Method and apparatus for collecting blood samples

This invention encompasses methods and apparatus for collecting blood samples. It has been discovered that p-aminobenzoyldiethylaminoethanol (procaine) is unexpectedly potent in stabilizing blood samples against platelet release reactions. Methods and apparatus of this invention are particularly useful for collecting blood samples for subsequent platelet factor 4 determination.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to apparatus and methods for collecting blood which 
is to be used in screening tests for activation of the coagulation system. 
Platelet Factor 4 (PF4) is a small molecular weight protein that is 
secreted from the .alpha.-granules of platelets when cells undergo the 
release reaction. This phenomenon occurs when the platelets become 
activated subsequent to contact with subendothelial tissue and a variety 
of other physiological agents including thrombin, adenosine diphosphate 
and epinephrine. PF4 in a blood sample is measured using classical 
radioimmunoassay techniques. Unfortunately classical blood collection 
techniques result in activatin of the release mechanism in the collected 
blood sample. Great care must be exercised to insure that the release 
reaction measured is a result of activating the release reaction in vivo 
and not in vitro. 
2. Prior Art 
The prior art discloses a wide variety of techniques and approaches for 
stabilizing collected blood samples against platelet release reactions. 
Careful handling of blood samples is required to inhibit the platelet 
release reaction. Generally the blood is collected by venipuncture 
techniques using standard EDTA (ethylenediaminetetraacetic acid) 
containing vacuum blood collection containers. The blood is mixed with the 
EDTA in the container by gentle inversion and the container is placed in 
an ice bath. After thirty minutes the sample is centrifuged at 
2500.times.g and 2.degree.-4.degree. C. for twenty minutes. Centrifugation 
must take place within two hours after collection. The plasma is separated 
and analyzed directly or it can be stored at 2.degree.-4.degree. C. for 24 
hours or at -20.degree. C. for up to three months. 
Thrombosis Research, 6, 543-548 (1975) describes collection of blook in the 
presence of EDTA and theophylline but requires centrifugation at 0.degree. 
C. 
Thrombosis Research, 12, 851-861 (1978) describes the collection of blood 
for PF4 analysis in the presence of platelet release inhibitor EDTA, 
prostoglandin E, and theophylline. This procedure also requires 
inconvenient centrifugation at low temperature. 
Niemetz, et al, Proc. Soc. Exp. Biol. Med, 128, 658 (1968) describes the 
effects of various anesthetics on clot retraction, adenosine diphosphate 
and thrombin-induced platelet aggregation. In those studies it was found 
that p-aminobenzoyldiethylaminoethanol hydrochloride (Procaine) and 
related homologs such as p-aminobenzoyldibutylaminoethanol (Butacaine) and 
p-butylaminobenzoyldimethylaminoethanol (Tetracaine) inhibited 
thrombin-induced platelet aggregation. Butacaine and tetracaine inhibited 
ADP-induced platelet aggregation while Procaine did not. Butacaine, 
tetracaine and procaine inhibited thrombin-induced platelet aggregation. 
Unexpectedly it has now been found that procaine and not butacaine and 
tetracaine inhibit the release of platelet factor 4 during the collection 
process. 
BRIEF DESCRIPTION OF THE INVENTION 
This invention describes apparatus and methods for inhibiting the platelet 
release reaction caused by in vitro handling of blood samples and 
therefore provides a blood sample where plasma components such as platelet 
factor 4 are a result of in vivo release. It has been discovered that 
p-aminobenzoyldiethylaminoethanol and the biologically acceptable acid 
addition salts thereof (Procaine) are unexpectedly effective in inhibiting 
the platelet release reaction and greatly facilitates handling of blood 
samples at room temperature for subsequent analysis for platelet release 
components such as platelet factor 4.

DETAILED DESCRIPTION OF THE INVENTION 
p-Aminobenzoyldiethylaminoethanol and the biologically acceptable acid 
addition salts thereof have been found to be particularly effective in 
inhibiting the platelet release reaction. Generally a final concentration 
in the blood of about 10-30 millimolar are effective in inhibiting the 
platelet release reaction with about 15 millimolar being a preferred 
effective amount. Biologically acceptable acid addition salts refers to a 
large variety of organic and mineral acid salts such as hydrochloric, 
hydrobromic, hydroiodic, sulfuric, phosphoric, nitric, acetic, benzoic, 
toluenesulfonic, formic acids and the like which are useful in preparing 
addition salts which do not destroy biological activity of proteins such 
as platelet factor 4. 
In a preferred embodiment 2-chloroadenosine in concentrations of about 
0.05-1 millimolar, preferably about 0.1 millimolar is used in combination 
with p-aminobenzoyldiethylaminoethanol hydrochloride to provide a 
particularly effective stabilizing reagent. 
These reagents are conveniently used in a conventional partially evacuated 
blood collection device containing anticoagulants such as 
ethylenediaminetetraacetic acid (EDTA). Thus conventional blood collecting 
devices can be injected with effective amounts of the above-described 
platelet release inhibiting agents or the device can be manufactured to 
contain these ingredients initially. 
In a broad context the invention encompasses a partially evacuated blood 
collection device containing an effective amount of 
p-aminobenzoyldiethylaminoethanol or the biologically acceptable acid 
addition salts thereof to inhibit the platelet release reaction. In 
particular the invention encompasses a partially evacuated siliconized 
stoppered glass tube containing EDTA as an anticoagulant and effective 
amounts of 2-chloroadenosine and p-aminobenzoyldiethylaminoethanol or the 
biologically acceptable acid addition salts thereof to inhibit the 
platelet release reaction. 
A most preferred embodiment is a 10 ml partially evacuated stoppered glass 
tube for blood collecting containing 0.6 ml of a reagent comprising 2.50% 
EDTA, 0.025% 2-chloroadenosine, 6.60% p-aminobenzoyldiethylaminoethanol 
hydrochloride, 0.3% sodium chloride having the pH adjusted to 6.5 with 
sodium hydroxide. The above-listed percents are weight/volume percents. 
Methods and devices of the present invention are particularly useful in 
platelet factor 4 determinations, in that sample collection and handling 
are greatly facilitated; for example, centrifugation at low temperature is 
not required. 
Using standard venipuncture techniques for multiple sampling two blood 
samples are drawn. The first is drawn to clear the system of activating 
factors, and the second is drawn into the above-identified most preferred 
embodiment. The sample is mixed by gentle inversion and the tube 
transferred to an ice bath to incubate for at least thirty minutes (no 
longer than two hours). The tube is then centrifuged at room temperature 
at 2500.times.g for twenty minutes or 1500.times.g for thirty minutes with 
a plasma separator. The plasma is transferred to a plastic tube with a 
plastic pipette. The sample may be stored at 2.degree.-4.degree. C. for 24 
hours or at -20.degree. C. for up to three months. 
Platelet factor 4 is determined by conventional radioimmunoassay 
techniques. Using methods and devices of this invention platelet factor 4 
normal levels are less than 5 ng/ml. Patients with coronary artery 
disease, post-heart valve surgery, post-coronary bypass surgery, 
post-myocardial infarction, and diabetes have elevated levels of platelet 
factor 4 ranging as high as 100 ng/ml.