Test combination and method of detecting fibrin monomers in blood

A test reagent for detecting fibrin monomers in blood, comprises an antibiotic of a pyrrole amidine series as a precipitant, introduced in a buffer solution. A method of detecting fibrin monomers in blood, comprises the steps of mixing venous blood with an anticoagulant, centrifuging the mixture of the venous blood with the anticoagulant, removing a citrate plasma, using an antibiotic of a pyrrole amidine series as a precipitant, and evaluating a precipitation reaction which takes place thereby.

BACKGROUND OF THE INVENTION 
The present invention relates to a test reagent for detecting fibrin 
monomers and a method of detecting the same in human blood plasma. 
The appearance of fibrin monomers in blood is a proof of an activation of 
the hemostasis system. This is a clinically frequently occurring symptom 
of many basic illnesses. Therefore, after the hypercoagulation stage, 
locally caused thromboses or generally coagulation pathologies can develop 
with resulting heavy bleeding. The possible early detection of coagulating 
activity is a prerequisite for an effective therapy. 
Several principles for detecting the fibrin monomers are known in the art. 
The oldest methods include the ethanol-gelatin test in accordance with 
GODAL (H. GODAL and U. ABILGAARD, Scand. J. Haem. 3, 1966, 342) and the 
protamine sulfate test in accordance with LIPPINSKI (B. LIPPINSKI and K. 
WAROWSKI, Thromb. Diath. Haem. 20, 1968, 44). These methods are easy to 
carry out. However, they show an insufficient sensitivity and specificity. 
A specific test is the agglutination test with fibrin monomer loaded 
erythrocytes in accordance with LARGO (R. LARGO et al., Blood 47, 1976, 
991). Furthermore, the test suitable for the diagnosing of von 
Willebrand's disease based on the precipitator Ristocetin-A (Ristomycin-A) 
is used for detecting of fibrin monomers (K. WATANABE and J. L. TULLIS, 
Amer. J. Clin. Pathol. 70, 1978, 691; G. PFLIEGLER et al. Abstr. Int. 
Congr. ISH-ISBT, Budapest, Aug. 1982, p. 380). Numerous comparative tests 
have shown that with the above listed precipitators, an nonspecific 
precipitation with fibrin cleavage products takes place, and with 
increasing thrombocyte content of the plasma used for detection, falsely 
positive reactions are obtained so that the reliability of the test is not 
guaranteed. In addition to the above methods, also various further methods 
which are connected with expensive analysis techniques are known (affinity 
chromatography gel-filtration). 
SUMMARY OF THE INVENTION 
Accordingly, it is an object of the present invention to provide a new test 
reagent for detecting fibrin monomers in human blood. 
It is also an object of the present invention to provide, on the basis of 
the proposed new test reagent, an economical method of detecting fibrin 
monomers by precipitation. 
In keeping with these objects and with others which will become apparent 
hereinafter, one feature of the present invention resides, briefly stated, 
in a test reagent which contains an antibiotic of the pyrrole-amidine 
series as a precipitator introduced in a buffer solution. 
In accordance with an advantageous feature of the present invention, in the 
new test reagent such pyrrole-amidine series antibiotics as netropsin, 
distamycin, anthelvencin or analogs introduced in HEPES-buffer are used. 
In accordance with a further advantageous feature of the present invention 
the selected pyrrole-amidine series antibiotic is used in its salt form, 
for example substantially as its hydrochloride. 
Still another feature of the present invention is a method of detecting 
fibrin monomers in human blood, in accordance with which human blood is 
mixed with an anticoagulant in a known manner, then centrifuged and the 
citrate plasma is mixed for precipitation with the above mentioned new 
test reagent. Then precipitation reaction, or in other words the 
precipitate sedimented on the bottom of the test vessel, is analyzed in a 
known manner. 
In accordance with still a further feature of the inventive method, the 
plasma with the precipitant or in other words with the test reagent in 
accordance with the present invention is maintained under room temperature 
and subsequently centrifuged. 
When the test composition is made and the method is performed in accordance 
with the present invention, a very cost favorable precipitant can be used 
such as for example netropsin hydrochloride, and high diagnostic 
reliability is guaranteed. 
DESCRIPTION OF THE PREFERRED EMBODIMENTS 
In accordance with the present invention a test reagent contains an 
antibiotic of the pyrrole-amidine series raw as a precipitant, introduced 
in a buffer solution. Advantageously, the pyrrole-amidine series 
antibiotic can be netropsin, distamycin, and anthelvencin or analogs, and 
the buffer solution can be a HEPES-buffer. The selected pyrrole-amidine 
series antibiotic can be in its salt form, for example as a hydrochloride. 
In accordance with the method of the invention, human blood is mixed in a 
known manner with an anticoagulant, then centrifuged, and the citrate 
plasma is mixed for precipitation with the above described new test 
reagent. Then, the precipitation reaction, or in other words the 
precipitate sedimented, is analyzed. The plasma provided with the 
precipitant or in other words with the test reagent of the invention is 
held under room temperature for the evaluation, and then centrifuged.

The advantages of the inventive solution for detecting fibrin monomers is 
illustrated by two examples presented hereinbelow: 
EXAMPLE I 
Venous blood is recovered exactly in accordance with the rules applicable 
for coagulation tests and citrate plasma is prepared in accordance with 
conventional methods. 
Netropsin-HCl is dissolved in accordance with the requirements in a 
concentration of 15 mg netropsin/ml with HEPES-buffer (0.15 molar, pH 7.5) 
so that a test reagent Netropsin RL is produced. 
0.4 ml citrate plasma is mixed with 0.1 ml netropsin-RL in a centrifuge 
tube and is retained for 30 minutes at room temperature. Then this tube is 
centrifuged for 5 minutes with maximum 50 r.p.m. The precipitate formed on 
the bottom is evaluated in accordance with the following diagram: 
______________________________________ 
flaky loose precipitation 
+ 
fibrous deposits ++ 
compact sediments +++ 
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The probes evaluated as negative in this manner are transferred to black 
small block dishes and again evaluated A negative evaluation of course 
corresponds to fibrin monomers not being detected, while the more positive 
the evaluation the stronger is the indication of fibrin monomers. 
A positive control for comparison is carried for each measuring row. 
EXAMPLE II 
The preparation, performance and evaluation of the precipitation is carried 
out as in the Example I. The precipitant in this example is, however, 
distamycin-HCl, which is dissolved in a concentration of 6 mg/ml in a 
HEPES-buffer (test reagent Distamycin RL). The sensitivity of this 
precipitation amounts to approximately 50%, as compared with netropsin. 
With the utilization of the inventive test reagent Netropsin RL and the 
respective methods, the following results were obtained in clinical 
laboratory tests: 
Examination of the testing method for its analytical usefulness. 
In vitro produced fibrin monomer containing plasma showed clearly a 
positive reaction. 
From split products of fibrinogens and fibrins (e-FDP, 1-FDP, e-fdp, 1-fdp) 
recovered in individual preparation, only the fraction of the early fibrin 
cleavage product (e-fdp) is detected. Since e-fdp is also a product of a 
thrombin-caused fibrinogen conversion, the specificity of the test is not 
effected. From a concentration of the low molecular salt products fragment 
D (0.3 g/1) and fragment E (0.15 g/1) a weakening of the precipitation is 
observed. 
A fibrinogen-dependent influence on the test result could be not found till 
a plasma fibrinogen concentration of 19 g/l is reached. 
An influence of heparin on the precipitation could not be observed. 
There was no dependency of the precipitation on thrombocyte content of the 
used plasma. 
There was no influence of plasma expanders upon dextran- and gelatin base. 
The test was not influenced by changes in the albumin, alpha-globulin, 
immunglobulin-G and the total albumin concentration of the plasma. 
Examinations for specificity of the test method 
The examination of 40 hemostatically healthy hospitalized patients using 
conventional blood taking techniques for the coagulation test produces a 
negative test result in all cases (specificity=1.0). 
Examination of the Sensitivity of the Test Method 
40 patients with clinically and laboratory-diagnostically reliably 
disseminated intravascular coagulation were subjected to the above 
described test, and in 37 cases a positive result was achieved. Thereby 
the method can be considered with a sensitivity of 0.93 as a suitable 
laboratory parameter for diagnosing of the disseminated intravascular 
coagulation. 
It will be understood that each of the elements described above, or two or 
more together, may also find a useful application in other types of test 
combinations and methods differing from the types described above. 
While the invention has been illustrated and described as embodied in a 
test reagent and method it is not intended to be limited to the details 
shown, since various modifications and structural changes may be made 
without departing in any way from the spirit of the present invention. 
Without further analysis, the foregoing will so fully reveal the gist of 
the present invention that others can, by applying current knowledge, 
readily adapt it for various applications without omitting features that, 
from the standpoint of prior art, fairly constitute essential 
characteristics of the generic or specific aspects of this invention.