U.S. patent number 3,902,964 [Application Number 05/362,454] was granted by the patent office on 1975-09-02 for method of and apparatus for chemically separating plasma or serum from formed elements of blood.
This patent grant is currently assigned to U.S. Medical Research and Development, Inc.. Invention is credited to Donald J. Greenspan.
United States Patent |
3,902,964 |
Greenspan |
September 2, 1975 |
**Please see images for:
( Certificate of Correction ) ** |
Method of and apparatus for chemically separating plasma or serum
from formed elements of blood
Abstract
A process for separating plasma or serum from formed elements of
the blood by adding a positively charged polymer and a lectin to a
tube into which blood is drawn to effect precipitation of the
formed elements of the plasma or serum.
Inventors: |
Greenspan; Donald J.
(Riverside, NJ) |
Assignee: |
U.S. Medical Research and
Development, Inc. (Riverside, NJ)
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Family
ID: |
26902375 |
Appl.
No.: |
05/362,454 |
Filed: |
May 21, 1973 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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207577 |
Dec 13, 1971 |
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847469 |
Aug 4, 1969 |
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Current U.S.
Class: |
435/269; 435/13;
436/177 |
Current CPC
Class: |
A61K
35/14 (20130101); A61K 35/16 (20130101); G01N
33/491 (20130101); G01N 33/5002 (20130101); B01D
21/01 (20130101); Y10T 436/25375 (20150115) |
Current International
Class: |
A61K
35/14 (20060101); G01N 33/50 (20060101); G01N
33/49 (20060101); G01n 033/16 () |
Field of
Search: |
;195/1,13.5R,127
;23/258.5,23B ;210/538 |
Foreign Patent Documents
Other References
August, A., Am. J. Med. Tech., Vol. 31, pp. 271-278,
(1965)..
|
Primary Examiner: Tanenholtz; Alvin E.
Attorney, Agent or Firm: Woodcock, Washburn, Kurtz &
Mackiewicz
Parent Case Text
RELATED APPLICATIONS
This is a continuation-in-part of application Ser. No. 207,577
filed Dec. 13, 1971, now abandoned which is a continuation of
application Ser. No. 847,469 filed Aug. 4, 1969 and now abondoned.
Claims
What is claimed is:
1. A method of separating serum from the formed elements in a blood
sample comprising the steps of:
combining a lectin hemagglutinin, a positively charged polymer and
a blood sample in a container;
agglutinating the formed elements into clumps with said lectin and
said positively charged polymer such that each of a plurality of
formed elements in each clump are agglutinated with another formed
element with a polymeric seam therebetween and agglutinated with a
further formed element with a lectin link therebetween; and
precipitating said clumps to the bottom of the container to create
a layer of plasma or serum above said formed elements.
2. The method of claim 1 wherein said clumps of formed elements are
combined to form a substantially integral mass after
precipitating.
3. The method of claim 2 wherein said clumps drag fibrin downwardly
therewith while precipitating.
4. A method of separating formed elements of the blood from serum
comprising the steps of:
combining a blood sample with a lectin hemagglutinin and a
positively charged polymer in a container;
agglutinating the formed elements of the blood with said
hemagglutinin and said positively charged polymer; and
precipitating the agglutinated formed elements of the blood so as
to form a layer of serum above 99% of the formed elements of the
blood in a given length of time, said layer having a total volume
greater then the sum of the volume in a layer of serum above 99% of
all elements in a blood sample of the same size combined with said
lectin alone after the same given length of time plus the volume in
a layer of serum above 99% of all elements of the blood in a blood
sample of the same size combined with said positively charged
polymer after the same given length of time.
5. A method of separating formed elements of the blood from serum
comprising the steps of:
combining a blood sample with a lectin hemagglutinin and a
positively charged polymer;
agglutinating the formed elements of the blood with said lectin and
said positively charged polymer; and
precipitating the agglutinated formed elements of the blood at a
rate in excess of the sum of the rates of precipitation achieved
with said lectin alone in said blood sample and said positively
charged polymer in said blood sample.
6. The method of separating plasma from formed elements of blood
comprising the steps of:
collecting a sample of blood and placing the sample in a blood
collection container; and
reacting said blood sample with a lectin hemagglutinin and a
positively charged polymer whereby the formed blood elements
separate from the plasma.
7. The method according to claim 6 wherein said positively charged
polymer is Polybrene.
8. The method according to claim 6 wherein said hemagglutinin is
Bacto Phytohemagglutinin P.
9. The method of claim 6 wherein the hemagglutinin and the
positively charged polymer are simultaneously reacted with the
blood.
10. The method according to claim 6 wherein said blood collection
container is hollow cylindrical in configuration and has a
longitudinal axis and comprising the additional step of disposing
said blood collection container with its longitudinal axis in a
substantially horizontal plane to enhance precipitation of the
formed blood elements from the plasma.
11. The method according to claim 6 to produce serum, including the
further step of adding a substance for inhibiting the formation of
fibrin.
12. The method according to claim 11 wherein said substance is
thrombin.
13. In combination, a blood collection container containing a
hemagglutinin and a positively charged polymer, said container
having volumetric capacity sufficient to hold more than 30 cubic
millimeters of blood.
14. The combination according to claim 13 wherein the container
also contains a substance for inhibiting the formation of
fibrin.
15. The combination of claim 13 wherein the said container is a
vacuum type blood collection tube.
Description
BACKGROUND OF THE INVENTION
This invention relates to the separation of formed elements of the
blood from plasma or serum.
At the present time, the separation of formed blood elements, e.g.,
red blood cells, from plasma or serum is accomplished almost
entirely by mechanical separation techniques. Of the various
mechanical separation techniques, centrifuging is most popular.
However, mechanical separation techniques including centrifuging
are cumbersome in that they require rather elaborate mechanical
apparatus. Moreover, mechanical separation techniques may provide
somewhat less than optimum plasma or serum yields. In addition,
mechanical separation techniques can result in faulty plasma or
serum analysis due to the time involved in transporting a blood
sample to a centrifuging apparatus thereby delaying the separation
of plasma or serum from the formed elements of the blood.
Ideally, separation of the cells from the plasma or serum should
take place immediately thereby decreasing the amount of serum or
plasma contamination by cell breakdown.
Chemically assisted mechanical separation techniques have been
explored. One such technique, which is disclosed in British Pat.
No. 969,741 -- Brewer, involves the use of "agglutination,
coagulation or precipitation" of solid components of blood due to
the presence of a phytohemagglutinin and mechanical stirring.
It has been found that the phytohemagglutinin alone, in the absence
of stirring, will result in very slow precipitation of the blood
cells which is believed to be due to the fact that clumps of blood
cells which are formed are small and not particularly dense. Even
with stirring, it is believed that any separation of serum from
blood cells is too slow to be clinically feasible. More
importantly, the stirring of the blood sample will itself cause
cell breakdown and contaminate the results of serum or plasma
analysis.
The use of "lectins" as hemagglutinins was described by Irvin E.
Liener in a paper presented for discussion at the Seed Protein
Conference sponsored by the Southern Utilization Research and
Development Division, Agricultural Research Service, United States
Department of Agriculture, New Orleans, La., Jan. 21-24, 1963 and
printed in Economic Botany as an article entitled "Seed
Hemagglutinins". In this paper, there is considerable discussion of
a theorized agglutinating mechanism involving receptor sites on the
blood cells to which the lectin attaches. In this manner, two blood
cells may be attached or clumped where their receptor sites are
linked by the lectin.
Another chemically assisted mechanical separation technique has
involved the use of an agglutinating agent comprising a positively
charged polymer such as Polybrene (hexadimethrine bromide). Such a
use of Polybrene is disclosed in an article entitled "A New Technic
for Separation of Human Leukocytes" by Parvitz Lalezari, appearing
in Blood, vol. 19, No. 1, January, 1962 where the blood sample
containing cells agglutinated by the Polybrene is centrifuged to
effect precipitation. In an article by Anne August, appearing in
the American Journal of Medical Technology, "The Effect of
Antiheparin Drugs on Human Blood", vol. 31, No. 4, July-August,
1965, Polybrene is also discussed as a means for inducing
agglutination of blood cells.
Precipitation of the blood agglutinated with Polybrene into fairly
large clumps is very slow without centrifuging. Moreover, the total
amount of plasma or serum which may be obtained with Polybrene is
relatively small. As a result, the total volume of serum which may
be obtained in a given length of time is small as compared with the
volume obtained utilizing the phytohemagglutinin described by
Brewer. Moreover, the longer the blood cells remain in the plasma,
the greater the contamination of the plasma so as to adversely
affect the plasma analysis. In addition, a good deal of debris will
remain in suspension in the plasma after precipitation. Any attempt
to increase the concentration of Polybrene beyond a certain point
for speeding precipitation will result in lysis of the blood cells.
The reason for this lysis is not completely understood although it
is believed that large concentrations of the positively charged
polymer may result in collisions between the polymer and the cells
which rupture the membrane of the cells. It is also possible that
the large concentration of the positively charged polymer creates
such substantial deformation of the cells as described by A.
Katchalsky et al. in Biochimica Et Biophysica Acta, "Basic
Polyelectrolytes in Red Blood Cells", vol. 33, pp. 120-139, 1959,
that the cells actually rupture.
SUMMARY OF THE INVENTION
It is a general object of this invention to provide an improved
method of chemically precipitating formed blood elements from
plasma or serum.
It is a further object of this invention to employ a chemical
separation technique which will not adversely effect the analysis
of the plasma or serum.
It is a further object of this invention to employ a chemical
separation technique which assures separation of the formed
elements of the blood from the serum or plasma within a short
period of time.
It is also an object of this invention to employ a chemical
separation technique which substantially prevents contamination of
the plasma or serum.
It is still a further object of this invention to employ a chemical
separation technique which yields substantial amounts of plasma or
serum which has been separated from the formed elements of the
blood.
In accordance with these and other objects, two chemical reagents
are utilized to cause agglutination of the formed elements of the
blood and precipitation of these elements from the plasma or serum
of the blood. The reagents comprise a positively charged polymer
(basic polyelectrolyte) such as Polybrene, and a lectin
(hemagglutinin plant extract), such as Bacto Phytohemagglutinin P.
which are added to a blood collection tube either during the
manufacturing of the tube or at a later time by, for example,
conventional needle insertion techniques. The blood taken from the
donor is received within the tube and the plasma is separated out
as a result of the action of the above-mentioned chemicals with the
blood. The separation mechanism occurs spontaneously within
one-half hour or less.
In accordance with one important aspect of the invention, the
chemical reagents create large three dimensional dense clumps of
blood cells which readily precipitate to the bottom of the
collection tube. These clumps are desirable in that they are not
easily broken apart by jarring the blood sample.
One explanation for the density, three-dimensionality and
durability of these clumps, which is believed to be correct, is
that a lectin has an increased agglutinating ability in the
presence of a positively charged polymer. Since the formed elements
of the blood are all negatively charged, they tend to repel one
another and the probability of the lectin attached to a receptor
site on one cell coming into contact with a receptor site of
another cell to form a clump is lowered by this mutual repulsion.
However, when the positively charged polymer is added to the blood
sample and attracted to the negatively charged cells, the cells and
polymer form neutralized masses which permit the lectin to more
effectively agglutinate the blood cells since the cells are more
likely to come into sufficiently close contact to permit the lectin
to form links between the cells at receptor sites and cells within
a clump are able to move into a densely packed configuration due to
the absence of repulsion.
In addition to the neutralizing effect which allows the lectin to
more effectively perform its agglutinating function, the positively
charged polymer also provides its own agglutinating function. In
other words, the single cell within the clump may be linked to one
or more cells by the lectin while the same cell is linked to one or
more other cells by the positively charged polymer. As a result of
utilizing the two different types agglutinating aggulatinating
agents, each cell is able to be attached to a greater number of
cells than would be the case if only one of the two different types
of agglutinating agents were utilized. This agglutinating mechanism
also helps create increased density of cells in a clump and larger
three dimensional clumps.
The large three dimensional clumps are essentially large net-like
structures which are capable of dragging debris and fibrin down
through the plasma. As the clumps collect at the bottom of the
collection tube, a substantially integral structure is formed
wherein all of the individual clumps are connected to one
another.
Other objects and a fuller understanding of the invention will be
had by referring to the following description in conjunction with
the drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1a is an elevational view of a container immediately after
blood, lectin and a positively charged polymer have been combined
in accordance with this invention;
FIG. 1b is an elevational view of the container and the contents of
FIG. 1a approximately 15 minutes later;
FIG. 2a is an elevational view of a container immediately after
blood and a lectin have been combined;
FIG. 2b is an elevational view of the container and the contents of
FIG. 2a approximately 15 minutes later;
FIG. 3a is an elevational view of a container immediately after
blood and a positively charged polymer have been combined;
FIG. 3b is an elevational view of the container of FIG. 3a
approximately 15 minutes later;
FIG. 4 is a schematic illustration of a clump of blood cells
agglutinated in accordance with this invention;
FIG. 4a is a schematic illustration of a clump of blood cells
agglutinated with a positively charged polymer; and
FIG. 4b is a schematic illustration of a clump of blood cells
agglutinated with an organic hemagglutinin.
DESCRIPTION OF THE PREFERRED EMBODIMENT
In the preferred embodiment of my invention, 5 milligrams (5mgs.)
of Polybrene, a positively charged polymer (hexadimethrine
bromide), which is manufactured by Abbott Laboratories and 0.05cc
of Bacto Phytohemagglutinin P., a lectin (hemagglutinin plant
extract) manufactured by Difco Company, are added to a 10cc
Becton-Dickinson red top vacutainer (this tube initially contains
no chemical reagents). The Bacto Phytohemagglutinin P. is
manufactured in 5cc vials and is assayed according to potency, not
weight. It is understood that other vacuum tubes may be utilized in
practicing the present invention, and that the above-mentioned
chemical reactants can be added during the manufacturing of the
tube, or at a later time by, for example, conventional needle
insertion techniques.
When blood which is taken from a patient or donor (approximately
9cc's of blood) enters the tube containing the positively charged
polymer and the lectin, the formed blood elements are caused to
separate from the plasma. This separation occurs due to the
precipitation of large, dense, three dimensional clumps, which
precipitation occurs within one-half hour or less so as to limit
the contamination of the plasma or serum. One explanation for the
size, density and three dimensionality of these clumps as compared
with those obtained when lectin is utilized alone or Polybrene is
utilized alone, is that the positively charged polymer Polybrene
actually increases the agglutinating ability of the lectin. Since
the formed elements of the blood are negatively charged, they tend
to repel one another. The probability of the lectin attaching to a
receptor site and one of the cells coming into contact with a
receptor site of another cell to form a clump is lowered by this
mutual repulsion. However, with the Polybrene being present, the
negative cells are attracted to the Polybrene to create
substantially electrically neutralized blood cell-polymer
combinations. The lectin which attaches to the blood cell is then
better able to agglutinate the blood cells and to clump since it
does not have to overcome or otherwise work against the repulsion
forces between the negatively charged blood cells since the blood
cells have in effect been neutralized by the Polybrene. As a
result, large, dense, three dimensional clumps of red blood cells
are formed which rapidly precipitate to the bottom of the sample
container. It is also important to note that the clumps of blood
cells are very durable in that they do not break-up when the sample
container is shaken, jarred or otherwise stirred. It is believed
that this reflects the strength of the lectin agglutinating link
between blood cells which no longer has to overcome the repulsion
of the blood cells due to the neutralization by the Polybrene. The
very dense nature of the clumps is to be attributable to the fact
that the lectin is capable of upholding the blood cells in a
closely linked relationship since the cells are no longer repelling
one another again due to the neutralization by the positively
charged polymer.
In order to enhance the precipitation of the formed elements of the
blood, the long axis of the collection tube is placed parallel to
the surface of a counter top. After the separation has taken place,
the tube is gradually rotated manually to dislodge and put into
solution any cells that remain on the glass part of the tube or
stopper. A rotation of the tube also aids in filtering out the
fibrin in the plasma to leave serum. The long axis of the tube is
then placed perpendicular to the counter top.
When serum, rather than plasma, is desired, a substance can be
added to inhibit the formation of fibrin or destroy the formation.
A substance may also be added to cause clotting and precipitate the
fibrin once it is present. For example, Fibrinolysin may be added
to the collection tube to prevent the formation of fibrin, or a
clotting agent such as thrombin may be added to accelerate clotting
of the fibrin. The clumped blood cells which form a net-like
structure effectively drag the fibrin downwardly with the clump of
formed blood elements to in effect filter, at least to some degree,
the fibrin out of the plasma.
In accordance with this one very important aspect of this
invention, the use of a positively charged polymer in combination
with a lectin produces a very rapid precipitation of the formed
elements of the blood as compared with the rate of precipitation
achieved using a lectin alone or a positively charged polymer
alone. FIG. 1a shows a collection tube filled with a blood sample
substantially immediately after the blood sample has been combined
with Polybrene and Bacto Phytohemagglutinin P. Note that the formed
elements of the blood near the bottom of the tube 10 have begun to
precipitate as can be seen from the light region 12 which
represents plasma and a relatively low concentration of the formed
blood elements. Precipitation results from the clumping of the
formed blood elements, which clumps 14 have been formed on the wall
of the tube 10. Microscopic examination of the clumps 14 indicates
that they are substantially three dimensional structures comprising
densely packed blood cells. After 15 minutes have passed, a layer
12' of plasma is achieved about 99% of the precipitated formed
elements of the blood. In this interim, a substantial number of the
clumps on the wall of the tube 10 have slid down the wall through
the plasma 12' and into the precipitated elements at the bottom of
the tube 10. Note that the clumps 14' which remain on the wall of
the tube 10 are much larger than the clumps 14 which existed
immediately after the blood had been combined with the Polybrene
and the Bacto Phytohemagglutinin P.
FIG. 2a is a tube 20 filled with a blood sample immediately after
it has been combined with Bacto Phytohemagglutinin P. alone without
the Polybrene being present. Once again, a layer of plasma 22 has
begun to form near the top of the blood sample due to the immediate
precipitation within the sample as a result of the clumps of blood
elements which are created by the Bacto Phytohemagglutinin P. The
clumps 24 on the walls of the tube 20 indicate that the Bacto
Phytohemagglutinin P. produces much smaller clumps than those
produced by the combination of the Polybrene and the Bacto
Phytohemagglutinin P. Note that the rate of precipitation of the
formed blood elements in the blood sample is much slower as
depicted in FIG. 2b. The layer of plasma 22' above 99% of the
formed blood elements at the end of 15 minutes of precipitation has
approximately half the height and volume as that produced with the
combination of Bacto Phytohemagglutinin P. and Polybrene as
depicted in FIG. 1b. Note also that a very large number of clumps
24' remain on the walls of the tube 20 after 15 minutes of
precipitation. Furthermore, these clumps are of substantially the
same size of the clumps 24 shown in FIG. 2a. In other words, the
size of the clumps does not increase with time and the clumps do
not become large enough to slide under the influence of gravity
down the walls of the tube 20.
In a container 30 illustrated in FIG. 3a, Polybrene has just been
added to a blood sample and precipitation has begun due to the
agglutinating properties of the Polybrene which forms clumps 34 on
the wall of the container 30. However, after 15 minutes, very
little actual precipitation has occurred as shown in FIG. 3b
wherein a very shallow layer of plasma 32' has formed above 99% of
the blood cells. Note that this layer of plasma 32' formed by the
Polybrene alone is much more shallow than the layer of plasma 22'.
This is the case even though the initial clumps 34 and the
subsequently formed clumps 34' which are quite fragile are larger
than the clumps 24 and 24' which are formed with the use of the
Bacto Phytohemagglutinin P. alone. Thus, the size of the clumps
alone is not the key to precipitation of the blood cells to
maximize the amount of plasma which can be obtained in a given
amount of time.
Note further that the amount of plasma 12' obtained utilizing the
combined agglutinating substances of the invention as shown in FIG.
1b is much more than the sum of the plasma obtained from utilizing
either of those agglutinating agents alone as shown in FIGS. 2b and
3b. Actual measurements indicate that the amount of plasma 12'
obtained utilizing this invention is approximately twice the amount
of plasma obtained in utilizing the individual ingredients alone as
depicted in FIGS. 2b and 3b. In other words, the result achieved in
utilizing the combination of the Polybrene and the Bacto
Phytohemagglutinin P. is more than (approximately twice) the sum of
the results achieved when utilizing these two ingredients
separately.
This synergistic result indicates that the positively charged
polymer and the lectin are interacting with one another to increase
the rate of precipitation of the clumped blood cells. If the
polyelectrolyte and the organic lectin were not interacting but
merely performing their own, independent agglutinating functions,
the amount of plasma obtained in a given amount of time utilizing
the combination should be equal to the sum of the plasma obtained
when the polyelectrolyte and the organic hemagglutinin are utilized
alone.
An explanation of this interaction, which is believed to be correct
in view of analyses performed after observing the results of this
combination, will now be set forth with reference to FIG. 4 in
conjunction with FIGS. 4a and 4b. FIG. 4 depicts a fragile clump of
blood cells 40 which are linked on to one another by polymeric
seams schematically depicted by the symbol x. Note that each of the
blood cells 40 is linked to at least one other blood cell by the
polymeric seam x and those blood cells 40 in the middle of the
clump are linked to two or three blood cells. However, each blood
cell does have a substantial surface area which is not adjacent, in
contact or connected to any other blood cell. An actual photograph
of this type of polymeric bonding between the blood cells 40 is
shown on page 132 of the aforesaid Katchalsky et al article
entitled "interactions of Basic Polyelectrolytes with the Red Blood
Cell". Since each cell 40 is apparently capable of forming a link
over a limited surface area, the resulting clump is not
particularly dense and voids 42 between the interconnected cells 40
of clump clamp do exist. The apparent inability of the blood cells
to form polymeric bonds along more of the surface area of each
blood cell is believed to be due to the fact that each negatively
charged blood cell 40 is capable of being attracted to a limited
number of positively charged polymers before the overall charge on
the combination polymerblood cell is neutralized so as to prevent
the attraction of that particular blood cell to any other
positively charged polymers.
FIG. 4b depicts a somewhat smaller clump than that shown in FIG. 4a
wherein the agglutination is achieved by a lectin. As shown in FIG.
4b, four cells 44 are linked or bonded as depicted by the symbol o.
The nature of the link o is not particularly well understood but
one very plausible explanation has been offered in the aforesaid
Liener article which suggests that each blood cell includes a
limited number of receptor sites to which a hemagglutinin such as
Bacto Phytohemagglutinin P. may be attached. Thus for two cells to
attach themselves to one another utilizing an agglutinating agent
such as Bacto Phytohemagglutinin P., receptor sites on two
different cells, must come in close proximity with the lectin
therebetween. Then the clumping of two cells 44 can occur. With
this limitation on the ability of the cells 44 to clump, it is not
suprising that each of the clumps 42 as depicted in FIGS. 2a and 2b
is rather small particularly in view of the repulsion between
negatively charged blood cells. It is also not surprising that
large areas of each of the cells 44 are not liked with any other
cells due to the absence or scarcity of receptor sites to form
those links.
When the positively charged polymer utilized to form the clump
depicted in FIG. 4a is combined with the lectin hemagglutinin which
forms the clump depicted in FIG. 4b, a dramatic difference is
observed in the nature of the clumps which are formed as depicted
in FIG. 4. As previously mentioned, it is believed that the
positively charged polymer is attracted to the negatively charged
elements or cells so as to neutralize those cells and thereby
permit the lectin which becomes attached to these cells to more
effectively agglutinate these cells since the negative charge on
the blood cells has been neutralized to preclude repulsion between
these cells. As shown in FIG. 4, the large, dense clump of blood
cells 46 is formed by a substantial number of individual blood
cells which are completely surrounded by other blood cells and
attached thereto by a plurality of lectin links o. Increased number
of lectin links is, as previously mentioned, due to the presence of
the positively charged polymer which neutralizes the negative
charge on the cells to preclude or at least substantially reduce
the repulsion therebetween. In addition, the positively charged
polymer forms bonds or seams x between blood cells in the clump so
as to perform the dual function of increasing the agglutinating
ability of the lectin while also providing its own agglutinating
function. When the clump in FIG. 4 is analyzed, it will be seen
that a substantial number of the blood cells in the clump have both
a lectin link o and a polymer seam x with other blood cells.
As mentioned previously, the size of the clumps is not the only
factor which determines the rate and extent of precipitation. If it
were, the positively charged polymer alone would precipitate more
rapidly than the organic lectin alone which would be inconsistent
with the experimental results depicted in FIGS. 2b and 3b. In this
connection, it is believed that the large, spider-like clumps of
blood cells, an example of which is depicted in FIG. 4a, create a
type of structure in the blood sample which is not only slow to
precipitate but actually self-supporting due to the formation of
fibrin so as to account for the rather shallow layer of plasma 32'
which is obtained when utilizing the positively charged polymer
alone as depicted in FIG. 3b. In contrast, the combination of the
positively charged polymer and the lectin achieves a dense clump
which precipitates rapidly and collapses into a rather limited area
so as to obtain a rather deep layer of plasma 12' as depicted in
FIG. 1b due to the inhibition of fibrin formation. This explanation
is borne out by microscopic examination of the blood cells at the
bottom of the tube 10 when the combination of the positively
charged polymer and the lectin is utilized. In particular, the
blood cells at the bottom of the tube 10 as depicted in FIG. 1b
seems to be a substantially unitary or integral mass of compact
blood cells which cling together when the tube 10 is tipped,
shaken, jarred or otherwise disturbed. In contrast, clumps of blood
cells precipitated to the bottom of containers 20 and 30 are not
part of an integral mass and separate from one another when the
tube is disturbed.
In accordance with another important aspect of the invention, rapid
precipitation of the blood cells is achieved while preserving the
integrity of the tests results and analysis of the plasma.
The chart reproduced below represents actual test results of my
system (M.S.) compared with the presently used conventional
centrifuged system (C.S.). A random selection of blood donors was
used, and the blood samplings from the centrifuged system were
obtained by using the standard Becton-Dickinson tubes and reagents.
In my system, Polybrene and Bacto Phytohemagglutinin P. were put
into a Becton-Dickinson red top vacutainer tube which initially
contained no reagents. Approximately one-half hour to 3 hours after
withdrawing the blood, the plasma from my tube and the standard
tube was removed. Most of the tests were performed with the
Technicon Auto-analyzer; the remainder being performed manually.
TEST C.S. M.S. TEST C.S. M.S.
______________________________________ Glucose 96 99 Chloride 104
107 (C.S. - 122 126 100 103 Gray top 96 96 97 99 tube used) 111 112
109 112 141 143 111 112 BUN 23 24 Sodium 133 137 20 20 137 134 16
15 139 138 22 22 140 142 21 22 137 136 31 31 Total 6.9 6.9 Potas-
4.3 4.2 Protein 6.1 6.5 sium 4.0 4.0 7.0 7.2 3.0 2.9 6.8 6.9 4.6
4.7 6.7 6.5 4.2 4.4 Albumin 5.3 5.0 Calcium 10.4 10.3 4.6 4.4 9.6
9.6 4.8 4.7 9.8 9.8 4.9 4.9 9.4 9.5 5.0 4.9 9.0 9.1 SGPT 12 12
Phos- 4.1 3.9 8 9 phorus 3.3 3.2 9 8 3.9 3.7 24 26 2.5 2.5
Cholesterol 238 228 Uric 6.9 6.9 324 314 Acid 4.7 4.6 267 257 10.6
10.3 299 289 5.6 5.5 CO.sub.2 26.5 26.5 Creati- 0.8 0.8 27.5 26.0
nine 0.8 0.8 27.5 26.0 1.9 1.9 23.0 22.5 1.4 1.4 26.0 25.0 1.0 1.1
Alk. Phos. 5.8 5.6 T-4 6.1 6.2 8.8 8.6 6.3 6.5 6.4 6.2 7.1 7.1 7.6
7.4 6.6 6.2 Serum Preg- Positive Positive P.B.I. 7.7 7.9 nancy Test
Negative Negative 6.8 6.9 Positive Positive 7.2 7.4 5.6 5.9 L.E.
Latex Negative Negative T-3 1.12 1.00 Positive Positive 1.07 .98
Negative Negative 1.04 1.07 Negative Negative Heterophile Positive
Positive Negative Negative Negative Negative Negative Negative
Positive Positive C. Reactive Negative Negative Protein Positive
Positive Negative Negative R. A. Latex Negative Negative Fixation
Negative Negative Positive Positive
______________________________________
The results reproduced in the above chart clearly indicate the
reliability of the method of my invention as compared with the
standard centrifuged separation method or any other presently
employed method.
In the foregoing, two specific chemical substances have been
mentioned for use in combination in practicing the invention,
namely, the positively charged polymer or basic polyelectrolyte
Polybrene (hexadimethrine bromide) and the organic hemagglutinin,
Bacto Phytohemagglutinin P. It will of course be appreciated that
other substances may be utilized in practicing the invention. For
example, another polyelectrolyte such as polylysine hydrobromide,
polyornithine hydrochloride, polyarginine sulfate, polyvinyl amine
hydrobromide and protamine sulfate may be utilized. Other lectins
suitable for use in the invention include ricin (extract from the
castor bean), Concanavalin A (globular type proteins isolated in
crystal form from jack bean meal as described by J. D. Sumner,
Journal of Biological Chemistry, vol, 37, page 137 (1919)) and
soybean hemagglutinin.
In the case of Polybrene and Bacto Phytohemagglutinin P. specific
concentrations have been described These concentrations may vary
but in general, it is preferred that a concentration be utilized
which is equal to 50-95% of the concentration which will lyse the
blood cells. This permits you to maximize the clumping and the
precipitation without hemolysis. Thus it will be appreciated that
the actual physical amounts of the different substances may very.
For example, ricin is a very powerful agglutinating agent and very
small amounts of ricin can result in hemolysis. Also, it will be
appreciated that the invention may be utilized with blood samples
of various sizes in excess of 30 cubic millimeters.
Although a specific embodiment of the invention has been described
and others have been suggested, it will be understood that the
invention embraces these and other embodiments as set forth in the
appended claims.
* * * * *