Kit for an initial screen for abnormal platelet condition comprising propyl gallate or tannin and a metal ion

Described is a novel screening method for detecting an abnormal platelet condition in blood, and a kit for use in such a method. An initial screen for an abnormal platelet condition in blood applies to a non-interfering test surface a platelet rich plasma specimen from the blood and an aqueous reagent including a hydroxy-substituted aromatic compound such as elagic acid, tannin or preferably propyl gallate and a metal ion such as Ni.sup.2+, Co.sup.2+, Fe.sup.3+, Cu.sup.+ or Cu.sup.2+ in concentrations sufficient to cause platelet aggregation in a normal platelet rich plasma sample upon agitation, lightly agitating the platelet rich plasma specimen in contact with the reagent, and visually detecting for the presence of platelet aggregates in the specimen.

BACKGROUND OF THE INVENTION 
The present invention resides in the field of assays to determine the 
capacity of blood platelets to aggregate. 
Although the causes of abnormal blood clotting are many, one cause which 
has been of particular importance is an abnormal platelet condition which 
renders the platelets unable to aggregate, a phenomenon essential to 
normal clotting. This abnormal platelet condition can be either a 
deficiency in the number of platelets in the patient's blood, or a defect 
in the platelets. 
As an example, it has been discovered that full-term pregnancy 
pre-eclamptic women often have prolonged bleeding times. This has been 
attributed to low platelet counts, and also in some instances is thought 
to be due to abnormalities in platelets. See, J. Ramanathan et al., 
Anesthesiol., 1989, 71, 188-91. Additionally, in the area of quality 
control, a recent article points up the need for a sensitive test which 
can be used to differentiate platelet concentrates which retain functional 
integrity after storage from those which do not, and explains that there 
is presently no available method for accomplishing this. T. Hervig et al., 
Clin. Chem., 1990, 36, No. 1, pp. 28 -31. 
Prior known and used tests for screening for abnormal platelet conditions 
are expensive and complicated. For example, the test currently used 
clinically is known as a platelet aggregation test. Generally, this test 
is performed by using expensive reagents such as ristocetin, collagen, 
adenosine diphosphate, or other reagents. Since these reagents work 
poorly, a sensitive optical instrument is used to follow the reaction. 
These tests, because of their inherent insensitivity, yield little 
clinically useful results. Needless to say, this test is not only costly, 
but also time consuming. Moreover, it cannot be conducted in the 
physician's office, necessitating sending samples to specialized 
laboratories for testing. 
What is therefore needed is an initial screen test for determining the 
capacity of a patient's platelets to aggregate which is simple, 
inexpensive, and which can be conducted rapidly in a physician's office. 
Depending on the outcome of this initial screen, further, more 
quantitative testing for platelet aggregation may or may not be indicated. 
The present invention addresses this need. 
SUMMARY OF THE PREFERRED EMBODIMENT 
One preferred embodiment of the invention provides an initial screen test 
for an abnormal platelet condition in blood. This screen comprises the 
steps of applying a platelet rich plasma specimen from the blood to a 
non-interfering test surface, contacting the platelet rich plasma specimen 
with an aqueous reagent including a hydroxy-substituted aromatic compound 
and a metal ion in sufficient concentrations to cause platelet aggregation 
in a normal platelet rich plasma specimen upon agitation, agitating the 
platelet rich plasma specimen in contact with the reagent, and visually 
detecting for the presence of platelet aggregates in the specimen. 
Another preferred embodiment of the invention provides a kit for an initial 
screen for an abnormal platelet condition in blood. The kit comprises a 
sealed vial containing preferrably a lyophilized, or alternatively a 
liquid, an aqueous reagent including a hydroxy-substituted aromatic 
compound and a metal ion in sufficient concentrations to cause platelet 
aggregation in a normal platelet rich plasma specimen upon agitation; an 
article including a plastic or siliconized glass surface for holding a 
platelet rich plasma specimen from the blood; and a member for holding the 
vial and the article in a relationship to prevent breakage. The preferred 
kit also includes printed matter for instructing a user to (1) contact a 
platelet rich plasma specimen with the reagent, (2) agitate the specimen 
in contact with the reagent, and (3) visually detect for the presence of 
platelet aggregates in the specimen upon its agitation. 
The invention provides a simple, reproducable and reliable initial screen 
for an abnormal platelet condition. This screen can be readily conducted 
in a physician's office without need for specialized equipment or 
prolonged sample analysis. Additional objects and advantages of the 
invention will be apparent from the following description. 
DESCRIPTION OF THE PREFERRED EMBODIMENTS 
For the purposes of promoting an understanding of the principles of the 
invention, reference will now be made to a preferred embodiment thereof 
and specific language will be used to describe the same. It will 
nevertheless be understood that no limitation of the scope of the 
invention is thereby intended, such alterations, further modifications 
applications of the principles of the invention being contemplated as 
would normally occur to one skilled in the art to which the invention 
pertains. 
As mentioned above, one preferred embodiment of the invention relates to an 
initial screen for an abnormal platelet condition in blood. A platelet 
rich plasma specimen from the blood is applied to a non-interfering test 
surface. The specimen is contacted with an aqueous reagent including a 
hydroxy-substituted aromatic compound and a divalent metal ion. The 
specimen in contact with the reagent is agitated on the test surface, and 
a visual detection is made for the presence of platelet aggregates in the 
specimen. 
In this regard, as used herein, the term "abnormal platelet condition" 
means a deficiency in the number of platelets in a blood sample or a 
defect in the platelets that substantially reduces or eliminates their 
ability to aggregate. The term "non-interfering test surface" means a 
surface that is inert to the platelets in the platelet rich plasma 
specimen and which will not itself cause them to adhere to the surface or 
aggregate. The term "hydroxy-substituted aromatic compound" means a 
compound having an aromatic ring directly attached to a hydroxyl group. 
The aromatic ring is preferably a carbocycle such as a phenyl ring. 
The platelet rich plasma specimen may be conventionally obtained. For 
example, it may be obtained by centrifuging a whole blood sample so as to 
separate platelet rich plasma in an upper layer. The platelet rich plasma 
specimen may then be drawn from this upper layer. These and other facets 
of drawing and preparing platelet rich plasma samples for testing are well 
within the abilities of those of ordinary skill in the pertinent art, and 
need not be further detailed here. 
The reagent is an aqueous medium including a hydroxy-substituted aromatic 
compound and a metal ion. As representative metal ions, there may be 
mentioned metal ions such as Cu.sup.2+, Ni.sup.2+, Co.sup.2+, Fe.sup.3+, 
and Cu.sup.+. Among these, Ni.sup.2+ is preferred. The metal ion is 
preferably included in the reagent at a level of about 10.sup.-9 to 
10.sup.-4 M, and more preferably about 10.sup.-4 to 10.sup.-5 M. These 
metal ions are also preferably provided by their water soluble halogen or 
sulfate salts, such as cupric sulfate, nickel chloride, or cobalt 
chloride. 
As representative hydroxy-substituted aromatic compounds, there may be 
mentioned ellagic acid, propyl gallate and tannin. Among these, propyl 
gallate is preferred. Preferably, the hydroxy-substituted aromatic 
compound is included in the reagent, in a molar concentration of about 
10.sup.-2 to about 10.sup.-9. Where ellagic acid is used, it is more 
preferably included at a level of about 10.sup.-4 to about 10.sup.-5 M. 
When propyl gallate is included, its more preferred level is about 
10.sup.-2 to about 10.sup.-3 M, and the more preferred level of tannin is 
about 10.sup.-5 to about 10.sup.-1 weight %, and even more preferably 
about 0.005 weight %. 
In addition to the above ingredients, the reagent also preferably includes 
a suitable buffer to maintain physiological pH. Many acceptable buffers of 
this type are known, including for instance 4-(2-hydroxyethyl)- 
1-piperazine-ethanesulfonic acid (also known as HEPES) and 
Tris(hydroxymethyl)aminomethane (also known as TRIS). The applicant's 
preferred buffer, however, is TRIS, which is most preferably included in a 
concentration of about 0.01M. 
The reagent is preferably a solution. Thus, the hydroxy-substituted 
aromatic compound and metal ion are fully solubilized, and the activity of 
the reagent, or at least a substantial percentage thereof, preferably 
about 85% or greater, and more preferably about 90-100%, is retained even 
after filtering through a 0.45 micron MILLIPORE filter. Accordingly, when 
herein a reagent is described as being a "solution", this means that a 
reagent's activity is substantially the same before and after filtering, 
such as through a 0.45 micron MILLIPORE filter. This retention of activity 
is evidenced by the similar aggregation results for plasma which are 
obtained using the reagent before, and after filtration. 
For additional information as to reagents suitable for use in this 
invention, reference can be made to my prior U.S. patent application, Ser. 
No. 07/510,178, filed Apr. 17, 1990, which is hereby incorporated herein 
by reference in its entirety. 
The article including the non-interfering surface is preferably a plastic 
or siliconized test tube or a plastic or siliconized glass slide, although 
other suitable, economically produced articles capable of holding the 
plasma specimen while agitating will also be suitable. In another 
preferred embodiment of the invention, this article is included in a kit 
which also includes a vial containing the reagent, with the vial and the 
article being retained in a relationship to prevent breakage, such as 
during handling or shipping. It will be understood the such a kit can also 
include additional vials of reagent and/or additional articles upon which 
to conduct the screen, and printed matter for instructing a user of the 
kit as to the screening method. 
In order to promote a further understanding of the invention and its 
preferred embodiments, the following specific Examples are provided. It 
will be understood that these examples are illustrative and not limiting 
in nature. 
[A] PREATION OF SUITABLE REAGENTS (Also referred to as "Activators"): 
The following examples illustrate the preparation of reagents which are 
suitable for use in the screen of the invention.

EXAMPLE 1 
Ellagic Acid and Copper II Activator 
0.18 g tetramethylammonium hydroxide were dissolved in 1,000 ml water. 
0.034 g ellagic acid were then dissolved in this tetramethylammonium 
hydroxide solution, whereafter 1.0 ml 0.1M cupric sulfate were added and 
the resulting solution mixed for 10 minutes with teflon coated stirring 
bar on a stir-plate. 1.2 g TRIS buffer were then dissolved into the 
solution. The resulting solution was clear and free from any visible 
particulate or other suspended matter. 
EXAMPLE 2 
Ellagic Acid and Nickel Activator 
Example 1 was repeated except 1.0 ml 0.1M nickel chloride was used instead 
of the 1.0 ml 0.1M cupric sulfate to form an ellagic acid/nickel 
coagulation activator which was also a clear solution free from visible 
particulate or other suspended matter. 
EXAMPLE 3 
Ellagic Acid and Cobalt Activator 
Example 1 was repeated except 1.0 ml 0.1M cobalt chloride was used instead 
of the 1.0 ml 0.1M cupric sulfate, and the TRIS buffer was not added. A 
clear ellagic acid/cobalt coagulation activator solution was formed. This 
unbuffered activator performs similarly to the buffered activators in 
Examples 1 and 2. The buffer further stabilizes the activators over time. 
EXAMPLE 4 
Propyl Gallate and Copper II Activator 
0.5 g propyl gallate were dissolved in 1,000 ml water. 1.0 ml 0.1M cupric 
sulfate were then added, whereafter the solution was mixed for 10 minutes 
with teflon-coated stir bar. 1.2 g TRIS buffer were then dissolved in this 
solution, which was thereafter stirred for an additional 10 minutes. A 
propyl gallate/copper coagulation activator was formed as a clear solution 
without any visible suspended materials. 
EXAMPLE 5 
Propyl Gallate and Nickel Activator 
Example 4 was repeated except 1.0 ml 0.1M nickel chloride was used instead 
of the cupric sulfate. A clear solution-form coagulation activator with 
propyl gallate and nickel was formed. 
EXAMPLE 6 
Propyl Gallate and Cobalt Activator 
Example 4 was repeated except 1.0 ml 0.1M cobalt chloride was used the 
place of the cupric sulfate to form a propyl gallate/cobalt solution-form 
coagulation activator. 
EXAMPLE 7 
Tannin and Copper II Activator 
0.5 g tannin were dissolved in 1,000 ml reagent water. 1.0 ml 0.1M cupric 
sulfate were added whereafter the resulting solution was mixed for 10 
minutes with a teflon coated stirring bar on a stir-plate. Then, 1.2 g 
TRIS buffer were added, and the resulting solution mixed for 10 minutes 
with a teflon coated stir-bar. A clear solution coagulation activator with 
tannin and copper was thus formed containing no visible suspended matter. 
EXAMPLE 8 
Tannin and Nickel Activator 
Example 7 was repeated except 1.0 ml 0.1M Nickel chlorine was used in the 
place of the cupric sulfate thus forming a solution-form coagulation 
activator with tannin and nickel. 
EXAMPLE 9 
Tannin and Cobalt Activator 
Example 7 was repeated except 0.1 ml 0.1M cobalt chloride was used instead 
of the cupric sulfate. Also, 2.5 g HEPES hemi sodium salt were added 
instead of the TRIS, to thus make a coagulation activator in solution form 
containing tannin and cobalt. 
[B] SCREENS FOR NORMAL AND ABNORMAL PLATELET CONDITION 
EXAMPLES 10 [I] and 10 [II] 
Screens for Abnormal Platelet Condition 
[I] TESTS ON NORMAL BLOOD 
Several platelet rich plasma specimens were obtained from blood known not 
to have an abnormal platelet condition. Each of these specimens was 
identically tested by placing the specimen (100 .mu.l) on a siliconized 
glass slide and subsequently adding the activator of Example 5 (100 .mu.l) 
to the specimen. The specimen was then agitated by gently rocking the 
glass slide, and tile appearance of platelet aggregates was noted. No 
stirring member was immersed in the fluid. (In other experiments, attempts 
to achieve admixture and agitation by use of a stirring member immersed in 
the fluid required larger sample sizes and substantially adversely 
affected the results.) The platelet aggregates appear as white 
agglomerates in the sample, and are readily detected visually. One example 
of a time for formation of the aggregates would be 60 seconds with normal 
samples. Similar results are obtained when the other activators 
exemplified above are employed. 
[II] SCREENING OF SAMPLES HAVING ABNORMAL PLATELET CONDITION 
Several screens were run on platelet rich plasma specimens known to have an 
abnormal platelet condition. The tests were run identically to those in 
Example 10 [I] above. The results show that the abnormal platelet 
conditions result in a failure to visually detect platelet aggregates in a 
period of three minutes. Similar results are obtained when the other 
activators illustrated in the Examples above are employed. 
EXAMPLES 11 -15 
[I] TESTS ON NORMAL BLOOD 
Instead of the propyl gallate and nickle chloride activator of Example 5, 
collagen, adrenalin, adenosine diphosphate, arachadonic acid, and 
ristocetin (each without additional metal ions) were alternately used in 
the procedure of Example 10 [I]. Results were much superior to mechanical 
platelet aggregometer tests in both time, cost, and sensitivity. However, 
the sensitivity was less than half that of Example 10 [I], and the tests 
took longer. 
While the invention has been described in detail in the foregoing 
description, the same is to be considered as illustrative and not 
restrictive in character, it being understood that only the preferred 
embodiments have been described, and that all changes and modifications 
that come within the spirit of the invention are desired to be protected.