same time as the RBC are being removed. 
Another possibility is the use of either one or more filters (filter 1 
and/or 2) to remove VLDL and LDL, while at the same time separating the 
red blood cells (see FIG. 1a). 
In a preferred embodiment, a membrane comprising a divalent cation 
containing anionic polymer, which minimizes red blood cell lysis and 
efficiently separates the VLDL and LDL from the HDL, is employed. 
Illustrative of such polyanion products are dextran sulfate having 
magnesium as the counterion, or sodium heparin having divalent manganese 
as the counterion. Of particular interest, therefore, are polymers of at 
least about 5,000 molecular weight, usually at least about 50,000 
molecular weight, particularly heparin (5-20 kDal) or dextran (25-500 
kDal) which are sulfuric acid derivatives, sulfonates or sulfates, in 
conjunction with divalent cations, particularly magnesium and manganese. 
The polymers may be covalently or non-covalently bound to a porous 
substrate, so long as the polymers are retained by the porous substrate 
during the subject procedure. Various substrates may be employed, such as 
glass fiber, paper, synthetic membranes, or the like. The various 
substrates may be activated for covalent bonding by a variety of 
conventional agents, such as silane, e.g., aminopropyltriethoxysilane, 
with glass fiber, activated, e.g., carbodiimide-activated, paper or 
synthetic membranes, or the like. The amount of polymer bound to the 
membrane may be readily determined by determining the efficiency with 
which the HDL is separated from the VLDL and LDL and the loss of HDL on 
the membrane. The polymers may be bound to any or all of the porous 
filtration substrates. That is in a preferred embodiment, the VLDL and LDL 
precipitating reagent may be bound to polymeric membrane, filter 1, filter 
2, membrane 3 or it may be bound to the sample receiving pad. The 
precipitating reagents may be bound onto any combinations of 2 or 3 or all 
porous substrates. (See FIG. 1) 
Usually, the amount of polymer will be about 0.001 g to 0.5 g per cm.sup.2. 
The membrane will generally have a thickness in the range of about 10 
.mu.m to 1000 .mu.m and a pore size in the range of about 0.1 to 50 .mu.m. 
Instead of using precipitating reagents, affinity reagents may be employed, 
such as monoclonal or polyclonal antibodies specific for apolipoproteins 
B, C, and E. The use of the antibodies also allows the determination of 
LDL uncontaminated with HDL, by employing antibodies specific for the apo 
A protein on the LDL, which will allow passage of the HDL through the 
membrane. 
The antibodies may be bound to the membranes described above by analogous 
techniques, using carbodiimide, cyanogen bromide, diazo compounds, 
activated olefins for reaction with thiol groups, or the like. The amount 
of antibodies bound to the membrane will vary with the affinity of the 
antibody, the number of active sites, the molecular weight of the 
antibody, e.g., IgM or IgG, or the like. Usually, the amount of antibody 
will be in the range of about 0.1 .mu.g to 100 .mu.g per cm.sup.2. These 
antibodies may be immobilized on the polymeric membrane, filters 1 or 2 or 
membrane 3 or on the sample receiving pad (FIG. 1). The polymeric membrane 
may be positioned above, or in between filters 1 and 2, or membrane 3. 
There may be one or more additional membranes to separate the sample 
receiving pad from the LDL removing membrane, to control flow, to further 
remove particulate matter, or for other purposes. These membranes will not 
be reactive, generally having pore sizes in the range of about 0.5 .mu.m 
to 1.0 .mu.m. 
Illustrative membranes which may find use for removal of red blood cells 
include S&S Glass 30, Whatman GFD, S&S 3662, Pall glass fiber membranes, 
Sartorius cellulose acetate, Filterite polysulfone asymmetric membrane, 
Ultrabind 450, Nucleopore, etc. Membranes which find use for removal of 
LDL are illustrated by Whatman GFD, Whatman 31ET or Ultrabind 450. 
The sample is received by a sample pad. The sample pad may serve a number 
of functions. The sample pad, in conjunction with other components, may 
serve to measure the volume of the sample. The sample pad will usually 
have a volume of about 1 to 60 .mu.l and a thickness of about 0.1 to 5 mm. 
In addition, the sample pad may have one or more reactants, particularly 
enzyme reactants bound to the pad. The sample pad will initially be 
protected from contact with the quantitation strip. Various mechanisms may 
be employed to provide a barrier for contact between the sample pad and 
the quantitation strip, which barrier may be moved to allow for contact 
between the sample pad and the quantitation strip. 
The sample pad may conveniently be a bibulous membrane, which will absorb 
the plasma sample and serve as a bridge for transport of eluent from an 
eluent source through the sample pad to conversion pad and subsequently to 
the quantitation strip and allow for substantially quantitative transport 
of cholesterol (including cholesterol precursors) or an enzymatic reaction 
product of cholesterol from the reaction pad to the quantitation strip. 
While it is preferred that one or more of the membranes serve to remove 
VLDL and LDL, the reagent may be present solely on the sample pad, solely 
on the membrane for removing RBCs, combinations thereof, or on all or some 
of the components of the device in which the plasma contacts before 
undergoing substantial enzymatic reaction. Where the enzymes are on the 
pad, the reagent for removing the VLDL and LDL will not be restricted to 
the sample pad. 
The cholesterol is measured by reaction with a combination of enzymes, 
cholesterol esterase and cholesterol oxidase. The esterase provides for 
hydrolysis of cholesterol esters to cholesterol, while cholesterol oxidase 
provides for oxidation of cholesterol with oxygen to hydrogen peroxide. 
The enzyme may be present on the pad, may be upstream from the pad in a 
reaction zone prior to the quantitation region of the quantitation strip, 
or a combination thereof. The enzymes may but are not required to be 
non-diffusively bound to the surface, so that the enzymes will 
substantially remain at the site where they are positioned prior to the 
beginning of the assay. 
The sample pad may be protected from acting as a bridge and contacting the 
quantitation strip in a variety of ways. In one embodiment, a removable 
plastic barrier may be inserted between the sample pad, the quantitation 
strip, and the strip providing the source of eluent. The plastic barrier 
may include a mesh strip, e.g., Nitex.RTM., 100-500.mu., for wiping the 
pad of excess sample. In another embodiment, the pad may be at a site 
distant from the quantitation strip and the eluent providing strip, where 
after receiving the sample, the sample pad is moved into contact with the 
two strips. In both of these cases, various wiping means may be employed 
to remove excess sample from the pad, so that a substantially reproducible 
amount of sample will be absorbed by the pad. 
The eluent-supplying strip may be of any convenient bibulous material which 
can be dipped into an eluent and wick the eluent up to the sample pad, 
when the sample pad is in contact with the eluent source strip. Various 
papers may find use, such as cellulose strips, e.g., chromatography paper, 
silica on a support, alumina on a support, polymeric membranes, such as 
nitrocellulose and nylon. Generally, the eluent source strip will be not 
less than about 0.5 cm and not more than about 2.5 cm. 
The quantitation strip may serve, as already indicated, in providing a 
reaction zone, where the cholesterol esters and enzymes are present which 
will react with the cholesterol to produce the reactant, hydrogen 
peroxide. Thus, a zone may be provided where the cholesterol will react to 
produce hydrogen peroxide, which will then be transported by the eluent to 
the quantitation region. The quantitation region is characterized by 
having one member of a dye couple bound to the region at a concentration 
which allows for a reasonable dynamic range associated with the 
concentration range of interest of HDL. For the most part, the 
concentration range of interest is from about 20 to 100 mg/dL of 
HDL-cholesterol, so that one wishes to have at least about 10 mm, per 25 
mg/dL change in concentration, preferably at least about 7 mm per 25 mg/dL 
and not more than about 20 mm, usually not more than about 15 mm per 25 
mg/dL. 
The quantitation strip will generally have dimensions of about 5 mm to 130 
mm and may be of the same or different material from the eluent source 
strip. Passively adsorbed to the strip will be one member of a dye couple, 
such as MBTH or AAP. The coupling member may be any of a variety of 
substrates for horseradish peroxidase, which can couple with the other 
member bound to the strip. Illustrative coupling members include 
N,N-dimethylaniline; 5-(N-methylanilino pentanoyl ethylene diamine; 
N-ethylaniline; N-ethylmeta-toluidine; 2-(N-ethyl-meta-toluidino)ethanol; 
N-ethyl-N-(2-hydroxy-3-sulfopropyl)-meta-toluidine sodium salt; N-ethyl, 
N-sulfopropyl-meta-toluidine sodium salt; 2-(N-ethyl-m-toluidino)ethanol, 
N-ethyl meta-toluidine; 1,8-dihydroxy-3,6-dimethoxynaphthalene or analogs 
thereof. These compounds are characterized by being capable of reacting in 
the presence of a peroxidase with hydrogen peroxide to form a compound 
which may be coupled with MBTH to form an indamine dye, which allows for 
detection of a color front in relation to the amount of hydrogen peroxide 
produced from the cholesterol in the sample. 
The eluent will comprise the horseradish peroxidase, the coupling member, 
usually buffers, and other miscellaneous additives as appropriate, e.g., 
PVP, non-interfering proteins, detergents, particularly nonionic 
detergents, antifungal agents, etc. The concentration of the horseradish 
peroxidase will generally be in the range of about 0.29.mu./ml to 
1.6.mu./ml, usually about 2-14 .mu.g/ml. Buffers will generally provide a 
pH in the range of about 4 to 9, with a buffer concentration that will be 
sufficient to provide the necessary buffering, generally being in the 
range of about 10 to 500 mM. Illustrative buffers include phosphate, Tris, 
MOPSO, MOPS, borate, carbonate, etc. 
In carrying out the assay, a blood sample may be placed on the topmost 
membrane and gravity and capillary action provide the transport force. The 
blood will migrate through the red blood cell separating membrane(s), so 
that the sample is substantially free of red blood cells when it contacts 
the membrane for removal of cholesterol present as other than HDL. The 
plasma sample will pass through the reactant membrane, so as to remove LDL 
and VLDL and leave HDL as the only protein-bound cholesterol. In a second 
embodiment the red blood cell separating membranes and the treated or 
reactant membranes, which removes VLDL and LDL, are the same. The sample 
receiving pad may also be used as a reactant membrane, The sample will 
then migrate to the sample pad and be absorbed by the sample pad. When the 
sample pad is at least saturated, the excess fluid will be removed as 
described above by moving the pad or other instrumentality, which serves 
to wipe the pad of the excess fluid. The pad will now be in contact with 
the eluent source strip and the quantitation strip. The eluent will move 
through the eluent source strip, using the pad as a bridge and carry 
cholesterol, cholesterol esters, and/or enzymatic products of cholesterol 
up to be quantitation region, where the hydrogen peroxide will react with 
the coupling agent to activate the coupling agent to react with the other 
member of the dye bound to the quantitation strip in the quantitation 
region. The extent of the color produced from an arbitrary point at the 
beginning of or prior to the quantitation region may be related to the 
amount of HDL-cholesterol in the sample. By using known standards, the 
distance of the color front from some origin, can be directly related to 
the amount of HDL-cholesterol in the sample in a quantitative manner. 
Turning now to FIGS. 1a and 1b and 2, the subject invention will be further 
described. The separation device 10 of FIG. 1a has a well 12 in which is 
situated in the direction of flow, polymeric membrane 14, filter 1, 16, 
filter 2, 18 membrane 3, 20 and polycarbonate membrane 22. The separation 
and well sit over sample pad 24. 
The polymeric membrane 14 serves to remove red blood cells without lysis, 
while filters 1 and 2, 16 and 18, respectively, are activated to remove 
VLDL and LDL, while allowing HDL to pass through. To further improve 
filtering, membrane 20 is provided to further ensure removal of VLDL and 
LDL, where membrane 3 may be activated in the same or different way from 
filters 1 and 2. 
A polycarbonate membrane 22 serves to prevent contact between membrane 3, 
20 and sample pad 24. 
As depicted in FIG. 2 sample pad 24 is moved from a first position as shown 
in the FIG. 2 to a second position, as shown in FIG. 1b, where the sample 
pad 24 comes into contact with the conversion area 28 and wicking strip 26 
to act as bridge to allow flow of fluid from the wicking strip 26 through 
the sample pad 24, conversion area 28, to the quantitation strip 30. 
The sample on sample pad 24 is carried by wicking buffer from wicking strip 
26 into conversion area 28 in which enzymes are present to convert 
cholesterol and cholesterol esters to hydrogen peroxide. The wicking 
buffer then carries the hydrogen peroxide into the quantitation area 30, 
where the hydrogen peroxide reacts with dyes under catalysis with by 
horseradish peroxidase. The extent of color formation is related to the 
amount of HDL present as indicated by the amount of cholesterol present in 
the HDL. 
The measurement device may be fabricated from three injection molded parts 
or by any other convenient process. The parts comprise a base plate 40, a 
slide 42 and a clear cover plate, not shown. The base plate 40 consists of 
a cutout to accept the slide 42, a slot 46 with locating pins 48 into 
which the conversion area 28, quantitation strip 30 and wicking strip 26 
are precisely positioned, maintaining about a 2 mm gap 44 between them, 
and a well 50 designed to capture the released transport solution, e.g., 
wicking buffer. 
The slide 42 consists of a vented receptor site 52 into which the reagent 
24 pad is inserted and over which the separation device 10 is placed. An 
arm 54 with dual shearing designed to facilitate the release of the 
transport solution from a pouch which is housed in a well of the cover 
plate, and a snap 56 to lock the slide in place, once pulled are provided. 
The cover plate consists of a well 32, which houses a sealed foil pouch 
(not shown) containing the transport solution. The cover plate has an 
orifice for placement of the separation device 10 for the introduction of 
the sample. The cover plate also comprises a squeegee metering bar which 
serves to control the volume of sample absorbed by the sample pad 24. 
The following examples are offered by way of illustration and not by way of 
limitation. 
EXPERIMENTAL 
Experimental Details 
Standard Curve 
1. An assay is performed by using mylar laminated chromatography strips 
containing the quantitation region (70.times.5 mm), conversion region 
(7.times.5 mm), sample pad (7 .times.5 mm) and wicking strip (12.times.5 
mm). The wicking reagent is (0.1M) MOPSO buffered (pH 7.0) protein 
solution containing HRP (5 .mu.g/ml). The paper employed for the 
quantitation region is 0.1 mg/ml MBTH paper and human serum samples 
equivalent to 25, 50, 75 and 100 mg/dL of HDL were employed. 
The assay is carried out as follows: 
(a) 0.01 ml of plasma or serum is deposited on the sample pad; (b) wicking 
is initiated by allowing the lower portion of the assay strip to contact 
0.5 ml wicking reagent contained in a test tube. The assay is complete 
when the wicking solution reaches the end of the measurement region. The 
strips are then removed and the height to which the colored band is formed 
is plotted against cholesterol concentration. A substantially linear plot 
was obtained, with the migration height varying from about 15 mm to about 
40 mm. 
The assay was also performed with hydrogen peroxide calibrators equivalent 
to 25, 50, 75 and 100 mg/dL. The plot of migration height versus hydrogen 
peroxide incubation was substantially linear with migration height varying 
from about 22 mm to about 48 mm 
2. Gel Electrophoresis 
AccuMeter.RTM. cassettes A (FIG. 2) (see U.S. application Ser. No. 353,910 
filed May 18, 1989, now U.S. Pat. No. 4,959,) with treated and untreated 
membranes are used for this study. Filters 1, 2, membrane 3 and/or a 
combination of 1, 2, 3 or all may be treated with the precipitating 
reagents. 
The assay involves the application of whole blood or its plasma (0.04 ml) 
to the sample site on the cassette. Following a two minute incubation the 
sample pad is removed from the cassette and its plasma contents squeezed 
out (0.005 ml) and applied to an Agarose gel. After the last sample has 
been applied to the gel, 10 mins. are allowed for diffusion. The gel is 
placed into a Paragon electrophoresis cell and electrophoresed for 50 
mins. at 100 volts. Upon completion of electrophoresis, the gel is removed 
from the Paragon electrophoresis cell and placed into a fixative solution 
for 5 mins. 
The gel is removed from the fixative solution and dried until completely 
dry. The dried gel is processed in the following sequence: 
______________________________________ 
Lipoprotein working stain 
5 minutes 
Destain Solution I 3 dips 
Destain Solution II 3 dips 
Destain Solution III 5 minutes 
______________________________________ 
The gel is finally rinsed in a deionized water and then completely dried. 
Eight samples were electrophoresed: 1 whole blood (cassette with untreated 
membranes; 2 and 3 whole blood (cassette with treated membranes); 4 plasma 
(cassette with untreated membranes); 5 and 6 plasma (cassette with treated 
membranes); 7 and 8 supernatant of plasma following LDL and VLDL 
precipitation using phosphotungstate/Mg.sup.2+. With the exception of 
sample 1 and 4, using untreated cassettes which showed the presence of 
pre-.beta. and .beta. subunits, all of the samples only showed the 
presence of the .alpha. subunit. 
Cholesterol Analyses 
Cholesterol concentrations are determined following in situ precipitation 
in AccuMeter.RTM. cassettes (FIG. 2). AccuMeter cassettes with treated and 
untreated membranes are used for this study. Filters 1, 2, membrane 3 
and/or a combination of 1, 2, 3 or all may be treated with the 
precipitating reagents. Boehringer Mannheim Diagnostic high performance 
reagent (BMDHP) is used to measure the plasma cholesterol collected on the 
sample pad. BMD Preciset aqueous based cholesterol calibrators equivalent 
to 50, 100, 150, 200, and 300 mg/dL are used for this study. 
Whatman glass fibers GF/D(6.0 .mu.m and/or Whatman 31ET chromatography 
paper were used to prepare treated membranes. The precipitating reagent 
solutions were prepared as follows: (1) Into deionized water (50 ml) was 
dissolved 0.5 g dextran sulfate (Na salt, Mw 500 kD), followed by the slow 
addition of 4.26 g anhydrous MgCl.sub.2 with stirring, (2) Into 30 ml 
deionized water was dissolved 0.421 g heparin (Na salt, 176 USP units/mg) 
and 2.97 g MnCl.sub.2. 
The precipitating reagent (12 ml) was poured into a lasagne dish tilted at 
45.degree.. The membrane material (10 cm.times.7 cm) was slowly and 
uniformly passed through the reagent solution. (The GFD membrane was 
supported by Nitex nylon screen of the same dimension.) The membrane was 
then dried in an oven at 80.degree. C. for about 15 min being inverted 
occasionally for uniformity. Discs (5 mm dia.) were then cut. 
For solution phase precipitation, the dextran sulfate solution was prepared 
as described above, the heparin solution was twice as concentrated, and 
the phosphotungstate/Mg.sup.2+ solution was prepared by diluting 4:1 
BMD-HDL-cholesterol reagent (0.55M phosphotungstic acid; 25 mM MgCl.sub.2) 
in deionized water. 
The assay involves the application of plasma or serum (0.04 ml) to the 
sample site on the cassette. Following a two minute incubation, the slide 
is pulled and the sample receiving pad removed and placed in the BMDHP 
reagent (1 ml). The plasma or serum sample on the pad (0.005 ml) is 
extracted for one hour at room temperature. The solution is then measured 
at A500 nm. A standard curve is generated by pipetting BMD cholesterol 
calibrators (0.005 ml) into the BMDHP reagent (1 ml), incubating for 15 
mins. and then measuring the absorbance at 500 nm. 
LDL and VLDL precipitation was also performed in test tubes using the 
precipitating reagents and assay protocol listed below: 
______________________________________ 
Ppt Reagent Ppt Reagent Vol. 
Sample Vol. 
______________________________________ 
Phosphotungstate/ 
0.5 ml 0.2 ml 
MgCl.sub.2 (diluted 4:1 
in H.sub.2 O) 
Dextran SO.sub.4 /MgCl.sub.2 
0.05 ml 0.5 ml 
Heparin/MnCl.sub.2 
0.05 ml 0.5 ml 
______________________________________ 
I) Vortex to mix sample and incubate for 10 min at RT. 
II) Centrifuge at 2000.times.g for 10 min at RT. 
III) Remove supernatant for further analyses 
The supernatant was analyzed for cholesterol as shown for the cholesterol 
calibrators. 
Results 
______________________________________ 
Cholesterol mg/dL 
Untreated Treated Treated 
Sample Membrane Membrane 1 Membrane 2 
______________________________________ 
Whole Blood 1 
170 (182.0) 30.6 (68.0) 
54.5 (68.0) 
Plasma 1 178 (182.0) 31.5 (68) 61.0 (68.0) 
Plasma 2 129.9 (128.8) 
50.1 (52.1) 
-- 
Plasma 3 193.4 (204.3) 
53.4 (45.1) 
-- 
Plasma 4 267.3 (283.1) 
54.7 (84.7) 
-- 
Plasma 5 176.5 (191.0) 
71.8 (56.0) 
71.2 (56.0) 
______________________________________ 
Key: 
() Cholesterol concentrations as determined by a reference method in test 
tubes. Reference precipitation method used involves 
phosphotungstate/Mg.sup.2+. 
Treated membrane 1 = Dextran SO.sub.4 
Treated membrane 2 = Heparin/Mn.sup.2 
It is evident from the above results, that the subject methodology provides 
for a convenient, simple assay for HDL, so that one may be able to obtain 
both HDL-cholesterol and total cholesterol to have an appropriate 
evaluation of the risks for coronary heart disease and myocardial 
infarction. In addition, the methodology provides for substantially no 
measurements, washings, or other manipulative steps, other than providing 
a sample to the device. In this manner, untrained people or individuals 
interested in monitoring their cholesterol are able to make the necessary 
determinations. 
All publications and patent applications mentioned in this specification 
are indicative of the level of skill of those skilled in the art to which 
this invention pertains. All publications and patent applications are 
herein incorporated by reference to the same extent as if each individual 
publication or patent application was specifically and individually 
indicated to be incorporated by reference. 
The invention now being fully described, it will be apparent to one of 
ordinary skill in the art that many changes and modifications can be made 
thereto without departing from the spirit or scope of the appended claims.